Subcellular distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase in bovine and murine adrenocortical tissue: species differences in the localization of activity and immunoreactivity.

PubMed

Perry, J E; Ishii-Ohba, H; Stalvey, J R

1991-06-01

Key to the production of biologically active steroids is the enzyme 3 beta-hydroxysteroid dehydrogenase-isomerase. Some controversy has arisen concerning the subcellular distribution of this enzyme within steroidogenic cells. The distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase was assessed in subcellular fractions obtained from homogenates of rat, bovine, and mouse adrenal glands in two ways. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was quantitated by measuring the conversion of radiolabeled pregnenolone to radiolabeled progesterone in an aliquot of each of the fractions obtained. The presence of the enzyme was assessed by performing Western analyses on aliquots of each of the fractions obtained with the use of a specific polyclonal antiserum against 3 beta-hydroxysteroid dehydrogenase-isomerase, the characterization of which is described. In control experiments, the degree of contamination of the fractions was determined by assessing the presence of known subcellular fraction markers with Western analysis. In the bovine and mouse adrenal glands, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to be localized solely in the microsomal fraction, while in the rat, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to have dual subcellular distribution: the microsomes and the inner mitochondrial membrane. We conclude that there is a species difference in the subcellular distribution of this important steroidogenic enzyme and that this species difference may be related to the steroidogenic pathway preferred in that species.

Visualization of metallodrugs in single cells by secondary ion mass spectrometry imaging.

PubMed

Wu, Kui; Jia, Feifei; Zheng, Wei; Luo, Qun; Zhao, Yao; Wang, Fuyi

2017-07-01

Secondary ion mass spectrometry, including nanoscale secondary ion mass spectrometry (NanoSIMS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), has emerged as a powerful tool for biological imaging, especially for single cell imaging. SIMS imaging can provide information on subcellular distribution of endogenous and exogenous chemicals, including metallodrugs, from membrane through to cytoplasm and nucleus without labeling, and with high spatial resolution and chemical specificity. In this mini-review, we summarize recent progress in the field of SIMS imaging, particularly in the characterization of the subcellular distribution of metallodrugs. We anticipate that the SIMS imaging method will be widely applied to visualize subcellular distributions of drugs and drug candidates in single cells, exerting significant influence on early drug evaluation and metabolism in medicinal and pharmaceutical chemistry. Recent progress of SIMS applications in characterizing the subcellular distributions of metallodrugs was summarized.

Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

PubMed

Vespa, Alisa; Darmon, Alison J; Turner, Christopher E; D'Souza, Sudhir J A; Dagnino, Lina

2003-03-28

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies

PubMed Central

Thapa, Dharendra; Shepherd, Danielle L.

2014-01-01

Cardiac tissue contains discrete pools of mitochondria that are characterized by their subcellular spatial arrangement. Subsarcolemmal mitochondria (SSM) exist below the cell membrane, interfibrillar mitochondria (IFM) reside in rows between the myofibrils, and perinuclear mitochondria are situated at the nuclear poles. Microstructural imaging of heart tissue coupled with the development of differential isolation techniques designed to sequentially separate spatially distinct mitochondrial subpopulations have revealed differences in morphological features including shape, absolute size, and internal cristae arrangement. These findings have been complemented by functional studies indicating differences in biochemical parameters and, potentially, functional roles for the ATP generated, based upon subcellular location. Consequently, mitochondrial subpopulations appear to be influenced differently during cardiac pathologies including ischemia/reperfusion, heart failure, aging, exercise, and diabetes mellitus. These influences may be the result of specific structural and functional disparities between mitochondrial subpopulations such that the stress elicited by a given cardiac insult differentially impacts subcellular locales and the mitochondria contained within. The goal of this review is to highlight some of the inherent structural and functional differences that exist between spatially distinct cardiac mitochondrial subpopulations as well as provide an overview of the differential impact of various cardiac pathologies on spatially distinct mitochondrial subpopulations. As an outcome, we will instill a basis for incorporating subcellular spatial location when evaluating the impact of cardiac pathologies on the mitochondrion. Incorporation of subcellular spatial location may offer the greatest potential for delineating the influence of cardiac pathology on this critical organelle. PMID:24778166

Subcellular partitioning of metals in Aporrectodea caliginosa along a gradient of metal exposure in 31 field-contaminated soils.

PubMed

Beaumelle, Léa; Gimbert, Frédéric; Hedde, Mickaël; Guérin, Annie; Lamy, Isabelle

2015-07-01

Subcellular fractionation of metals in organisms was proposed as a better way to characterize metal bioaccumulation. Here we report the impact of a laboratory exposure to a wide range of field-metal contaminated soils on the subcellular partitioning of metals in the earthworm Aporrectodea caliginosa. Soils moderately contaminated were chosen to create a gradient of soil metal availability; covering ranges of both soil metal contents and of several soil parameters. Following exposure, Cd, Pb and Zn concentrations were determined both in total earthworm body and in three subcellular compartments: cytosolic, granular and debris fractions. Three distinct proxies of soil metal availability were investigated: CaCl2-extractable content dissolved content predicted by a semi-mechanistic model and free ion concentration predicted by a geochemical speciation model. Subcellular partitionings of Cd and Pb were modified along the gradient of metal exposure, while stable Zn partitioning reflected regulation processes. Cd subcellular distribution responded more strongly to increasing soil Cd concentration than the total internal content, when Pb subcellular distribution and total internal content were similarly affected. Free ion concentrations were better descriptors of Cd and Pb subcellular distribution than CaCl2 extractable and dissolved metal concentrations. However, free ion concentrations and soil total metal contents were equivalent descriptors of the subcellular partitioning of Cd and Pb because they were highly correlated. Considering lowly contaminated soils, our results raise the question of the added value of three proxies of metal availability compared to soil total metal content in the assessment of metal bioavailability to earthworm. Copyright © 2015 Elsevier B.V. All rights reserved.

In Situ Subcellular Imaging of Copper and Zinc in Contaminated Oysters Revealed by Nanoscale Secondary Ion Mass Spectrometry.

PubMed

Weng, Nanyan; Jiang, Haibo; Wang, Wen-Xiong

2017-12-19

Determining the in situ localization of trace elements at high lateral resolution levels in the biological system is very challenging, but critical for our understanding of metal sequestration and detoxification. Here, the cellular and subcellular distributions of Cu and Zn in contaminated oysters of Crassostrea hongkongensis were for the first time mapped using nanoscale secondary ion mass spectrometry (nanoSIMS). Three types of metal-containing cells were revealed in the gill and mantle of oysters, including Cu-specific hemocytes, Cu and Zn-containing granular hemocytes, and Cu and Zn-containing calcium cells. Obvious intercellular distribution of Cu was found in the gill tissue, indicating the potential role of hemolymph in the transportation of Cu in oysters. The distribution of Cu showed a strong colocalization with sulfur and nitrogen in Cu-specific hemocyte and intercellular hemolymph. In the Cu and Zn-containing granular hemocytes and calcium cells, the co-occurrence of Cu and Zn with phosphorus and calcium was also found. Different relationships of distributions between Cu/Zn and macronutrient elements (nitrogen, sulfur and phosphorus) implied the differential metal complexation in oysters. Interestingly, quantitative analysis of the ratios of 32 S - / 12 C 14 N - and 31 P - / 12 C 14 N - of metal-deposited sites suggested the dynamic process of transfer of Cu and Zn from the metabolized protein pool to a more thermodynamically stable and detoxified form.

Quantification of non-coding RNA target localization diversity and its application in cancers.

PubMed

Cheng, Lixin; Leung, Kwong-Sak

2018-04-01

Subcellular localization is pivotal for RNAs and proteins to implement biological functions. The localization diversity of protein interactions has been studied as a crucial feature of proteins, considering that the protein-protein interactions take place in various subcellular locations. Nevertheless, the localization diversity of non-coding RNA (ncRNA) target proteins has not been systematically studied, especially its characteristics in cancers. In this study, we provide a new algorithm, non-coding RNA target localization coefficient (ncTALENT), to quantify the target localization diversity of ncRNAs based on the ncRNA-protein interaction and protein subcellular localization data. ncTALENT can be used to calculate the target localization coefficient of ncRNAs and measure how diversely their targets are distributed among the subcellular locations in various scenarios. We focus our study on long non-coding RNAs (lncRNAs), and our observations reveal that the target localization diversity is a primary characteristic of lncRNAs in different biotypes. Moreover, we found that lncRNAs in multiple cancers, differentially expressed cancer lncRNAs, and lncRNAs with multiple cancer target proteins are prone to have high target localization diversity. Furthermore, the analysis of gastric cancer helps us to obtain a better understanding that the target localization diversity of lncRNAs is an important feature closely related to clinical prognosis. Overall, we systematically studied the target localization diversity of the lncRNAs and uncovered its association with cancer.

Separate responses of karyopherins to glucose and amino acid availability regulate nucleocytoplasmic transport

PubMed Central

Huang, Hsiao-Yun; Hopper, Anita K.

2014-01-01

The importin-β family members (karyopherins) mediate the majority of nucleocytoplasmic transport. Msn5 and Los1, members of the importin-β family, function in tRNA nuclear export. tRNAs move bidirectionally between the nucleus and the cytoplasm. Nuclear tRNA accumulation occurs upon amino acid (aa) or glucose deprivation. To understand the mechanisms regulating tRNA subcellular trafficking, we investigated whether Msn5 and Los1 are regulated in response to nutrient availability. We provide evidence that tRNA subcellular trafficking is regulated by distinct aa-sensitive and glucose-sensitive mechanisms. Subcellular distributions of Msn5 and Los1 are altered upon glucose deprivation but not aa deprivation. Redistribution of tRNA exportins from the nucleus to the cytoplasm likely provides one mechanism for tRNA nuclear distribution upon glucose deprivation. We extended our studies to other members of the importin-β family and found that all tested karyopherins invert their subcellular distributions upon glucose deprivation but not aa deprivation. Glucose availability regulates the subcellular distributions of karyopherins likely due to alteration of the RanGTP gradient since glucose deprivation causes redistribution of Ran. Thus nuclear–cytoplasmic distribution of macromolecules is likely generally altered upon glucose deprivation due to collapse of the RanGTP gradient and redistribution of karyopherins between the nucleus and the cytoplasm. PMID:25057022

Optogenetic stimulation of myelination (Conference Presentation)

NASA Astrophysics Data System (ADS)

Yang, In Hong; Lee, Hae Ung; Thakor, Nitish V.

2016-03-01

Myelination is governed by axon-glia interaction which is modulated by neural activity. Currently, the effects of subcellular activation of neurons which induce neural activity upon myelination are not well understood. To identify if subcellular neuronal stimulation can enhance myelination, we developed a novel system for focal stimulation of neural activity with optogenetic in a compartmentalized microfluidic platform. In our systems, stimulation for neurons in restricted subcellular parts, such as cell bodies and axons promoted oligodendrocyte differentiation and the myelination of axons the just as much as whole cell activation of neurons did. The number of premature O4 positive oligodendrocytes was reduced and the numbers of mature and myelin basic protein-positive oligodendrocytes was increased both by subcellular optogenetic stimulation.

Imaging trace element distributions in single organelles and subcellular features

NASA Astrophysics Data System (ADS)

Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

2016-02-01

The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells.

PubMed

Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

2014-02-18

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca(2+)] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells.

Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

PubMed Central

Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

2014-01-01

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells. PMID:24535122

Hepatic subcellular distribution of squalene changes according to the experimental setting.

PubMed

Martínez-Beamonte, Roberto; Alda, Olga; Sanclemente, Teresa; Felices, María J; Escusol, Sara; Arnal, Carmen; Herrera-Marcos, Luis V; Gascón, Sonia; Surra, Joaquín C; Osada, Jesús; Rodríguez-Yoldi, Mª Jesús

2018-02-22

Squalene is the main unsaponifiable component of virgin olive oil, the main source of dietary fat in Mediterranean diet, traditionally associated with a less frequency of cardiovascular diseases. In this study, two experimental approaches were used. In the first, New Zealand rabbits fed for 4 weeks with a chow diet enriched in 1% sunflower oil for the control group, and in 1% of sunflower oil and 0.5% squalene for the squalene group. In the second, APOE KO mice received either Western diet or Western diet enriched in 0.5% squalene for 11 weeks. In both studies, liver samples were obtained and analyzed for their squalene content by gas chromatography-mass spectrometry. Hepatic distribution of squalene was also characterized in isolated subcellular organelles. Our results show that dietary squalene accumulates in the liver and a differential distribution according to studied model. In this regard, rabbits accumulated in cytoplasm within small size vesicles, whose size was not big enough to be considered lipid droplets, rough endoplasmic reticulum, and nuclear and plasma membranes. On the contrary, mice accumulated in large lipid droplets, and smooth reticulum fractions in addition to nuclear and plasma membranes. These results show that the squalene cellular localization may change according to experimental setting and be a starting point to characterize the mechanisms involved in the protective action of dietary squalene in several pathologies.

Biodynamics of copper oxide nanoparticles and copper ions in an oligochaete - Part II: Subcellular distribution following sediment exposure

USGS Publications Warehouse

Thit, Amalie; Ramskov, Tina; Croteau, Marie-Noele; Selck, Henriette

2016-01-01

The use and likely incidental release of metal nanoparticles (NPs) is steadily increasing. Despite the increasing amount of published literature on metal NP toxicity in the aquatic environment, very little is known about the biological fate of NPs after sediment exposures. Here, we compare the bioavailability and subcellular distribution of copper oxide (CuO) NPs and aqueous Cu (Cu-Aq) in the sediment-dwelling worm Lumbriculus variegatus. Ten days (d) sediment exposure resulted in marginal Cu bioaccumulation in L. variegatus for both forms of Cu. Bioaccumulation was detected because isotopically enriched 65Cu was used as a tracer. Neither burrowing behavior or survival was affected by the exposure. Once incorporated into tissue, Cu loss was negligible over 10 d of elimination in clean sediment (Cu elimination rate constants were not different from zero). With the exception of day 10, differences in bioaccumulation and subcellular distribution between Cu forms were either not detectable or marginal. After 10 d of exposure to Cu-Aq, the accumulated Cu was primarily partitioned in the subcellular fraction containing metallothionein-like proteins (MTLP, ≈40%) and cellular debris (CD, ≈30%). Cu concentrations in these fractions were significantly higher than in controls. For worms exposed to CuO NPs for 10 d, most of the accumulated Cu was partitioned in the CD fraction (≈40%), which was the only subcellular fraction where the Cu concentration was significantly higher than for the control group. Our results indicate that L. variegatus handle the two Cu forms differently. However, longer-term exposures are suggested in order to clearly highlight differences in the subcellular distribution of these two Cu forms.

Separate responses of karyopherins to glucose and amino acid availability regulate nucleocytoplasmic transport.

PubMed

Huang, Hsiao-Yun; Hopper, Anita K

2014-09-15

The importin-β family members (karyopherins) mediate the majority of nucleocytoplasmic transport. Msn5 and Los1, members of the importin-β family, function in tRNA nuclear export. tRNAs move bidirectionally between the nucleus and the cytoplasm. Nuclear tRNA accumulation occurs upon amino acid (aa) or glucose deprivation. To understand the mechanisms regulating tRNA subcellular trafficking, we investigated whether Msn5 and Los1 are regulated in response to nutrient availability. We provide evidence that tRNA subcellular trafficking is regulated by distinct aa-sensitive and glucose-sensitive mechanisms. Subcellular distributions of Msn5 and Los1 are altered upon glucose deprivation but not aa deprivation. Redistribution of tRNA exportins from the nucleus to the cytoplasm likely provides one mechanism for tRNA nuclear distribution upon glucose deprivation. We extended our studies to other members of the importin-β family and found that all tested karyopherins invert their subcellular distributions upon glucose deprivation but not aa deprivation. Glucose availability regulates the subcellular distributions of karyopherins likely due to alteration of the RanGTP gradient since glucose deprivation causes redistribution of Ran. Thus nuclear-cytoplasmic distribution of macromolecules is likely generally altered upon glucose deprivation due to collapse of the RanGTP gradient and redistribution of karyopherins between the nucleus and the cytoplasm. © 2014 Huang and Hopper. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Mercury speciation and subcellular distribution in experimentally dosed and wild birds.

PubMed

Perkins, Marie; Barst, Benjamin D; Hadrava, Justine; Basu, Niladri

2017-12-01

Many bird species are exposed to methylmercury (MeHg) at levels shown to cause sublethal effects. Although MeHg sensitivity and assimilation can vary among species and developmental stages, the underlying reasons (such as MeHg toxicokinetics) are poorly understood. We investigated Hg distribution at the tissue and cellular levels in birds by examining Hg speciation in blood, brain, and liver and Hg subcellular distribution in liver. We used MeHg egg injection of white leghorn chicken (Gallus gallus domesticus), sampled at 3 early developmental stages, and embryonic ring-billed gulls (Larus delawarensis) exposed to maternally deposited MeHg. The percentage of MeHg (relative to total Hg [THg]) in blood, brain, and liver ranged from 94 to 121%, indicating little MeHg demethylation. A liver subcellular partitioning procedure was used to determine how THg was distributed between potentially sensitive and detoxified compartments. The distributions of THg among subcellular fractions were similar among chicken time points, and between embryonic chicken and ring-billed gulls. A greater proportion of THg was associated with metal-sensitive fractions than detoxified fractions. Within the sensitive compartment, THg was found predominately in heat-denatured proteins (∼42-46%), followed by mitochondria (∼15-18%). A low rate of MeHg demethylation and high proportion of THg in metal-sensitive subcellular fractions further indicates that embryonic and hatchling time points are Hg-sensitive developmental stages, although further work is needed across a range of additional species and life stages. Environ Toxicol Chem 2017;36:3289-3298. © 2017 SETAC. © 2017 SETAC.

Dynamic changes to survivin subcellular localization are initiated by DNA damage

PubMed Central

Asumen, Maritess Gay; Ifeacho, Tochukwu V; Cockerham, Luke; Pfandl, Christina; Wall, Nathan R

2010-01-01

Subcellular distribution of the apoptosis inhibitor survivin and its ability to relocalize as a result of cell cycle phase or therapeutic insult has led to the hypothesis that these subcellular pools may coincide with different survivin functions. The PIK kinases (ATM, ATR and DNA-PK) phosphorylate a variety of effector substrates that propagate DNA damage signals, resulting in various biological outputs. Here we demonstrate that subcellular repartitioning of survivin in MCF-7 cells as a result of UV light-mediated DNA damage is dependent upon DNA damage-sensing proteins as treatment with the pan PIK kinase inhibitor wortmannin repartitioned survivin in the mitochondria and diminished it from the cytosol and nucleus. Mitochondrial redistribution of survivin, such as was recorded after wortmannin treatment, occurred in cells lacking any one of the three DNA damage sensing protein kinases: DNA-PK, ATM or ATR. However, failed survivin redistribution from the mitochondria in response to low-dose UV occurred only in the cells lacking ATM, implying that ATM may be the primary kinase involved in this process. Taken together, this data implicates survivian’s subcellular distribution is a dynamic physiological process that appears responsive to UV light-initiated DNA damage and that its distribution may be responsible for its multifunctionality. PMID:20856848

Differential tolerance of two Gammarus pulex populations transplanted from different metallogenic regions to a polymetal gradient.

PubMed

Khan, Farhan R; Irving, Jennifer R; Bury, Nicolas R; Hogstrand, Christer

2011-03-01

The River Hayle, Cornwall, UK exhibits pronounced Cu and Zn concentration gradients which were used to compare the metal handling abilities of two populations of Gammarus pulex (Crustacea: Amphipoda). One population was native to the Hayle region (Drym) and presumably has been historically impacted by elevated Cu and Zn levels, whilst naïve gammarids were collected from the River Cray, Kent, UK. Both populations were subject to a 32 day in situ exposure at four R. Hayle sites (Drym, Godolphin, Relubbus and St. Erth). Mortality (LT50), Cu and Zn accumulation and sub-cellular distribution, and oxidative stress (malondialdehyde production) increased with the expected Cu and Zn bioavailabilities at the four sites (i.e. Godolphin>Relubbus>St. Erth>Drym). The naïve population experienced greater metal induced effects in terms of Cu and Zn accumulation, oxidative stress responses and lower LT50s. Analysis of Cu and Zn sub-cellular distribution, however, revealed no significant differences in metal handling. In both populations each metal was localised predominantly to the sub-cellular fraction containing metal bound to metallothionein-like proteins (MTLP) or that holding both metal-rich granules (MRG) and exoskeleton, MTLP and MRG binding being indicative of metal detoxification. However, a greater capacity for detoxified metal storage is not a mechanism implicated in the perceived tolerance of the historically impacted gammarids. Instead our results suggest that the historically impacted population was adapted for lower uptake of Cu and Zn leading to lower bioaccumulation, stress response and ultimately mortality. These results demonstrate not only the usefulness of the in situ methodology, but also that differences in population exposure history can cause significant differences in metal responses during exposure at higher concentrations. Copyright © 2011 Elsevier B.V. All rights reserved.

Subcellular Distribution of O-Acetylserine(thiol)lyase in Cauliflower (Brassica oleracea L.) Inflorescence.

PubMed

Rolland, N; Droux, M; Douce, R

1992-03-01

The subcellular localization of O-acetyiserine(thiol)lyase (EC 4.2.99.8) in nongreen tissue from higher plants has been studied using purified proplastids, mitochondria, and protoplasts from cauliflower (Brassica oleracea L.) buds as a source of subcellular fractions. O-Acetylserine(thiol)lyase has been detected in both organelles (proplastids and mitochondria) and a cytosolic extract obtained by protoplast fractionation. We confirmed these observations, demonstrating that a form of the enzyme different in global charge and separated from others by anion-exchange chromatography corresponded to each subcellular location. Our observations are consistent with the need for cysteine biosynthesis in each subcellular compartment where the synthesis of proteins occurs.

Subcellular Distribution of O-Acetylserine(thiol)lyase in Cauliflower (Brassica oleracea L.) Inflorescence

PubMed Central

Rolland, Norbert; Droux, Michel; Douce, Roland

1992-01-01

The subcellular localization of O-acetyiserine(thiol)lyase (EC 4.2.99.8) in nongreen tissue from higher plants has been studied using purified proplastids, mitochondria, and protoplasts from cauliflower (Brassica oleracea L.) buds as a source of subcellular fractions. O-Acetylserine(thiol)lyase has been detected in both organelles (proplastids and mitochondria) and a cytosolic extract obtained by protoplast fractionation. We confirmed these observations, demonstrating that a form of the enzyme different in global charge and separated from others by anion-exchange chromatography corresponded to each subcellular location. Our observations are consistent with the need for cysteine biosynthesis in each subcellular compartment where the synthesis of proteins occurs. ImagesFigure 1 PMID:16668766

Imaging trace element distributions in single organelles and subcellular features

DOE PAGES

Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; ...

2016-02-25

The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro-and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (whichmore » some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. In conclusion, it could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.« less

System dynamics of subcellular transport.

PubMed

Chen, Vivien Y; Khersonsky, Sonya M; Shedden, Kerby; Chang, Young Tae; Rosania, Gus R

2004-01-01

In pharmacokinetic experiments, interpretations often hinge on treating cells as a "black box": a single, lumped compartment or boundary. Here, a combinatorial library of fluorescent small molecules was used to visualize subcellular transport pathways in living cells, using a kinetic, high content imaging system to monitor spatiotemporal variations of intracellular probe distribution. Most probes accumulate in cytoplasmic vesicles and probe kinetics conform to a nested, two-compartment dynamical system. At steady state, probes preferentially partition from the extracellular medium to the cytosol, and from the cytosol to cytoplasmic vesicles, with hydrophobic molecules favoring sequestration. Altogether, these results point to a general organizing principle underlying the system dynamics of subcellular, small molecule transport. In addition to plasma membrane permeability, subcellular transport phenomena can determine the active concentration of small molecules in the cytosol and the efflux of small molecules from cells. Fundamentally, direct observation of intracellular probe distribution challenges the simple boundary model of classical pharmacokinetics, which considers cells as static permeability barriers.

Adult murine CNS stem cells express aquaporin channels.

PubMed

La Porta, Caterina A M; Gena, Patrizia; Gritti, Angela; Fascio, Umberto; Svelto, Maria; Calamita, Giuseppe

2006-02-01

Fluid homoeostasis is of critical importance in many functions of the CNS (central nervous system) as indicated by the fact that dysregulation of cell volume underlies clinical conditions such as brain oedema and hypoxia. Water balance is also important during neurogenesis as neural stem cells move considerable amounts of water into or out of the cell to rapidly change their volume during differentiation. Consistent with the relevance of water transport in CNS, multiple AQP (aquaporin) water channels have been recognized and partially characterized in brain cell function. However, the presence and distribution of AQPs in CNS stem cells has not yet been assessed. In the present study, we investigate the expression and subcellular localization of AQPs in murine ANSCs (adult neural stem cells). Considerable AQP8 mRNAs were found in ANSCs where, as expected, the transcript of two additional AQPs, AQP4 and AQP9, was also detected. Immunoblotting with subcellular membrane fractions of ANSCs showed predominant expression of AQP8 in the mitochondria-enriched fraction. This result was consistent with the spotted immunoreactivity profile encountered within the ANSCs by confocal immunofluorescence. AQP8 may have a role in mitochondrial volume regulation during ANSC differentiation. Recognition of AQPs in ANSCs is a step forward in our knowledge of water homoeostasis in the CNS and provides useful information for the purposes of stem cell technology.

Distinct Cellular and Subcellular Distributions of G Protein-Coupled Receptor Kinase and Arrestin Isoforms in the Striatum

PubMed Central

Bychkov, Evgeny; Zurkovsky, Lilia; Garret, Mika B.; Ahmed, Mohamed R.; Gurevich, Eugenia V.

2012-01-01

G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling. PMID:23139825

Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

PubMed

Bychkov, Evgeny; Zurkovsky, Lilia; Garret, Mika B; Ahmed, Mohamed R; Gurevich, Eugenia V

2012-01-01

G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

Distribution of polycyclic aromatic hydrocarbons in subcellular root tissues of ryegrass (Lolium multiflorum Lam.)

PubMed Central

2010-01-01

Background Because of the increasing quantity and high toxicity to humans of polycyclic aromatic hydrocarbons (PAHs) in the environment, several bioremediation mechanisms and protocols have been investigated to restore PAH-contaminated sites. The transport of organic contaminants among plant cells via tissues and their partition in roots, stalks, and leaves resulting from transpiration and lipid content have been extensively investigated. However, information about PAH distributions in intracellular tissues is lacking, thus limiting the further development of a mechanism-based phytoremediation strategy to improve treatment efficiency. Results Pyrene exhibited higher uptake and was more recalcitrant to metabolism in ryegrass roots than was phenanthrene. The kinetic processes of uptake from ryegrass culture medium revealed that these two PAHs were first adsorbed onto root cell walls, and they then penetrated cell membranes and were distributed in intracellular organelle fractions. At the beginning of uptake (< 50 h), adsorption to cell walls dominated the subcellular partitioning of the PAHs. After 96 h of uptake, the subcellular partition of PAHs approached a stable state in the plant water system, with the proportion of PAH distributed in subcellular fractions being controlled by the lipid contents of each component. Phenanthrene and pyrene primarily accumulated in plant root cell walls and organelles, with about 45% of PAHs in each of these two fractions, and the remainder was retained in the dissolved fraction of the cells. Because of its higher lipophilicity, pyrene displayed greater accumulation factors in subcellular walls and organelle fractions than did phenanthrene. Conclusions Transpiration and the lipid content of root cell fractions are the main drivers of the subcellular partition of PAHs in roots. Initially, PAHs adsorb to plant cell walls, and they then gradually diffuse into subcellular fractions of tissues. The lipid content of intracellular components determines the accumulation of lipophilic compounds, and the diffusion rate is related to the concentration gradient established between cell walls and cell organelles. Our results offer insights into the transport mechanisms of PAHs in ryegrass roots and their diffusion in root cells. PMID:20860818

The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela, E-mail: angela.pearson@iaf.inrs.ca

2013-09-15

Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs.more » Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.« less

BAG3 is involved in neuronal differentiation and migration.

PubMed

Santoro, Antonietta; Nicolin, Vanessa; Florenzano, Fulvio; Rosati, Alessandra; Capunzo, Mario; Nori, Stefania L

2017-05-01

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

Subcellular distribution of raffinose oligosaccharides and other metabolites in summer and winter leaves of Ajuga reptans (Lamiaceae).

PubMed

Findling, Sarah; Zanger, Klaus; Krueger, Stephan; Lohaus, Gertrud

2015-01-01

In Ajuga reptans, raffinose oligosaccharides accumulated during winter. Stachyose, verbascose, and higher RFO oligomers were exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. The evergreen labiate Ajuga reptans L. can grow at low temperature. The carbohydrate metabolism changes during the cold phase, e.g., raffinose family oligosaccharides (RFOs) accumulate. Additionally, A. reptans translocates RFOs in the phloem. In the present study, subcellular concentrations of metabolites were studied in summer and winter leaves of A. reptans to gain further insight into regulatory instances involved in the cold acclimation process and into the function of RFOs. Subcellular metabolite concentrations were determined by non-aqueous fractionation. Volumes of the subcellular compartments of summer and winter leaves were analyzed by morphometric measurements. The metabolite content varied strongly between summer and winter leaves. Soluble metabolites increased up to tenfold during winter whereas the starch content was decreased. In winter leaves, the subcellular distribution showed a shift of carbohydrates from cytoplasm to vacuole and chloroplast. Despite this, the metabolite concentration was higher in all compartments in winter leaves compared to summer leaves because of the much higher total metabolite content in winter leaves. The different oligosaccharides did show different compartmentations. Stachyose, verbascose, and higher RFO oligomers were almost exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. Apparently, the subcellular distribution of the RFOs differs because they fulfill different functions in plant metabolism during winter. Raffinose might function in protecting chloroplast membranes during freezing, whereas higher RFO oligomers may exert protective effects on vacuolar membranes. In addition, the high content of RFOs in winter leaves may also result from reduced consumption of assimilates.

Subcellular Nanoparticle Distribution from Light Transmission Spectroscopy

NASA Astrophysics Data System (ADS)

Deatsch, Alison; Sun, Nan; Johnson, Jeffrey; Stack, Sharon; Tanner, Carol; Ruggiero, Steven

We have measured the particle-size distribution (PSD) of subcellular structures in plant and animal cells. We have employed a new technique developed by our group, Light Transmission Spectroscopy-combined with cell fractionation-to accurately measure PSDs over a wide size range: from 10 nm to 3000nm, which includes objects from the size of individual proteins to organelles. To date our experiments have included cultured human oral cells and spinach cells. These results show a power-law dependence of particle density with particle diameter, implying a universality of the packing distribution. We discuss modeling the cell as a self-similar (fractal) body comprised of spheres on all size scales. This goal of this work is to obtain a better understanding of the fundamental nature of particle packing within cells in order to enrich our knowledge of the structure, function, and interactions of sub-cellular nanostructures across cell types.

Spatial mapping and profiling of metabolite distributions during germination

DOE PAGES

Feenstra, Adam D.; Alexander, Liza E.; Song, Zhihong; ...

2017-06-20

Germination is a highly complex process by which seeds begin to develop and establish themselves as viable organisms. In this paper, we utilize a combination of GC-MS, LC-fluorescence, and mass spectrometry imaging (MSI) approaches to profile and visualize the metabolic distributions of germinating seeds from two different inbreds of maize seeds, B73 and Mo17. GC and LC analyses demonstrate that the two inbreds are highly differentiated in their metabolite profiles throughout the course of germination, especially with regard to amino acids, sugar alcohols, and small organic acids. Crude dissection of the seed followed by GC-MS analysis of polar metabolites alsomore » revealed that many compounds were highly sequestered among the various seed tissue types. To further localize compounds, matrix-assisted laser desorption/ionization MSI is utilized to visualize compounds in fine detail in their native environments over the course of germination. Most notably, the fatty acyl chain-dependent differential localization of phospholipids and TAGs were observed within the embryo and radicle, showing correlation with the heterogeneous distribution of fatty acids. Furthermore, other interesting observations include unusual localization of ceramides on the endosperm/scutellum boundary, and subcellular localization of ferulate in the aleurone.« less

Specific serine-proline phosphorylation and glycogen synthase kinase 3β-directed subcellular targeting of stathmin 3/Sclip in neurons.

PubMed

Devaux, Sara; Poulain, Fabienne E; Devignot, Véronique; Lachkar, Sylvie; Irinopoulou, Theano; Sobel, André

2012-06-22

During nervous system development, neuronal growth, migration, and functional morphogenesis rely on the appropriate control of the subcellular cytoskeleton including microtubule dynamics. Stathmin family proteins play major roles during the various stages of neuronal differentiation, including axonal growth and branching, or dendritic development. We have shown previously that stathmins 2 (SCG10) and 3 (SCLIP) fulfill distinct, independent and complementary regulatory roles in axonal morphogenesis. Although the two proteins have been proposed to display the four conserved phosphorylation sites originally identified in stathmin 1, we show here that they possess distinct phosphorylation sites within their specific proline-rich domains (PRDs) that are differentially regulated by phosphorylation by proline-directed kinases involved in the control of neuronal differentiation. ERK2 or CDK5 phosphorylate the two proteins but with different site specificities. We also show for the first time that, unlike stathmin 2, stathmin 3 is a substrate for glycogen synthase kinase (GSK) 3β both in vitro and in vivo. Interestingly, stathmin 3 phosphorylated at its GSK-3β target site displays a specific subcellular localization at neuritic tips and within the actin-rich peripheral zone of the growth cone of differentiating hippocampal neurons in culture. Finally, pharmacological inhibition of GSK-3β induces a redistribution of stathmin 3, but not stathmin 2, from the periphery toward the Golgi region of neurons. Stathmin proteins can thus be either regulated locally or locally targeted by specific phosphorylation, each phosphoprotein of the stathmin family fulfilling distinct and specific roles in the control of neuronal differentiation.

Spatial distributions of AQP5 and AQP0 in embryonic and postnatal mouse lens development

PubMed Central

Petrova, Rosica S.; Schey, Kevin L.; Donaldson, Paul J.; Grey, Angus C.

2015-01-01

The expression of the water channel protein aquaporin (AQP)-5 in adult rodent and human lenses was recently reported using immunohistochemistry, molecular biology, and mass spectrometry techniques, confirming a second transmembrane water channel that is present in lens fibre cells in addition to the abundant AQP0 protein. Interestingly, the sub-cellular distribution and level of post-translational modification of both proteins changes with fibre cell differentiation and location in the adult rodent lens. This study compares the sub-cellular distribution of AQP0 and AQP5 during embryonic and postnatal fibre cell development in the mouse lens to understand how the immunolabelling patterns for both AQPs observed in adult lens are first established. Immunohistochemistry was used to map the cellular and sub-cellular distribution of AQP5 and AQP0 throughout the lens in cryosections from adult (6 weeks to 8 months) and postnatal (0-2 weeks) mouse lenses and in sections from paraffin embedded mouse embryos (E10-E19). All sections were imaged by fluorescence confocal microscopy. Using antibodies directed against the C-terminus of each AQP, AQP5 was abundantly expressed early in development, being found in the cytoplasm of cells of the lens vesicle and surrounding tissues (E10), while AQP0 was detected later (E11), and only in the membranes of elongating primary fibre cells. During the course of subsequent embryonic and postnatal development the pattern of cytoplasmic AQP5 and membranous AQP0 labelling was maintained until postnatal day 6 (P6). From P6 AQP5 labelling became progressively more membranous initially in the lens nucleus and then later in all regions of the lens, while AQP0 labelling was abruptly lost in the lens nucleus due to C-terminal truncation. Our results show that the spatial distribution patterns of AQP0 and AQP5 observed in the adult lens are established during a narrow window of post natal development (P6-P15) that precedes eye opening and coincides with regression of the hyaloid vascular system. Our results support the hypothesis that, in the older fibre cells, insertion of AQP5 into the fibre cell membrane may compensate for any change in the functionality of AQP0 induced by truncation of its C-terminal tail. PMID:25595964

Subcellular fractionation by differential and zonal centrifugation of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis

PubMed Central

Cartledge, T. G.; Lloyd, D.

1972-01-01

1. Homogenates were prepared from sphaeroplasts of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis and the distributions of marker enzymes were investigated after differential centrifugation. Cytochrome c oxidase and cytochrome c were sedimented almost completely at 105g-min, and this fraction also contained 37% of the catalase, 27% of the acid p-nitrophenyl phosphatase, 53 and 54% respectively of the NADH– and NADPH–cytochrome c oxidoreductases. 2. Zonal centrifugation indicated complex density distributions of the sedimentable portions of these enzymes and of adenosine triphosphatases and suggested the presence of two mitochondrial populations, as well as a bimodal distribution of peroxisomes and heterogeneity of the acid p-nitrophenyl phosphatase-containing particles. 3. Several different adenosine triphosphatases were distinguished in a post-mitochondrial supernatant that contained no mitochondrial fragments; these enzymes varied in their sensitivities to oligomycin and ouabain and their distributions were different from those of pyrophosphatase, adenosine phosphatase and adenosine pyrophosphatase. 4. The distribution of NADPH–cytochrome c oxidoreductase demonstrated that it cannot be used in S. carlsbergensis as a specific marker enzyme for the microsomal fraction. Glucose 6-phosphatase, inosine pyrophosphatase, cytochrome P-450 and five other enzymes frequently assigned to microsomal fractions of mammalian origin were not detected in yeast under these growth conditions. ImagesPLATE 2PLATE 1 (cont.)PLATE 1PLATE 2 (cont.) PMID:4400904

Matrix metalloproteinase-2 and -9 in the cerebellum of teleost fish: Functional implications for adult neurogenesis.

PubMed

Sîrbulescu, Ruxandra F; Ilieş, Iulian; Zupanc, Günther K H

2015-09-01

Matrix metalloproteinases (MMPs) are a family of highly conserved zinc-dependent proteases involved in both development and pathogenesis. The present study examines the role of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in adult neurogenesis, using the corpus cerebelli, a subdivision of the cerebellum, of knifefish (Apteronotus leptorhynchus) as a model system. Transcripts of five isoforms of these gelatinases were identified in the central nervous system of this species. Sequence similarity analysis and homology modeling indicated that functionally and structurally critical elements were highly conserved in knifefish gelatinases. Immunohistochemical staining revealed a differential distribution of MMP-2 and MMP-9 at both the cellular and subcellular level. MMP-2 expression was found mainly in Sox2-immunopositive stem/progenitor cells, both quiescent and mitotically active; and was localized in both the cytoplasmic compartment and the nucleus. By contrast, MMP-9 immunoreactivity was absent in neurogenic niches and displayed a more homogenous distribution, with low to moderate intensity levels, in the molecular and granular layers. MMP-9 expression appeared to be restricted to the extracellular space. In situ zymography indicated that gelatinase activity matched the cellular and subcellular distributions of the two MMPs. The observed patterns of gelatinase activity and expression support the hypothesis that MMP-2 is primarily involved in regulation of the activity of stem/progenitor cells that give rise to new granule neurons, whereas MMP-9 facilitates migration of the progeny of these cells by proteolysis of extracellular matrix proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Distribution of physostigmine and metabolites in brain subcellular fractions of the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

King, B.F.; Somani, S.M.

1987-10-26

The distribution of /sup 3/H-physostigmine (Phy) has been studied in the rat brain subcellular fractions at various time intervals following i.v. injection. /sup 3/H-Phy or its metabolites rapidly accumulate into the cytoplasm of cells and penetrates the intracellular compartments. Kinetic studies of the subcellular distribution of radioactivity (RA) per gm of rat brain following i.v. injection of /sup 3/H-Phy show peak concentrations at 30 min in all subcellular fractions with the exception of mitochondria. In the mitochondrial fraction the RA levels continue to rise from 4682 +/- 875 DPM/gm at 5 min to 27,474 +/- 2825 DPM/gm at 60 minmore » (P < .05). The cytosol contains the highest RA: 223,341 +/- 21,044 DPM/gm at 30 min which declined to 53,475 +/- 3756 DPM/gm at 60 min. RA in synaptosome, microsomes and myelin increases from 5 to 30 min, and declines at 60 min. In vitro studies did not show a greater uptake of RA by the mitochondrial or synaptosomal fractions. The finding of relatively high concentrations of RA in the mitochondrial fraction at 60 min increases the likelihood that Phy or its metabolites could interfere with the physiological function of the organelle. 21 references, 1 figure, 2 tables.« less

Estradiol-induced regulation of GLUT4 in 3T3-L1 cells: involvement of ESR1 and AKT activation.

PubMed

Campello, Raquel S; Fátima, Luciana A; Barreto-Andrade, João Nilton; Lucas, Thais F; Mori, Rosana C; Porto, Catarina S; Machado, Ubiratan F

2017-10-01

Impaired insulin-stimulated glucose uptake involves reduced expression of the GLUT4 (solute carrier family 2 facilitated glucose transporter member 4, SLC2A4 gene). 17β-estradiol (E 2 ) modulates SLC2A4 /GLUT4 expression, but the involved mechanisms are unclear. Although E 2 exerts biological effects by binding to estrogen receptors 1/2 (ESR1/2), which are nuclear transcriptional factors; extranuclear effects have also been proposed. We hypothesize that E 2 regulates GLUT4 through an extranuclear ESR1 mechanism. Thus, we investigated the effects of E 2 upon (1) subcellular distribution of ESRs and the proto-oncogene tyrosine-protein kinases (SRC) involvement; (2) serine/threonine-protein kinase (AKT) activation; (3) Slc2a4 /GLUT4 expression and (4) GLUT4 subcellular distribution and glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were cultivated or not with E 2 for 24 h, and additionally treated or not with ESR1-selective agonist (PPT), ESR1-selective antagonist (MPP) or selective SRC inhibitor (PP2). Subcellular distribution of ESR1, ESR2 and GLUT4 was analyzed by immunocytochemistry; Slc2a4 mRNA and GLUT4 were quantified by qPCR and Western blotting, respectively; plasma membrane GLUT4 translocation and glucose uptake were analyzed under insulin stimulus for 20 min or not. E 2 induced (1) translocation of ESR1, but not of ESR2, from nucleus to plasma membrane and AKT phosphorylation, effects mimicked by PPT and blocked by MPP and PP2; (2) increased Slc2a4 /GLUT4 expression and (3) increased insulin-stimulated GLUT4 translocation and glucose uptake. In conclusion, E 2 treatment promoted a SRC-mediated nucleus-plasma membrane shuttle of ESR1, and increased AKT phosphorylation, Slc2a4 /GLUT4 expression and plasma membrane GLUT4 translocation; consequently, improving insulin-stimulated glucose uptake. These results unravel mechanisms through which estrogen improves insulin sensitivity. © 2017 Society for Endocrinology.

Cadmium accumulation, sub-cellular distribution and chemical forms in rice seedling in the presence of sulfur.

PubMed

Zhang, Wen; Lin, Kuangfei; Zhou, Jian; Zhang, Wei; Liu, Lili; Zhang, Qianqian

2014-01-01

Changes in cadmium (Cd) accumulation, distribution, and chemical form in rice seedling in the joint presence of different concentrations of sulfur (S) remain almost unknown. Therefore, the indoor experiments were performed to determine the accumulation, sub-cellular distribution and chemical forms of Cd under three S levels in rice seedling for the first time. The result showed that Cd accumulation in rice roots was more than in shoots. Sub-cellular distribution of Cd in rice roots and shoots indicated that the largest proportion of Cd accumulated in cell walls and soluble fractions. As S supply increased, the proportion of Cd in cell walls reduced, while it increased in the soluble fractions. The majority of Cd existed in inorganic form, and then gradually changed to organic forms that included pectates and proteins with increased S supply. The results showed that S supply significantly influenced Cd accumulation, distribution, and chemical forms, suggesting that S might provide the material for the synthesis of sulfhydryl protein and thereby affect Cd stress on plants. These observations provided a basic understanding of potential ecotoxicological effects of joint Cd and S exposure in the environment. Copyright © 2013 Elsevier B.V. All rights reserved.

Subcellular localization of glycolytic enzymes and characterization of intermediary metabolism of Trypanosoma rangeli.

PubMed

Rondón-Mercado, Rocío; Acosta, Héctor; Cáceres, Ana J; Quiñones, Wilfredo; Concepción, Juan Luis

2017-09-01

Trypanosoma rangeli is a hemoflagellate protist that infects wild and domestic mammals as well as humans in Central and South America. Although this parasite is not pathogenic for human, it is being studied because it shares with Trypanosoma cruzi, the etiological agent of Chagas' disease, biological characteristics, geographic distribution, vectors and vertebrate hosts. Several metabolic studies have been performed with T. cruzi epimastigotes, however little is known about the metabolism of T. rangeli. In this work we present the subcellular distribution of the T. rangeli enzymes responsible for the conversion of glucose to pyruvate, as determined by epifluorescense immunomicroscopy and subcellular fractionation involving either selective membrane permeabilization with digitonin or differential and isopycnic centrifugation. We found that in T. rangeli epimastigotes the first six enzymes of the glycolytic pathway, involved in the conversion of glucose to 1,3-bisphosphoglycerate are located within glycosomes, while the last four steps occur in the cytosol. In contrast with T. cruzi, where three isoenzymes (one cytosolic and two glycosomal) of phosphoglycerate kinase are expressed simultaneously, only one enzyme with this activity is detected in T. rangeli epimastigotes, in the cytosol. Consistent with this latter result, we found enzymes involved in auxiliary pathways to glycolysis needed to maintain adenine nucleotide and redox balances within glycosomes such as phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, pyruvate phosphate dikinase and glycerol-3-phosphate dehydrogenase. Glucokinase, galactokinase and the first enzyme of the pentose-phosphate pathway, glucose-6-phosphate dehydrogenase, were also located inside glycosomes. Furthermore, we demonstrate that T. rangeli epimastigotes growing in LIT medium only consume glucose and do not excrete ammonium; moreover, they are unable to survive in partially-depleted glucose medium. The velocity of glucose consumption is about 40% higher than that of procyclic Trypanosoma brucei, and four times faster than by T. cruzi epimastigotes under the same culture conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Raman microspectroscopy of nucleus and cytoplasm for human colon cancer diagnosis.

PubMed

Liu, Wenjing; Wang, Hongbo; Du, Jingjing; Jing, Chuanyong

2017-11-15

Subcellular Raman analysis is a promising clinic tool for cancer diagnosis, but constrained by the difficulty of deciphering subcellular spectra in actual human tissues. We report a label-free subcellular Raman analysis for use in cancer diagnosis that integrates subcellular signature spectra by subtracting cytoplasm from nucleus spectra (Nuc.-Cyt.) with a partial least squares-discriminant analysis (PLS-DA) model. Raman mapping with the classical least-squares (CLS) model allowed direct visualization of the distribution of the cytoplasm and nucleus. The PLS-DA model was employed to evaluate the diagnostic performance of five types of spectral datasets, including non-selective, nucleus, cytoplasm, ratio of nucleus to cytoplasm (Nuc./Cyt.), and nucleus minus cytoplasm (Nuc.-Cyt.), resulting in diagnostic sensitivity of 88.3%, 84.0%, 98.4%, 84.5%, and 98.9%, respectively. Discriminating between normal and cancerous cells of actual human tissues through subcellular Raman markers is feasible, especially when using the nucleus-cytoplasm difference spectra. The subcellular Raman approach had good stability, and had excellent diagnostic performance for rectal as well as colon tissues. The insights gained from this study shed new light on the general applicability of subcellular Raman analysis in clinical trials. Copyright © 2017 Elsevier B.V. All rights reserved.

Mapping the subcellular distribution of biomolecules at the ultrastructural level by ion microscopy.

PubMed

Galle, P; Escaig, F; Dantin, F; Zhang, L

1996-05-01

Analytical ion microscopy, a method proposed and developed in 1960 by Casting and Slodzian at the Orsay University (France), makes it possible to obtain easily and rapidly analytical images representing the distribution in a tissue section of elements or isotopes (beginning from the three isotopes of hydrogen until to transuranic elements), even when these elements or isotopes are at a trace concentration of 1 ppm or less. This method has been applied to study the subcellular distribution of different varieties of biomolecules. The subcellular location of these molecules can be easily determined when the molecules contain in their structures a specific atom such as fluorine, iodine, bromine or platinum, what is the case of many pharmaceutical drugs. In this situation, the distribution of these specific atoms can be considered as representative of the distribution of the corresponding molecule. In other cases, the molecules must be labelled with an isotope which may be either radioactive or stable. Recent developments in ion microscopy allow the obtention of their chemical images at ultra structural level. In this paper we present the results obtained with the prototype of a new Scanning Ion Microscope used for the study of the intracellular distribution of different varieties of molecules: glucocorticoids, estrogens, pharmaceutical drugs and pyrimidine analogues.

Age distribution patterns of human gene families: divergent for Gene Ontology categories and concordant between different subcellular localizations.

PubMed

Liu, Gangbiao; Zou, Yangyun; Cheng, Qiqun; Zeng, Yanwu; Gu, Xun; Su, Zhixi

2014-04-01

The age distribution of gene duplication events within the human genome exhibits two waves of duplications along with an ancient component. However, because of functional constraint differences, genes in different functional categories might show dissimilar retention patterns after duplication. It is known that genes in some functional categories are highly duplicated in the early stage of vertebrate evolution. However, the correlations of the age distribution pattern of gene duplication between the different functional categories are still unknown. To investigate this issue, we developed a robust pipeline to date the gene duplication events in the human genome. We successfully estimated about three-quarters of the duplication events within the human genome, along with the age distribution pattern in each Gene Ontology (GO) slim category. We found that some GO slim categories show different distribution patterns when compared to the whole genome. Further hierarchical clustering of the GO slim functional categories enabled grouping into two main clusters. We found that human genes located in the duplicated copy number variant regions, whose duplicate genes have not been fixed in the human population, were mainly enriched in the groups with a high proportion of recently duplicated genes. Moreover, we used a phylogenetic tree-based method to date the age of duplications in three signaling-related gene superfamilies: transcription factors, protein kinases and G-protein coupled receptors. These superfamilies were expressed in different subcellular localizations. They showed a similar age distribution as the signaling-related GO slim categories. We also compared the differences between the age distributions of gene duplications in multiple subcellular localizations. We found that the distribution patterns of the major subcellular localizations were similar to that of the whole genome. This study revealed the whole picture of the evolution patterns of gene functional categories in the human genome.

Diazocyte development in the marine diazotrophic cyanobacterium Trichodesmium.

PubMed

Sandh, Gustaf; Xu, Linghua; Bergman, Birgitta

2012-02-01

The establishment of non-diazotrophic cultures of the filamentous marine cyanobacterium Trichodesmium erythraeum IMS101 enabled the first detailed investigation of the process leading to the development of its unique nitrogen-fixing cell type, the diazocyte. Trichome heterogeneity was apparent already within 3-8 h, while the differentiation of mature diazocytes, containing the nitrogenase enzyme, required 27 h after the removal of combined nitrogen. The distribution of 'pro-diazocytes' within the trichomes correlates with the localization of mature diazocytes, which suggests that pattern regulation is an early event during diazocyte development. The development was initially identified as changes in the subcellular ultrastructure, most notably the degradation of glycogen granules and gas vacuoles. These changes were preceded by the induced expression of the global nitrogen regulator ntcA at an early stage of combined nitrogen deprivation, followed by elevated expression of genes related to nitrogen metabolism and their corresponding proteins. The strongest induction (10-fold) was related to the transcription of the respiratory gene coxB2, apparent already at an early stage, which suggests an important role for respiration and the subsequent energy generation in the subcellular changes found, and in the creation of the reducing environment required for nitrogen fixation in diazocytes.

ALA-mediated PDT of melanoma tumors: light-sensitizer interactions determined by a novel spectral imaging system

NASA Astrophysics Data System (ADS)

Malik, Zvi; Dishi, M.

1995-05-01

The subcellular localization of endogenous protoporphyrin (endo- PP) during photosensitization in B-16 melanoma cells was analyzed by a novel spectral imaging system, the SpectraCube 1000. The melanoma cells were incubated with 5-aminolevulinic acid (ALA), and then the fluorescence of endo-PP was recorded in individual living cells by three modes: conventional fluorescence imaging, multipixel point by point fluorescence spectroscopy, and image processing, by operating a function of spectral similarity mapping and reconstructing new images derived from spectral information. The fluorescence image of ALA-treated cells revealed vesicular distribution of endo-PP all over the cytosol, with mitochondrial, lysosomal, as well as endoplasmic reticulum cisternael accumulation. Two main spectral fluorescence peaks were demonstrated at 635 and 705 nm, with intensities that differed from one subcellular site to another. Photoirradiation of the cells included point-specific subcellular fluorescence spectrum changes and demonstrated photoproduct formation. Spectral image reconstruction revealed the local distribution of a chosen spectrum in the photosensitized cells. On the other hand, B 16 cells treated with exogenous protoporphyrin (exo-PP) showed a dominant fluorescence peak at 670 nm and a minor peak at 630 nm. Fluorescence was localized at a perinuclear=Golgi region. Light exposure induced photobleaching and photoproduct-spectral changes followed by relocalization. The new localization at subcellular compartments showed pH dependent spectral shifts and photoproduct formation on a subcellular level.

Protein location prediction using atomic composition and global features of the amino acid sequence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cherian, Betsy Sheena, E-mail: betsy.skb@gmail.com; Nair, Achuthsankar S.

2010-01-22

Subcellular location of protein is constructive information in determining its function, screening for drug candidates, vaccine design, annotation of gene products and in selecting relevant proteins for further studies. Computational prediction of subcellular localization deals with predicting the location of a protein from its amino acid sequence. For a computational localization prediction method to be more accurate, it should exploit all possible relevant biological features that contribute to the subcellular localization. In this work, we extracted the biological features from the full length protein sequence to incorporate more biological information. A new biological feature, distribution of atomic composition is effectivelymore » used with, multiple physiochemical properties, amino acid composition, three part amino acid composition, and sequence similarity for predicting the subcellular location of the protein. Support Vector Machines are designed for four modules and prediction is made by a weighted voting system. Our system makes prediction with an accuracy of 100, 82.47, 88.81 for self-consistency test, jackknife test and independent data test respectively. Our results provide evidence that the prediction based on the biological features derived from the full length amino acid sequence gives better accuracy than those derived from N-terminal alone. Considering the features as a distribution within the entire sequence will bring out underlying property distribution to a greater detail to enhance the prediction accuracy.« less

The Ubiquitous Distribution of Late Embryogenesis Abundant Proteins across Cell Compartments in Arabidopsis Offers Tailored Protection against Abiotic Stress[C][W][OPEN

PubMed Central

Candat, Adrien; Paszkiewicz, Gaël; Neveu, Martine; Gautier, Romain; Logan, David C.; Avelange-Macherel, Marie-Hélène; Macherel, David

2014-01-01

Late embryogenesis abundant (LEA) proteins are hydrophilic, mostly intrinsically disordered proteins, which play major roles in desiccation tolerance. In Arabidopsis thaliana, 51 genes encoding LEA proteins clustered into nine families have been inventoried. To increase our understanding of the yet enigmatic functions of these gene families, we report the subcellular location of each protein. Experimental data highlight the limits of in silico predictions for analysis of subcellular localization. Thirty-six LEA proteins localized to the cytosol, with most being able to diffuse into the nucleus. Three proteins were exclusively localized in plastids or mitochondria, while two others were found dually targeted to these organelles. Targeting cleavage sites could be determined for five of these proteins. Three proteins were found to be endoplasmic reticulum (ER) residents, two were vacuolar, and two were secreted. A single protein was identified in pexophagosomes. While most LEA protein families have a unique subcellular localization, members of the LEA_4 family are widely distributed (cytosol, mitochondria, plastid, ER, and pexophagosome) but share the presence of the class A α-helix motif. They are thus expected to establish interactions with various cellular membranes under stress conditions. The broad subcellular distribution of LEA proteins highlights the requirement for each cellular compartment to be provided with protective mechanisms to cope with desiccation or cold stress. PMID:25005920

Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms

PubMed Central

Sipieter, François; Cappe, Benjamin; Gonzalez Pisfil, Mariano; Spriet, Corentin; Bodart, Jean-François; Cailliau-Maggio, Katia; Vandenabeele, Peter; Héliot, Laurent; Riquet, Franck B.

2015-01-01

Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues. PMID:26517832

Lysine Methylation of Nuclear Co-repressor Receptor Interacting Protein 140

PubMed Central

Huq, MD Mostaqul; Ha, Sung Gil; Barcelona, Helene; Wei, Li-Na

2009-01-01

Receptor interacting protein 140 (RIP140) undergoes extensive posttranslational modifications (PTMs), including phosphorylation, acetylation, arginine methylation, and pyridoxylation. PTMs affect its sub-cellular distribution, protein-protein interaction, and biological activity in adipocyte differentiation. Arginine methylation on Arg240, Arg650, and Arg948 suppresses the repressive activity of RIP140. Here we find that endogenous RIP140 in differentiated 3T3-L1 cells is also modified by lysine methylation. Three lysine residues, Lys591, Lys653, and Lys757 are mapped as potential methylation sites by mass spectrometry. Site-directed mutagenesis study shows that lysine methylation enhances its gene repressive activity. Mutation of lysine methylation sites enhances arginine methylation, while mutation on arginine methylation sites has little effect on its lysine methylation, suggesting a relationship between lysine methylation and arginine methylation. Kinetic analysis of PTMs of endogenous RIP140 in differentiated 3T3-L1 cells demonstrates sequential modifications on RIP140, initiated from constitutive lysine methylation, followed by increased arginine methylation later in differentiation. This study reveals a potential hierarchy of modifications, at least for lysine and arginine methylation, which bi-directionally regulate the functionality of a non-histone protein. PMID:19216533

Intracellular Mannose Binding Lectin Mediates Subcellular Trafficking of HIV-1 gp120 in Neurons

PubMed Central

Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, CL; Kaul, M; Singh, KK

2014-01-01

Human immunodeficiency virus -1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. PMID:24825317

Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

PubMed

Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, C L; Kaul, M; Singh, K K

2014-09-01

Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. Published by Elsevier Inc.

Comparison of cadmium absorption, translocation, subcellular distribution and chemical forms between two radish cultivars (Raphanus sativus L.).

PubMed

Xin, Juan; Zhao, Xiaohu; Tan, Qiling; Sun, Xuecheng; Hu, Chengxiao

2017-11-01

Cadmium (Cd) absorption and accumulation vary greatly not only among plant species but also among cultivars within the same species. In order to better understand the mechanisms of Cd absorption, transportation and distribution, we examined the differences of Cd absorption, translocation, subcellular distribution and chemical forms between L19, a Cd-tolerant genotype, and H4, a Cd-sensitive genotype, using kinetic analysis and soil culture experiment. Kinetic assays showed that the different Cd concentrations between the two cultivars might be ascribed to root absorption and translocation from root to shoot. The investigations of subcellular distribution and chemical forms verified that Cd concentrations of all subcellular fractions in H4 were all higher than in L19. Meanwhile, most of the Cd was associated with cell walls in the root of H4, but the Cd in the root of L19 and leaf of the two cultivars was mainly stored in soluble fraction, which could be one possible mechanism of tolerance to Cd toxicity. In addition, Cd fractions extracted by 1M NaCl and 2% HAC were predominant in root and leaf of both cultivars and the concentrations and proportions extracted by water and 80% ethanol in root and 1M NaCl in leaf were all higher in H4 than in L19. These results indicate that the Cd in H4 is more active than L19, which could be responsible for the sensitivity of H4 to Cd damage. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of molecular characteristics on cellular uptake, subcellular localization, and phototoxicity of Zn(II) N-alkylpyridylporphyrins.

PubMed

Ezzeddine, Rima; Al-Banaw, Anwar; Tovmasyan, Artak; Craik, James D; Batinic-Haberle, Ines; Benov, Ludmil T

2013-12-20

Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para > meta > ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs.

Effect of Molecular Characteristics on Cellular Uptake, Subcellular Localization, and Phototoxicity of Zn(II) N-Alkylpyridylporphyrins*

PubMed Central

Ezzeddine, Rima; Al-Banaw, Anwar; Tovmasyan, Artak; Craik, James D.; Batinic-Haberle, Ines; Benov, Ludmil T.

2013-01-01

Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para > meta > ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs. PMID:24214973

A draft map of the mouse pluripotent stem cell spatial proteome

PubMed Central

Christoforou, Andy; Mulvey, Claire M.; Breckels, Lisa M.; Geladaki, Aikaterini; Hurrell, Tracey; Hayward, Penelope C.; Naake, Thomas; Gatto, Laurent; Viner, Rosa; Arias, Alfonso Martinez; Lilley, Kathryn S.

2016-01-01

Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms. Capturing the subcellular proteome in a single experiment has proven challenging, with studies focusing on specific compartments or assigning proteins to subcellular niches with low resolution and/or accuracy. Here we introduce hyperLOPIT, a method that couples extensive fractionation, quantitative high-resolution accurate mass spectrometry with multivariate data analysis. We apply hyperLOPIT to a pluripotent stem cell population whose subcellular proteome has not been extensively studied. We provide localization data on over 5,000 proteins with unprecedented spatial resolution to reveal the organization of organelles, sub-organellar compartments, protein complexes, functional networks and steady-state dynamics of proteins and unexpected subcellular locations. The method paves the way for characterizing the impact of post-transcriptional and post-translational modification on protein location and studies involving proteome-level locational changes on cellular perturbation. An interactive open-source resource is presented that enables exploration of these data. PMID:26754106

Determining the distribution of probes between different subcellular locations through automated unmixing of subcellular patterns.

PubMed

Peng, Tao; Bonamy, Ghislain M C; Glory-Afshar, Estelle; Rines, Daniel R; Chanda, Sumit K; Murphy, Robert F

2010-02-16

Many proteins or other biological macromolecules are localized to more than one subcellular structure. The fraction of a protein in different cellular compartments is often measured by colocalization with organelle-specific fluorescent markers, requiring availability of fluorescent probes for each compartment and acquisition of images for each in conjunction with the macromolecule of interest. Alternatively, tailored algorithms allow finding particular regions in images and quantifying the amount of fluorescence they contain. Unfortunately, this approach requires extensive hand-tuning of algorithms and is often cell type-dependent. Here we describe a machine-learning approach for estimating the amount of fluorescent signal in different subcellular compartments without hand tuning, requiring only the acquisition of separate training images of markers for each compartment. In testing on images of cells stained with mixtures of probes for different organelles, we achieved a 93% correlation between estimated and expected amounts of probes in each compartment. We also demonstrated that the method can be used to quantify drug-dependent protein translocations. The method enables automated and unbiased determination of the distributions of protein across cellular compartments, and will significantly improve imaging-based high-throughput assays and facilitate proteome-scale localization efforts.

Intracellular And Subcellular Partitioning Of Nickel In Aureococcus Anophagefferens

NASA Astrophysics Data System (ADS)

Wang, B.; Axe, L.; Wei, L.; Bagheri, S.; Michalopoulou, Z.

2008-12-01

Brown tides are caused by Aureococcus anophagefferens, a species of Pelagophyceae, and have been observed in NY/NJ waterways effecting ecosystems by attenuating light, changing water color, reducing eelgrass beds, decreasing shellfisheries, and further impacting the food web by reducing phytoplankton. Although the impact of macronutrients and iron on A. anophagefferens has been well studied, contaminants, and specifically trace metals have not. In long-term experiments designed to investigate the growth and toxicity, Cd, Cu, Ni, and Zn exposure was evaluated over 10-13 to 10-7 M for the free metal ion. While growth was inhibited or terminated from exposure to Cd and Cu, nickel addition ([Ni2+]: 10-11.23 to 10-10.23 M) promoted A. anophagefferens growth. Short-term experiments are being conducted to better understand mechanistically nickel speciation and distribution. Both total intracellular and subcellular metal concentrations are being assessed with radio-labeled 63Ni. Subcellular fractions are defined as metal-sensitive fractions (MSF) constituting organelles, cell debris, and heat-denatured protein [HDP] and biologically detoxified metal comprising heat-stabilized protein [HSP] and metal-rich granules [MRG]. Based on subcellular distribution, aqueous [Ni2+] concentrations, and A. anophagefferens growth rates, potential reaction pathways promoting A. anophagefferens growth can be addressed.

PF-4/CXCL4 and CXCL4L1 exhibit distinct subcellular localization and a differentially regulated mechanism of secretion.

PubMed

Lasagni, Laura; Grepin, Renaud; Mazzinghi, Benedetta; Lazzeri, Elena; Meini, Claudia; Sagrinati, Costanza; Liotta, Francesco; Frosali, Francesca; Ronconi, Elisa; Alain-Courtois, Nathalie; Ballerini, Lara; Netti, Giuseppe Stefano; Maggi, Enrico; Annunziato, Francesco; Serio, Mario; Romagnani, Sergio; Bikfalvi, Andreas; Romagnani, Paola

2007-05-15

PF-4/CXCL4 is a member of the CXC chemokine family, which is mainly produced by platelets and known for its pleiotropic biological functions. Recently, the proteic product of a nonallelic variant gene of CXCL4 was isolated from human platelets and named as CXCL4L1. CXCL4L1 shows only 4.3% amino acid divergence in the mature protein, but exhibits a 38% amino acid divergence in the signal peptide region. We hypothesized that this may imply a difference in the cell type in which CXCL4L1 is expressed or a difference in its mode of secretion. In different types of transfected cells, CXCL4 and CXCL4L1 exhibited a distinct subcellular localization and a differential regulation of secretion, CXCL4 being stored in secretory granules and released in response to protein kinase C activation, whereas CXCL4L1 was continuously synthesized and secreted through a constitutive pathway. A protein kinase C-regulated CXCL4 secretion was observed also in lymphocytes, a cell type expressing mainly CXCL4 mRNA, whereas smooth muscle cells, which preferentially expressed CXCL4L1, exhibited a constitutive pathway of secretion. These results demonstrate that CXCL4 and CXCL4L1 exhibit a distinct subcellular localization and are secreted in a differentially regulated manner, suggesting distinct roles in inflammatory or homeostatic processes.

Interaction of HSP20 with a viral RdRp changes its sub-cellular localization and distribution pattern in plants.

PubMed

Li, Jing; Xiang, Cong-Ying; Yang, Jian; Chen, Jian-Ping; Zhang, Heng-Mu

2015-09-11

Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1-296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellular distribution of sHSPs.

Subcellular distribution of delta 5-3 beta-hydroxy steroid dehydrogenase in the granulosa cells of the domestic fowl (Gallus domesticus).

PubMed Central

Armstrong, D G

1979-01-01

1. The distribution of 3 beta-hydroxy steroid dehydrogenase was examined in the subcellular fractions of granulosa cells collected from the ovary of the domestic fowl. 2. 3 beta-hydroxy steroid dehydrogenase activity was observed in the mitochondrial (4000g for 20min) and microsomal (105 000g for 120min) fractions. 3. Approximately three times more 3 beta-hydroxy steroid dehydrogenase activity was associated with the cytochrome oxidase activity (a mitochondrial marker enzyme) in anteovulatory-follicle granulosa cells than with that of the postovulatory follicle. 4. Comparison of the latent properties of mitochondrial 3 beta-hydroxy steroid dehydrogenase with those of cytochrome oxidase and isocitrate dehydrogenase indicated that 3 beta-hydroxy steroid dehydrogenase is located extramitochondrially. 5. This apparent distribution of 3 beta-hydroxy steroid dehydrogenase is explained on the basis that the mitochondrial activity is either an artefact caused by a redistribution in the subcellular location of the enzyme, occurring during homogenization, or by the existence of a functionally heterogeneous endoplasmic reticulum that yields particles of widely differing sedimentation properties. PMID:518548

Critical behavior of subcellular density organization during neutrophil activation and migration.

PubMed

Baker-Groberg, Sandra M; Phillips, Kevin G; Healy, Laura D; Itakura, Asako; Porter, Juliana E; Newton, Paul K; Nan, Xiaolin; McCarty, Owen J T

2015-12-01

Physical theories of active matter continue to provide a quantitative understanding of dynamic cellular phenomena, including cell locomotion. Although various investigations of the rheology of cells have identified important viscoelastic and traction force parameters for use in these theoretical approaches, a key variable has remained elusive both in theoretical and experimental approaches: the spatiotemporal behavior of the subcellular density. The evolution of the subcellular density has been qualitatively observed for decades as it provides the source of image contrast in label-free imaging modalities (e.g., differential interference contrast, phase contrast) used to investigate cellular specimens. While these modalities directly visualize cell structure, they do not provide quantitative access to the structures being visualized. We present an established quantitative imaging approach, non-interferometric quantitative phase microscopy, to elucidate the subcellular density dynamics in neutrophils undergoing chemokinesis following uniform bacterial peptide stimulation. Through this approach, we identify a power law dependence of the neutrophil mean density on time with a critical point, suggesting a critical density is required for motility on 2D substrates. Next we elucidate a continuum law relating mean cell density, area, and total mass that is conserved during neutrophil polarization and migration. Together, our approach and quantitative findings will enable investigators to define the physics coupling cytoskeletal dynamics with subcellular density dynamics during cell migration.

Critical behavior of subcellular density organization during neutrophil activation and migration

PubMed Central

Baker-Groberg, Sandra M.; Phillips, Kevin G.; Healy, Laura D.; Itakura, Asako; Porter, Juliana E.; Newton, Paul K.; Nan, Xiaolin; McCarty, Owen J.T.

2015-01-01

Physical theories of active matter continue to provide a quantitative understanding of dynamic cellular phenomena, including cell locomotion. Although various investigations of the rheology of cells have identified important viscoelastic and traction force parameters for use in these theoretical approaches, a key variable has remained elusive both in theoretical and experimental approaches: the spatiotemporal behavior of the subcellular density. The evolution of the subcellular density has been qualitatively observed for decades as it provides the source of image contrast in label-free imaging modalities (e.g., differential interference contrast, phase contrast) used to investigate cellular specimens. While these modalities directly visualize cell structure, they do not provide quantitative access to the structures being visualized. We present an established quantitative imaging approach, non-interferometric quantitative phase microscopy, to elucidate the subcellular density dynamics in neutrophils undergoing chemokinesis following uniform bacterial peptide stimulation. Through this approach, we identify a power law dependence of the neutrophil mean density on time with a critical point, suggesting a critical density is required for motility on 2D substrates. Next we elucidate a continuum law relating mean cell density, area, and total mass that is conserved during neutrophil polarization and migration. Together, our approach and quantitative findings will enable investigators to define the physics coupling cytoskeletal dynamics with subcellular density dynamics during cell migration. PMID:26640599

Imaging Subcellular Structures in the Living Zebrafish Embryo.

PubMed

Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

2016-04-02

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation

PubMed Central

Neve, Jonathan; Burger, Kaspar; Li, Wencheng; Hoque, Mainul; Patel, Radhika; Tian, Bin; Gullerova, Monika; Furger, Andre

2016-01-01

Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3′ UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type–specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3′ UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice. PMID:26546131

Techniques for the Cellular and Subcellular Localization of Endocannabinoid Receptors and Enzymes in the Mammalian Brain.

PubMed

Cristino, Luigia; Imperatore, Roberta; Di Marzo, Vincenzo

2017-01-01

This chapter attempts to piece together knowledge about new advanced microscopy techniques to study the neuroanatomical distribution of endocannabinoid receptors and enzymes at the level of cellular and subcellular structures and organelles in the brain. Techniques ranging from light to electron microscopy up to the new advanced LBM, PALM, and STORM super-resolution microscopy will be discussed in the context of their contribution to define the spatial distribution and organization of receptors and enzymes of the endocannabinoid system (ECS), and to better understand ECS brain functions. © 2017 Elsevier Inc. All rights reserved.

Subcellular distribution of trace elements and liver histology of landlocked Arctic char (Salvelinus alpinus) sampled along a mercury contamination gradient.

PubMed

Barst, Benjamin D; Rosabal, Maikel; Campbell, Peter G C; Muir, Derek G C; Wang, Xioawa; Köck, Günter; Drevnick, Paul E

2016-05-01

We sampled landlocked Arctic char (Salvelinus alpinus) from four lakes (Small, 9-Mile, North, Amituk) in the Canadian High Arctic that span a gradient of mercury contamination. Metals (Hg, Se, Tl, and Fe) were measured in char tissues to determine their relationships with health indices (relative condition factor and hepatosomatic index), stable nitrogen isotope ratios, and liver histology. A subcellular partitioning procedure was employed to determine how metals were distributed between potentially sensitive and detoxified compartments of Arctic char livers from a low- and high-mercury lake (Small Lake and Amituk Lake, respectively). Differences in health indices and metal concentrations among char populations were likely related to differences in feeding ecology. Concentrations of Hg, Se, and Tl were highest in the livers of Amituk char, whereas concentrations of Fe were highest in Small and 9-Mile char. At the subcellular level we found that although Amituk char had higher concentrations of Tl in whole liver than Small Lake char, they maintained a greater proportion of this metal in detoxified fractions, suggesting an attempt at detoxification. Mercury was found mainly in potentially sensitive fractions of both Small and Amituk Lake char, indicating that Arctic char are not effectively detoxifying this metal. Histological changes in char livers, mainly in the form of melano-macrophage aggregates and hepatic fibrosis, could be linked to the concentrations and subcellular distributions of essential or non-essential metals. Copyright © 2016 Elsevier Ltd. All rights reserved.

Extraction protocol and liquid chromatography/tandem mass spectrometry method for determining micelle-entrapped paclitaxel at the cellular and subcellular levels: Application to a cellular uptake and distribution study.

PubMed

Zheng, Nan; Lian, Bin; Du, Wenwen; Xu, Guobing; Ji, Jiafu

2018-01-01

Paclitaxel-loaded polymeric micelles (PTX-PM) are commonly used as tumor-targeted nanocarriers and display outstanding antitumor features in clinic, but its accumulation and distribution in vitro are lack of investigation. It is probably due to the complex micellar system and its low concentration at the cellular or subcellular levels. In this study, we developed an improved extraction method, which was a combination of mechanical disruption and liquid-liquid extraction (LLE), to extract the total PTX from micelles in the cell lysate and subcellular compartments. An ultra-performance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) method was optimized to detect the low concentration of PTX at cellular and subcellular levels simultaneously, using docetaxel as internal standard (IS). The method was proved to release PTX totally from micelles (≥95.93%) with a consistent and reproducible extraction recovery (≥75.04%). Good linearity was obtained at concentrations ranging from 0.2 to 20ng/mL. The relative error (RE%) for accuracy varied from 0.68 to 7.56%, and the intra- and inter-precision (relative standard deviation, RSD%) was less than 8.64% and 13.14%, respectively. This method was fully validated and successfully applied to the cellular uptake and distribution study of PTX-loaded PLGA-PEG micelles in human breast cancer cells (MCF-7). Copyright © 2017 Elsevier B.V. All rights reserved.

Inner membrane fusion mediates spatial distribution of axonal mitochondria

PubMed Central

Yu, Yiyi; Lee, Hao-Chih; Chen, Kuan-Chieh; Suhan, Joseph; Qiu, Minhua; Ba, Qinle; Yang, Ge

2016-01-01

In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, we developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. We found that stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distributions and morphology differentially. Knockdown of dOpa1 also impaired axonal transport of mitochondria. But the changed spatial distributions of mitochondria resulted primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, caused similar transport velocity impairment but different spatial distributions. Together, our data reveals that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution. PMID:26742817

Evolution of alanine:glyoxylate aminotransferase 1 peroxisomal and mitochondrial targeting. A survey of its subcellular distribution in the livers of various representatives of the classes Mammalia, Aves and Amphibia.

PubMed

Danpure, C J; Fryer, P; Jennings, P R; Allsop, J; Griffiths, S; Cunningham, A

1994-08-01

As part of a wider study on the molecular evolution of alanine:glyoxylate aminotransferase 1 (AGT1) intracellular compartmentalization, we have determined the subcellular distribution of immunoreactive AGT1, using postembedding protein A-gold immunoelectron microscopy, in the livers of various members of the classes Mammalia, Aves, and Amphibia. As far as organellar distribution is concerned, three categories could be distinguished. In members of the first category (type I), all, or nearly all, of the immunoreactive AGT1 was concentrated within the peroxisomes. In the second category (type II), AGT1 was found more evenly distributed in both peroxisomes and mitochondria. In the third category (type III), AGT1 was localized mainly within the mitochondria with much lower, but widely variable, amounts in the peroxisomes. Type I animals include the human, two great apes (gorilla, orangutan), two Old World monkeys (anubis baboon, Japanese macaque), a New World monkey (white-faced Saki monkey), a lago, morph (European rabbit), a bat (Seba's short-tailed fruit bat), two caviomorph rodents (guinea pig, orange-rumped agouti), and two Australian marsupials (koala, Bennett's wallaby). Type II animals include two New World monkeys (common marmoset, cotton-top tamarin), three prosimians (brown lemur, fat-tailed dwarf lemur, pygmy slow loris), five rodents (a hybrid crested porcupine, Colombian ground squirrel, laboratory rat, laboratory mouse, golden hamster), an American marsupial (grey short-tailed opossum), and a bird (raven). Type III animals include the large tree shrew, three insectivores (common Eurasian mole, European hedgehog, house shrew), four carnivores (domestic cat, ocelot, domestic dog, polecat ferret), and an amphibian (common frog). In addition to these categories, some animals (e.g. guinea pig, common frog) possessed significant amounts of cytosolic AGT1. Whereas the subcellular distribution of AGT1 in some orders (e.g. Insectivora and Carnivora) did not appear to vary markedly between the different members, in other orders (e.g. Primates, Rodentia and Marsupialia) it fluctuated widely between the different species. Phylogenetic analysis indicates that the subcellular distribution of AGT1 has changed radically on numerous occasions during the evolution of mammals. The new observations presented in this paper are compatible with our previous demonstration of a relationship between AGT1 subcellular distribution and either present or putative ancestral dietary habit, and our previous suggestion that the molecular evolution of the AGT gene has been markedly influenced by dietary selection pressure.

Spatial Distribution of Cellular Function: The Partitioning of Proteins between Mitochondria and the Nucleus in MCF7 Breast Cancer Cells

PubMed Central

Qattan, Amal T.; Radulovic, Marko; Crawford, Mark; Godovac-Zimmermann, Jasminka

2014-01-01

Concurrent proteomics analysis of the nuclei and mitochondria of MCF7 breast cancer cells identified 985 proteins (40% of all detected proteins) present in both organelles. Numerous proteins from all five complexes involved in oxidative phosphorylation (e.g., NDUFA5, NDUFB10, NDUFS1, NDUF2, SDHA, UQRB, UQRC2, UQCRH, COX5A, COX5B, MT-CO2, ATP5A1, ATP5B, ATP5H, etc.), from the TCA-cycle (DLST, IDH2, IDH3A, OGDH, SUCLAG2, etc.), and from glycolysis (ALDOA, ENO1, FBP1, GPI, PGK1, TALDO1, etc.) were distributed to both the nucleus and mitochondria. In contrast, proteins involved in nuclear/mitochondrial RNA processing/translation and Ras/Rab signaling showed different partitioning patterns. The identity of the OxPhos, TCA-cycle, and glycolysis proteins distributed to both the nucleus and mitochondria provides evidence for spatio-functional integration of these processes over the two different subcellular organelles. We suggest that there are unrecognized aspects of functional coordination between the nucleus and mitochondria, that integration of core functional processes via wide subcellular distribution of constituent proteins is a common characteristic of cells, and that subcellular spatial integration of function may be a vital aspect of cancer. PMID:23051583

Noninvasive identification of subcellular organization and nuclear morphology features associated with leukemic cells using light-scattering spectroscopy

NASA Astrophysics Data System (ADS)

Hsiao, Austin; Hunter, Martin; Greiner, Cherry; Gupta, Sharad; Georgakoudi, Irene

2011-03-01

Leukemia is the most common and deadly cancer among children and one of the most prevalent cancers among adults. Improvements in its diagnosis and monitoring of leukemic patients could have a significant impact in their long-term treatment. We demonstrate that light-scattering spectroscopy (LSS)-based approaches could serve as a tool to achieve this goal. Specifically, we characterize the light scattering properties of leukemic (NALM-6) cells and compare them to those of normal lymphocytes and granulocytes in the 440-710 nm range, over +/-4 deg about the exact backscattering direction. We find that the LSS spectra are well described by an inverse power-law wavelength dependence, with a power exponent insensitive to the scattering angle but significantly higher for leukemic cells than for normal leukocytes. This is consistent with differences in the subcellular morphology of these cells, detected in differential interference contrast images. Furthermore, the residual light-scattering signal, extracted after subtracting the inverse power-law fit from the data, can be analyzed assuming a Gaussian distribution of spherical scatterers using Mie theory. This analysis yields scatterer sizes that are consistent with the diameters of cell nuclei and allows the detection of the larger nuclei of NALM-6 cells compared to those of lymphocytes and granulocytes.

A novel representation for apoptosis protein subcellular localization prediction using support vector machine.

PubMed

Zhang, Li; Liao, Bo; Li, Dachao; Zhu, Wen

2009-07-21

Apoptosis, or programmed cell death, plays an important role in development of an organism. Obtaining information on subcellular location of apoptosis proteins is very helpful to understand the apoptosis mechanism. In this paper, based on the concept that the position distribution information of amino acids is closely related with the structure and function of proteins, we introduce the concept of distance frequency [Matsuda, S., Vert, J.P., Ueda, N., Toh, H., Akutsu, T., 2005. A novel representation of protein sequences for prediction of subcellular location using support vector machines. Protein Sci. 14, 2804-2813] and propose a novel way to calculate distance frequencies. In order to calculate the local features, each protein sequence is separated into p parts with the same length in our paper. Then we use the novel representation of protein sequences and adopt support vector machine to predict subcellular location. The overall prediction accuracy is significantly improved by jackknife test.

Distribution of Single-Wall Carbon Nanotubes in the Xenopus laevis Embryo after Microinjection

PubMed Central

Holt, Brian D.; Shawky, Joseph H.; Dahl, Kris Noel; Davidson, Lance A.; Islam, Mohammad F.

2016-01-01

Single-wall carbon nanotubes (SWCNTs) are advanced materials with the potential for a myriad of diverse applications, including biological technologies and largescale usage with the potential for environmental impacts. SWCNTs have been exposed to developing organisms to determine their effects on embryogenesis, and results have been inconsistent arising, in part, from differing material quality, dispersion status, material size, impurity from catalysts, and stability. For this study, we utilized highly purified SWCNT samples with short, uniform lengths (145 ± 17 nm) well dispersed in solution. To test high exposure doses, we microinjected > 500 μg mL-1 SWCNT concentrations into the well-established embryogenesis model, Xenopus laevis, and determined embryo compatibility and sub-cellular localization during development. SWCNTs localized within cellular progeny of the microinjected cells, but heterogeneously distributed throughout the target-injected tissue. Co-registering unique Raman spectral intensity of SWCNTs with images of fluorescently labelled sub-cellular compartments demonstrated that even at the regions of highest SWCNT concentration, there were no gross alterations to sub-cellular microstructures, including filamentous actin, endoplasmic reticulum and vesicles. Furthermore, SWCNTs did not aggregate or localize to the perinuclear sub-cellular region. Combined, these results suggest that purified and dispersed SWCNTs are not toxic to X. laevis animal cap ectoderm and may be suitable candidate materials for biological applications. PMID:26510384

Influences of calcium silicate on chemical forms and subcellular distribution of cadmium in Amaranthus hypochondriacus L.

NASA Astrophysics Data System (ADS)

Lu, Huanping; Li, Zhian; Wu, Jingtao; Shen, Yong; Li, Yingwen; Zou, Bi; Tang, Yetao; Zhuang, Ping

2017-01-01

A pot experiment was conducted to investigate the effects of calcium silicate (CS) on the subcellular distribution and chemical forms of cadmium (Cd) in grain amaranths (Amaranthus hypochondriacus L. Cv. ‘K112’) grown in a Cd contaminated soil. Results showed that the dry weight and the photosynthetic pigments contents in grain amaranths increased significantly with the increasing doses of CS treatments, with the highest value found for the treatment of CS3 (1.65 g/kg). Compared with the control, application of CS4 (3.31 g/kg) significantly reduced Cd concentrations in the roots, stems and leaves of grain amaranths by 68%, 87% and 89%, respectively. At subcellular level, CS treatment resulted in redistribution of Cd, higher percentages of Cd in the chloroplast and soluble fractions in leaves of grain amaranths were found, while lower proportions of Cd were located at the cell wall of the leaves. The application of CS enhanced the proportions of pectate and protein integrated forms of Cd and decreased the percentages of water soluble Cd potentially associated with toxicity in grain amaranths. Changes of free Cd ions into inactive forms sequestered in subcellular compartments may indicate an important mechanism of CS for alleviating Cd toxicity and accumulation in plants.

Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

NASA Astrophysics Data System (ADS)

Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

2017-10-01

Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

Univariate and multivariate methods for chemical mapping of cervical cancer cells

NASA Astrophysics Data System (ADS)

Duraipandian, Shiyamala; Zheng, Wei; Huang, Zhiwei

2012-01-01

Visualization of cells and subcellular organelles are currently carried out using available microscopy methods such as cryoelectron microscopy, and fluorescence microscopy. These methods require external labeling using fluorescent dyes and extensive sample preparations to access the subcellular structures. However, Raman micro-spectroscopy provides a non-invasive, label-free method for imaging the cells with chemical specificity at sub-micrometer spatial resolutions. The scope of this paper is to image the biochemical/molecular distributions in cells associated with cancerous changes. Raman map data sets were acquired from the human cervical carcinoma cell lines (HeLa) after fixation under 785 nm excitation wavelength. The individual spectrum was recorded by raster-scanning the laser beam over the sample with 1μm step size and 10s exposure time. Images revealing nucleic acids, lipids and proteins (phenylalanine, amide I) were reconstructed using univariate methods. In near future, the small pixel to pixel variations will also be imaged using different multivariate methods (PCA, clustering (HCA, K-means, FCM)) to determine the main cellular constitutions. The hyper-spectral image of cell was reconstructed utilizing the spectral contrast at different pixels of the cell (due to the variation in the biochemical distribution) without using fluorescent dyes. Normal cervical squamous cells will also be imaged in order to differentiate normal and cancer cells of cervix using the biochemical changes in different grades of cancer. Based on the information obtained from the pseudo-color maps, constructed from the hyper-spectral cubes, the primary cellular constituents of normal and cervical cancer cells were identified.

A persistent change in subcellular distribution of calcineurin following fluid percussion injury in the rat.

PubMed

Kurz, Jonathan E; Hamm, Robert J; Singleton, Richard H; Povlishock, John T; Churn, Severn B

2005-06-28

Calcineurin, a neuronally enriched, calcium-stimulated phosphatase, is an important modulator of many neuronal processes, including several that are physiologically related to the pathology of traumatic brain injury. The effect of moderate, central fluid percussion injury on the subcellular distribution of this important neuronal enzyme was examined. Animals were sacrificed at several time points post-injury and calcineurin distribution in subcellular fractions was assayed by Western blot analysis and immunohistochemistry. A persistent increase in calcineurin concentration was observed in crude synaptoplasmic membrane-containing fractions. In cortical fractions, calcineurin immunoreactivity remained persistently increased for 2 weeks post-injury. In hippocampal homogenates, calcineurin immunoreactivity remained increased for up to 4 weeks. Finally, immunohistochemical analysis of hippocampal slices revealed increased staining in the apical dendrites of CA1 neurons. The increased staining was greatest in magnitude 24 h post-injury; however, staining was still more intense than control 4 weeks post-injury. The data support the conclusion that fluid percussion injury results in redistribution of the enzyme in the rat forebrain. These changes have broad physiological implications, possibly resulting in altered cellular excitability or a greater likelihood of neuronal cell death.

Arsenic accumulation in livers of pinnipeds, seabirds and sea turtles: subcellular distribution and interaction between arsenobetaine and glycine betaine.

PubMed

Fujihara, Junko; Kunito, Takashi; Kubota, Reiji; Tanabe, Shinsuke

2003-12-01

Concentrations of total arsenic and individual arsenic compounds were determined in liver samples of pinnipeds (northern fur seal Callorhinus ursinus and ringed seal Pusa hispida), seabirds (black-footed albatross Diomedea nigripes and black-tailed gull Larus crassirostris) and sea turtles (hawksbill turtle Eretmochelys imbricata and green turtle Chelonia mydas). Among these species, the black-footed albatross contained the highest hepatic arsenic concentration (5.8+/-3.7 microg/g wet mass). Arsenobetaine was the major arsenic species found in the liver of all these higher tropic marine animals. To investigate the cause of high accumulation of arsenobetaine, subcellular distribution of arsenic and relationship between arsenobetaine and glycine betaine concentrations were examined in the livers of these animals. There was no relationship between total arsenic concentration and its subcellular distribution in liver tissues. However, a significant negative correlation was found between arsenobetaine and glycine betaine concentrations in the liver of six species examined. This result may indicate that arsenobetaine is accumulated in these marine animals as an osmolyte along with glycine betaine, which is a predominant osmolyte in marine animals because the chemical structure and properties of arsenobetaine are similar to those of glycine betaine.

Superresolution Imaging of Aquaporin-4 Cluster Size in Antibody-Stained Paraffin Brain Sections

PubMed Central

Smith, Alex J.; Verkman, Alan S.

2015-01-01

The water channel aquaporin-4 (AQP4) forms supramolecular clusters whose size is determined by the ratio of M1- and M23-AQP4 isoforms. In cultured astrocytes, differences in the subcellular localization and macromolecular interactions of small and large AQP4 clusters results in distinct physiological roles for M1- and M23-AQP4. Here, we developed quantitative superresolution optical imaging methodology to measure AQP4 cluster size in antibody-stained paraffin sections of mouse cerebral cortex and spinal cord, human postmortem brain, and glioma biopsy specimens. This methodology was used to demonstrate that large AQP4 clusters are formed in AQP4−/− astrocytes transfected with only M23-AQP4, but not in those expressing only M1-AQP4, both in vitro and in vivo. Native AQP4 in mouse cortex, where both isoforms are expressed, was enriched in astrocyte foot-processes adjacent to microcapillaries; clusters in perivascular regions of the cortex were larger than in parenchymal regions, demonstrating size-dependent subcellular segregation of AQP4 clusters. Two-color superresolution imaging demonstrated colocalization of Kir4.1 with AQP4 clusters in perivascular areas but not in parenchyma. Surprisingly, the subcellular distribution of AQP4 clusters was different between gray and white matter astrocytes in spinal cord, demonstrating regional specificity in cluster polarization. Changes in AQP4 subcellular distribution are associated with several neurological diseases and we demonstrate that AQP4 clustering was preserved in a postmortem human cortical brain tissue specimen, but that AQP4 was not substantially clustered in a human glioblastoma specimen despite high-level expression. Our results demonstrate the utility of superresolution optical imaging for measuring the size of AQP4 supramolecular clusters in paraffin sections of brain tissue and support AQP4 cluster size as a primary determinant of its subcellular distribution. PMID:26682810

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: I. Fractionation procedure and characterization of the subcellular fractions

PubMed Central

Noe, BD; Baste, CA; Bauer, GE

1977-01-01

Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82 percent of the total cellular IRI and 89(percent) of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93 percent of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagons found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level. PMID:328517

Mating changes the subcellular distribution and the functionality of estrogen receptors in the rat oviduct.

PubMed

Orihuela, Pedro A; Zuñiga, Lidia M; Rios, Mariana; Parada-Bustamante, Alexis; Sierralta, Walter D; Velásquez, Luis A; Croxatto, Horacio B

2009-11-30

Mating changes the mode of action of 17beta-estradiol (E2) to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER) are required. This change was designated as intracellular path shifting (IPS). Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta) in oviductal epithelial cells of rats on day 1 of cycle (C1) or pregnancy (P1) using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb) and calbindin 9 kDa (s100g) in rats on C1 or P1 treated with selective agonists for ESR1 (PPT) or ESR2 (DPN). The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats. Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c-fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats. Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct.

Grouping annotations on the subcellular layered interactome demonstrates enhanced autophagy activity in a recurrent experimental autoimmune uveitis T cell line.

PubMed

Jia, Xiuzhi; Li, Jingbo; Shi, Dejing; Zhao, Yu; Dong, Yucui; Ju, Huanyu; Yang, Jinfeng; Sun, Jianhua; Li, Xia; Ren, Huan

2014-01-01

Human uveitis is a type of T cell-mediated autoimmune disease that often shows relapse-remitting courses affecting multiple biological processes. As a cytoplasmic process, autophagy has been seen as an adaptive response to cell death and survival, yet the link between autophagy and T cell-mediated autoimmunity is not certain. In this study, based on the differentially expressed genes (GSE19652) between the recurrent versus monophasic T cell lines, whose adoptive transfer to susceptible animals may result in respective recurrent or monophasic uveitis, we proposed grouping annotations on a subcellular layered interactome framework to analyze the specific bioprocesses that are linked to the recurrence of T cell autoimmunity. That is, the subcellular layered interactome was established by the Cytoscape and Cerebral plugin based on differential expression, global interactome, and subcellular localization information. Then, the layered interactomes were grouping annotated by the ClueGO plugin based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. The analysis showed that significant bioprocesses with autophagy were orchestrated in the cytoplasmic layered interactome and that mTOR may have a regulatory role in it. Furthermore, by setting up recurrent and monophasic uveitis in Lewis rats, we confirmed by transmission electron microscopy that, in comparison to the monophasic disease, recurrent uveitis in vivo showed significantly increased autophagy activity and extended lymphocyte infiltration to the affected retina. In summary, our framework methodology is a useful tool to disclose specific bioprocesses and molecular targets that can be attributed to a certain disease. Our results indicated that targeted inhibition of autophagy pathways may perturb the recurrence of uveitis.

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

PubMed Central

Becker, Jordan T.

2017-01-01

ABSTRACT Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans. In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5′ packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy. PMID:28053097

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly.

PubMed

Becker, Jordan T; Sherer, Nathan M

2017-03-15

Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis -acting RNA regulatory elements: the 5' packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins ( gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy. Copyright © 2017 American Society for Microbiology.

Predicting protein subcellular locations using hierarchical ensemble of Bayesian classifiers based on Markov chains.

PubMed

Bulashevska, Alla; Eils, Roland

2006-06-14

The subcellular location of a protein is closely related to its function. It would be worthwhile to develop a method to predict the subcellular location for a given protein when only the amino acid sequence of the protein is known. Although many efforts have been made to predict subcellular location from sequence information only, there is the need for further research to improve the accuracy of prediction. A novel method called HensBC is introduced to predict protein subcellular location. HensBC is a recursive algorithm which constructs a hierarchical ensemble of classifiers. The classifiers used are Bayesian classifiers based on Markov chain models. We tested our method on six various datasets; among them are Gram-negative bacteria dataset, data for discriminating outer membrane proteins and apoptosis proteins dataset. We observed that our method can predict the subcellular location with high accuracy. Another advantage of the proposed method is that it can improve the accuracy of the prediction of some classes with few sequences in training and is therefore useful for datasets with imbalanced distribution of classes. This study introduces an algorithm which uses only the primary sequence of a protein to predict its subcellular location. The proposed recursive scheme represents an interesting methodology for learning and combining classifiers. The method is computationally efficient and competitive with the previously reported approaches in terms of prediction accuracies as empirical results indicate. The code for the software is available upon request.

Comparison of GLUT1, GLUT3, and GLUT4 mRNA and the subcellular distribution of their proteins in normal human muscle

NASA Technical Reports Server (NTRS)

Stuart, C. A.; Wen, G.; Gustafson, W. C.; Thompson, E. A.

2000-01-01

Basal, "insulin-independent" glucose uptake into skeletal muscle is provided by glucose transporters positioned at the plasma membrane. The relative amount of the three glucose transporters expressed in muscle has not been previously quantified. Using a combination of qualitative and quantitative ribonuclease protection assay (RPA) methods, we found in normal human muscle that GLUT1, GLUT3, and GLUT4 mRNA were expressed at 90 +/- 10, 46 +/- 4, and 156 +/- 12 copies/ng RNA, respectively. Muscle was fractionated by DNase digestion and differential sedimentation into membrane fractions enriched in plasma membranes (PM) or low-density microsomes (LDM). GLUT1 and GLUT4 proteins were distributed 57% to 67% in LDM, whereas GLUT3 protein was at least 88% in the PM-enriched fractions. These data suggest that basal glucose uptake into resting human muscle could be provided in part by each of these three isoforms.

Modelling Spatially Regulated β-Catenin Dynamics and Invasion in Intestinal Crypts

PubMed Central

Murray, Philip J.; Kang, Jun-Won; Mirams, Gary R.; Shin, Sung-Young; Byrne, Helen M.; Maini, Philip K.; Cho, Kwang-Hyun

2010-01-01

Experimental data (e.g., genetic lineage and cell population studies) on intestinal crypts reveal that regulatory features of crypt behavior, such as control via morphogen gradients, are remarkably well conserved among numerous organisms (e.g., from mouse and rat to human) and throughout the different regions of the small and large intestines. In this article, we construct a partial differential equation model of a single colonic crypt that describes the spatial distribution of Wnt pathway proteins along the crypt axis. The novelty of our continuum model is that it is based upon assumptions that can be directly related to processes at the cellular and subcellular scales. We use the model to predict how the distributions of Wnt pathway proteins are affected by mutations. The model is then extended to investigate how mutant cell populations can invade neighboring crypts. The model simulations suggest that cell crowding caused by increased proliferation and decreased cell loss may be sufficient for a mutant cell population to colonize a neighboring healthy crypt. PMID:20682248

Prostaglandin D2 Regulates SOX9 Nuclear Translocation during Gonadal Sex Determination in Tammar Wallaby, Macropus eugenii.

PubMed

Chen, Yu; Yu, Hongshi; Pask, Andrew J; Shaw, Geoff; Renfree, Marilyn B

2017-01-01

Sex determination and sexual differentiation pathways are highly conserved between marsupials and eutherians. There are 2 different pathways of prostaglandin D2 (PGD2) synthesis: prostaglandin D synthase (PTGDS) and haematopoietic prostaglandin D synthase (HPGDS). PGD2 regulates the subcellular localization of SOX9 during gonadal sexual differentiation. To investigate the function of PGD2 in the tammar gonad, we cultured undifferentiated male gonads in the presence of the HPGDS inhibitor HQL-79 and female gonads with exogenous PGD2 to mimic activation of the PTGDS-PGD2 pathway. Tammar PTGDS and HPGDS have only 50% similarity with mouse and human orthologues, but functional domains are conserved. The expression of SOX9 was unchanged by the treatments in cultured gonads, but its subcellular localization was markedly affected. SOX9 remained cytoplasmic in the Sertoli cells of testes treated with HQL-79. Treated testes developed a thickened ovary-like surface epithelium. In contrast, SOX9 became nuclear in the granulosa cells of developing ovaries treated with PGD2 and the surface epithelium was thin, as in testes. These results demonstrate that PGD2 regulates the subcellular localization of SOX9 and subsequent gonadal development in the developing marsupial gonads, as it does in mice, and that it must have been an ancestral mechanism. © 2017 S. Karger AG, Basel.

The association of GSK3 beta with E2F1 facilitates nerve growth factor-induced neural cell differentiation.

PubMed

Zhou, Fangfang; Zhang, Long; Wang, Aijun; Song, Bo; Gong, Kai; Zhang, Lihai; Hu, Min; Zhang, Xiufang; Zhao, Nanming; Gong, Yandao

2008-05-23

It is widely acknowledged that E2F1 and GSK3beta are both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3beta in cell differentiation has yet to be discovered. Here, we provide evidence that in the differentiation of PC12 cells induced by nerve growth factor (NGF), GSK3beta was increased at both the mRNA and protein levels, whereas E2F1 at these two levels was decreased. Both wild-type GSK3beta and its kinase-defective mutant GSK3beta KM can inhibit E2F1 by promoting its ubiquitination through physical interaction. In addition, the colocalization of GSK3beta and E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. At the tissue level, GSK3beta colocalized and interacted with E2F1 in mouse hippocampus. Furthermore, GSK3beta facilitated neurite outgrowth by rescuing the promoter activities of Cdk inhibitors p21 and p15 from the inhibition caused by E2F1. To summarize, our findings suggest that GSK3beta can promote the ubiquitination of E2F1 via physical interaction and thus inhibit its transcription activity in a kinase activity independent manner, which plays an important role in the NGF-induced PC12 differentiation.

Learning from Heterogeneous Data Sources: An Application in Spatial Proteomics

PubMed Central

Breckels, Lisa M.; Holden, Sean B.; Wojnar, David; Mulvey, Claire M.; Christoforou, Andy; Groen, Arnoud; Trotter, Matthew W. B.; Kohlbacher, Oliver; Lilley, Kathryn S.; Gatto, Laurent

2016-01-01

Sub-cellular localisation of proteins is an essential post-translational regulatory mechanism that can be assayed using high-throughput mass spectrometry (MS). These MS-based spatial proteomics experiments enable us to pinpoint the sub-cellular distribution of thousands of proteins in a specific system under controlled conditions. Recent advances in high-throughput MS methods have yielded a plethora of experimental spatial proteomics data for the cell biology community. Yet, there are many third-party data sources, such as immunofluorescence microscopy or protein annotations and sequences, which represent a rich and vast source of complementary information. We present a unique transfer learning classification framework that utilises a nearest-neighbour or support vector machine system, to integrate heterogeneous data sources to considerably improve on the quantity and quality of sub-cellular protein assignment. We demonstrate the utility of our algorithms through evaluation of five experimental datasets, from four different species in conjunction with four different auxiliary data sources to classify proteins to tens of sub-cellular compartments with high generalisation accuracy. We further apply the method to an experiment on pluripotent mouse embryonic stem cells to classify a set of previously unknown proteins, and validate our findings against a recent high resolution map of the mouse stem cell proteome. The methodology is distributed as part of the open-source Bioconductor pRoloc suite for spatial proteomics data analysis. PMID:27175778

Mutations in the C-terminal region affect subcellular localization of crucian carp herpesvirus (CaHV) GPCR.

PubMed

Wang, Jun; Gui, Lang; Chen, Zong-Yan; Zhang, Qi-Ya

2016-08-01

G protein-coupled receptors (GPCRs) are known as seven transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization. Crucian carp herpesvirus (CaHV) was recently isolated from infected fish with acute gill hemorrhage. CaHV GPCR of 349 amino acids (aa) was identified based on amino acid identity. A series of variants with truncation/deletion/substitution mutation in the C-terminal (aa 315-349) were constructed and expressed in fathead minnow (FHM) cells. The roles of three key C-terminal regions in subcellular localization of CaHV GPCR were determined. Lysine-315 (K-315) directed the aggregation of the protein preferentially at the nuclear side. Predicted N-myristoylation site (GGGWTR, aa 335-340) was responsible for punctate distribution in periplasm or throughout the cytoplasm. Predicted phosphorylation site (SSR, aa 327-329) and GGGWTR together determined the punctate distribution in cytoplasm. Detection of organelles localization by specific markers showed that the protein retaining K-315 colocalized with the Golgi apparatus. These experiments provided first evidence that different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs. The study provided valuable information and new insights into the precise interactions between herpesvirus and fish cells, and could also provide useful targets for antiviral agents in aquaculture.

Synchronization by Daytime Restricted Food Access Modulates the Presence and Subcellular Distribution of β-Catenin and Its Phosphorylated Forms in the Rat Liver

PubMed Central

De Ita-Pérez, Dalia Luz; Díaz-Muñoz, Mauricio

2017-01-01

β-catenin, the principal effector of the Wnt pathway, is also one of the cadherin cell adhesion molecules; therefore, it fulfills signaling and structural roles in most of the tissues and organs. It has been reported that β-catenin in the liver regulates metabolic responses such as gluconeogenesis and histological changes in response to obesity-promoting diets. The function and cellular location of β-catenin is finely modulated by coordinated sequences of phosphorylation–dephosphorylation events. In this article, we evaluated the levels and cellular localization of liver β-catenin variants, more specifically β-catenin phosphorylated in serine 33 (this phosphorylation provides recognizing sites for β-TrCP, which results in ubiquitination and posterior proteasomal degradation of β-catenin) and β-catenin phosphorylated in serine 675 (phosphorylation that enhances signaling and transcriptional activity of β-catenin through recruitment of different transcriptional coactivators). β-catenin phosphorylated in serine 33 in the nucleus shows day–night fluctuations in their expression level in the Ad Libitum group. In addition, we used a daytime restricted feeding (DRF) protocol to show that the above effects are sensitive to food access-dependent circadian synchronization. We found through western blot and immunohistochemical analyses that DRF protocol promoted (1) higher total β-catenins levels mainly associated with the plasma membrane, (2) reduced the presence of cytoplasmic β-catenin phosphorylated in serine 33, (3) an increase in nuclear β-catenin phosphorylated in serine 675, (4) differential co-localization of total β-catenins/β-catenin phosphorylated in serine 33 and total β-catenins/β-catenin phosphorylated in serine 675 at different temporal points along day and in fasting and refeeding conditions, and (5) differential liver zonation of β-catenin variants studied along hepatic acinus. In conclusion, the present data comprehensively characterize the effect food synchronization has on the presence, subcellular distribution, and liver zonation of β-catenin variants. These results are relevant to understand the set of metabolic and structural liver adaptations that are associated with the expression of the food entrained oscillator (FEO). PMID:28220106

Prion subcellular fractionation reveals infectivity spectrum, with a high titre-low PrPres level disparity

PubMed Central

2012-01-01

Background Prion disease transmission and pathogenesis are linked to misfolded, typically protease resistant (PrPres) conformers of the normal cellular prion protein (PrPC), with the former posited to be the principal constituent of the infectious 'prion'. Unexplained discrepancies observed between detectable PrPres and infectivity levels exemplify the complexity in deciphering the exact biophysical nature of prions and those host cell factors, if any, which contribute to transmission efficiency. In order to improve our understanding of these important issues, this study utilized a bioassay validated cell culture model of prion infection to investigate discordance between PrPres levels and infectivity titres at a subcellular resolution. Findings Subcellular fractions enriched in lipid rafts or endoplasmic reticulum/mitochondrial marker proteins were equally highly efficient at prion transmission, despite lipid raft fractions containing up to eight times the levels of detectable PrPres. Brain homogenate infectivity was not differentially enhanced by subcellular fraction-specific co-factors, and proteinase K pre-treatment of selected fractions modestly, but equally reduced infectivity. Only lipid raft associated infectivity was enhanced by sonication. Conclusions This study authenticates a subcellular disparity in PrPres and infectivity levels, and eliminates simultaneous divergence of prion strains as the explanation for this phenomenon. On balance, the results align best with the concept that transmission efficiency is influenced more by intrinsic characteristics of the infectious prion, rather than cellular microenvironment conditions or absolute PrPres levels. PMID:22534096

Cadmium resistance in an oligochaete and its effect on cadmium trophic transfer to an omnivorous shrimp

USGS Publications Warehouse

Wallace, W.G.; Lopez, G.R.; Levinton, J.S.

1998-01-01

It has been demonstrated that the deposit-feeding oligochaete Limnodrilus hoffmeisteri inhabiting Foundry Cove (FC), a severely cadmium (Cd)-contaminated cove located on the Hudson River, New York, USA, has evolved resistance to Cd. In this study we investigate how this resistance influences Cd trophic transfer from this oligochaete to the grass shrimp Palaemonetes pugio. Cadmium-resistant worms collected from FC and nonresistant worms collected from an adjacent unpolluted site were investigated for differences in Cd tolerance, accumulation, subcellular distribution and bioavailability to shrimp. FC worms were more tolerant of Cd, surviving twice as long as worms from the unpolluted site during a toxicity bioassay. The 7 d concentration factor of Cd-resistant worms was 4 times greater than that of nonresistant worms (2020 vs 577). There were also differences between worm populations with respect to subcellular Cd distributions. Cd-resistant worms produced metallothionein-like proteins (MT) as well as metal-rich granules (MRG) for Cd storage and detoxification; nonresistant worms only produced MT. These differences in subcellular Cd distributions led to large differences in Cd bioavailability to shrimp; shrimp fed Cd-resistant worms absorbed 21% of the ingested Cd, while those fed nonresistant worms absorbed roughly 4 times that amount (~75%). These absorption efficiencies were in good agreement with the proportions of Cd bound to the worm's most biologically available subcellular fractions (i.e. the cytosol and organelles). Although Cd-resistant worms predominantly stored the toxic metal in biologically unavailable MRG, their increased accumulation of Cd would still result in substantial trophic transfer to shrimp because of the storage of Cd in the biologically available fractions. This work demonstrates that the evolution of Cd resistance can have profound implications for Cd bioavailability and cycling within aquatic ecosystems.

Hepatic Subcellular Compartmentation of Cytoplasmic Phosphoenolpyruvate Carboxykinase Determined by Immunogold Electron Microscopy

NASA Astrophysics Data System (ADS)

Gao, Kuixiong; Cardell, Emma Lou; Morris, Randal E.; Giffin, Bruce F.; Cardell, Robert R.

1995-08-01

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting gluconeogenic enzyme and in liver occurs in a lobular gradient from periportal to pericentral regions. The subcellular distribution of cytoplasmic PEPCK molecules within hepatocytes and its relationship to organelles have not been determined previously. In this study, we have used immunogold electron microscopy to evaluate the subcellar distribution of the enzyme, in addition to brightfield and epipolarized light microscopy. Cryosections (10 [mu]m) of perfusion-fixed rat liver were collected on silanated slides and immunostained using goat anti-rat PEPCK followed by 5-nm gold-labeled secondary and tertiary antibodies. Additionally, free-floating vibratome sections (25, 50, and 100 [mu]m) of perfusion-immersion-fixed rat liver were immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled secondary antibody, with and without silver enhancement. The immunogold labeled sections from both procedures were embedded in epoxy resin for the preparation of thin sections for electron microscopy. The results showed that the gold-labeled antibodies penetrated the entire thickness of cryosections, resulting in a high signal for PEPCK, but membranes in general, the smooth endoplasmic reticulum in particular, were not identifiable as electron dense unit membranes. On the other hand, the vibratome sections of well-fixed tissue allowed good visualization of the ultrastructure of cellular organelles, with the smooth endoplasmic reticulum appearing as vesicles and tubules with electron dense unit membranes; however, the penetration of the gold-labeled antibody was limited to cells at the surface of the vibratome sections. In both procedures, PEPCK, as indicated by gold particles, is predominantly in the glycogen areas of the cytosome and not in mitochondria, nuclei, Golgi apparatus, or other cell organelles. Hepatocytes in periportal regions have a compact subcellular distribution of PEPCK shown by gold particles; hepatocytes in pericentral regions have a diffuse subcellular distribution of PEPCK and thus more scattered gold particles. When normal serum replaced the first antibody in the immunogold staining procedures, the background was very low.

Effect of subcellular distribution on nC₆₀ uptake and transfer efficiency from Scenedesmus obliquus to Daphnia magna.

PubMed

Chen, Qiqing; Hu, Xialin; Yin, Daqiang; Wang, Rui

2016-06-01

The potential uptake and trophic transfer ability of nanoparticles (NPs) in aquatic organisms have not been well understood yet. There has been an increasing awareness of the subcellular fate of NPs in organisms, but how the subcellular distribution of NPs subsequently affects the trophic transfer to predator remains to be answered. In the present study, the food chain from Scenedesmus obliquus to Daphnia magna was established to simulate the trophic transfer of fullerene aqueous suspension (nC60). The nC60 contaminated algae were separated into three fractions: cell wall (CW), cell organelle (CO), and cell membrane (CM) fractions, and we investigated the nC60 uptake amounts and trophic transfer efficiency to the predator through dietary exposure to algae or algal subcellular fractions. The nC60 distribution in CW fraction of S. obliquus was the highest, following by CO and CM fractions. nC60 uptake amounts in D. magna were found to be mainly relative to the NPs' distribution in CW fraction and daphnia uptake ability from CW fraction, whereas the nC60 trophic transfer efficiency (TE) were mainly in accordance with the transfer ability of NPs from the CO fraction. CW fed group possessed the highest uptake amount, followed by CO and CM fed groups, but the presence of humic acid (HA) significantly decreased the nC60 uptake from CW fed group. The CO fed groups acquired high TE values for nC60, while CM fed groups had low TE values. Moreover, even though CW fed group had a high TE value; it decreased significantly with the presence of HA. This study contributes to the understanding of fullerene NPs' dietary exposure to aquatic organisms, suggesting that NPs in different food forms are not necessarily equally trophically available to the predator. Copyright © 2016 Elsevier Inc. All rights reserved.

Single-Molecule Localization Microscopy allows for the analysis of cancer metastasis-specific miRNA distribution on the nanoscale

PubMed Central

Tezcan, Kerem Can; Schaufler, Wladimir; Bestvater, Felix; Patil, Nitin; Birk, Udo; Hafner, Mathias; Altevogt, Peter; Cremer, Christoph; Allgayer, Heike

2015-01-01

We describe a novel approach for the detection of small non-coding RNAs in single cells by Single-Molecule Localization Microscopy (SMLM). We used a modified SMLM–setup and applied this instrument in a first proof-of-principle concept to human cancer cell lines. Our method is able to visualize single microRNA (miR)-molecules in fixed cells with a localization accuracy of 10–15 nm, and is able to quantify and analyse clustering and localization in particular subcellular sites, including exosomes. We compared the metastasis-site derived (SW620) and primary site derived (SW480) human colorectal cancer (CRC) cell lines, and (as a proof of principle) evaluated the metastasis relevant miR-31 as a first example. We observed that the subcellular distribution of miR-31 molecules in both cell lines was very heterogeneous with the largest subpopulation of optically acquired weakly metastatic cells characterized by a low number of miR-31 molecules, as opposed to a significantly higher number in the majority of the highly metastatic cells. Furthermore, the highly metastatic cells had significantly more miR-31-molecules in the extracellular space, which were visualized to co-localize with exosomes in significantly higher numbers. From this study, we conclude that miRs are not only aberrantly expressed and regulated, but also differentially compartmentalized in cells with different metastatic potential. Taken together, this novel approach, by providing single molecule images of miRNAs in cellulo can be used as a powerful supplementary tool in the analysis of miRNA function and behaviour and has far reaching potential in defining metastasis-critical subpopulations within a given heterogeneous cancer cell population. PMID:26561203

Tissue-specific accumulation of cadmium in subcellular compartments of eastern oysters Crassostrea virginica Gmelin (Bivalvia: Ostreidae).

PubMed

Sokolova, I M; Ringwood, A H; Johnson, C

2005-09-10

Cadmium distribution was studied in different subcellular fractions of gill and hepatopancreas tissues of eastern oysters Crassostrea virginica. Oysters were exposed for up to 21 days to low sublethal Cd concentrations (25 microg L(-1)). Gill and hepatopancreas tissues were sampled and divided into organelle fractions and cytosol by differential centrifugation. Organelle content of different fractions was verified by activities of marker enzymes, citrate synthase and acid phosphatase for mitochondria and lysosomes, respectively. In both tissue types, there was a significant accumulation of cadmium in cytosol reaching 230-350 ng mg(-1) protein. Among organelles, mitochondria were the main target for Cd bioaccumulation in gills (250-300 ng mg(-1) protein), whereas in hepatopancreas tissues, the highest cadmium accumulation occurred in lysosomes (90-94 ng mg(-1) protein). Although 75-83% of total cadmium burden was associated with the cytosol reflecting high volume fraction of this compartment, Cd concentrations in organelle fractions reached levels that could cause dysfunction of mitochondria and lysosomes. Organ- and organelle-specific patterns of cadmium bioaccumulation support our previous in vivo studies, which showed adverse effects of cadmium exposures on mitochondrial oxidation in gills and on the lysosomal system of hepatopancreas. This may have important implications for the development of biomarkers of effect for heavy metals and for understanding the mechanisms of toxic effects of metals.

Changes in subcellular distribution of ependymins in goldfish brain induced by learning.

PubMed

Schmidt, R

1987-06-01

Goldfish were trained for 4 h to swim with an attached polystyrene foam float and tested for retention 3 days later. Intracerebroventricular injection of anti-ependymin antisera was shown to prevent long-term memory formation of this vestibulomotor learning task, as reported previously. In further experiments, fish were killed 4-14 h after the start of training. The brains were dissected, incubated in an isoosmolar solution for collection of proteins of the brain extracellular fluid (ECF), homogenized, and fractionated by differential centrifugation. The ECF, a supernatant fraction enriched in cytoplasmic constituents (S3), and various particulate subcellular fractions were analyzed for their ependymin contents by radioimmunoassay. No statistically significant changes that might be induced by the learning were revealed in any of the particulate fractions. Steady-state concentrations of ependymins in the cytoplasm, however, increased temporarily by 39% in fish that had mastered the training task as compared with nonlearning animals (passive and active controls). In the ECF, the specific concentration of ependymins first decreased to 88% of control levels (4-5 h after the start of training), but later on, it increased to 138% (8-14 h). Apparently, ependymins present in the ECF are used during biochemical reactions of memory consolidation. The resulting decrease in extracellular ependymin concentrations might trigger their resynthesis in the cytoplasm and lead to an increased release of these glycoproteins into the ECF.

Dynamic Virus-Dependent Subnuclear Localization of the Capsid Protein from a Geminivirus

PubMed Central

Wang, Liping; Tan, Huang; Wu, Mengshi; Jimenez-Gongora, Tamara; Tan, Li; Lozano-Duran, Rosa

2017-01-01

Viruses are intracellular parasites with a nucleic acid genome and a proteinaceous capsid. Viral capsids are formed of at least one virus-encoded capsid protein (CP), which is often multifunctional, playing additional non-structural roles during the infection cycle. In animal viruses, there are examples of differential localization of CPs associated to the progression of the infection and/or enabled by other viral proteins; these changes in the distribution of CPs may ultimately regulate the involvement of these proteins in different viral functions. In this work, we analyze the subcellular localization of a GFP- or RFP-fused CP from the plant virus Tomato yellow leaf curl virus (TYLCV; Fam. Geminiviridae) in the presence or absence of the virus upon transient expression in the host plants Nicotiana benthamiana and tomato. Our findings show that, in agreement with previous reports, when the CP is expressed alone it localizes mainly in the nucleolus and weakly in the nucleoplasm. Interestingly, the presence of the virus causes the sequential re-localization of the CP outside of the nucleolus and into discrete nuclear foci and, eventually, into an uneven distribution in the nucleoplasm. Expression of the viral replication-associated protein, Rep, is sufficient to exclude the CP from the nucleolus, but the localization of the CP in the characteristic patterns induced by the virus cannot be recapitulated by co-expression with any individual viral protein. Our results demonstrate that the subcellular distribution of the CP is a dynamic process, temporally regulated throughout the progression of the infection. The regulation of the localization of the CP is determined by the presence of other viral components or changes in the cellular environment induced by the virus, and is likely to contribute to the multifunctionality of this protein. Bearing in mind these observations, we suggest that viral proteins should be studied in the context of the infection and considering the temporal dimension in order to comprehensively understand their roles and effects in the interaction between virus and host. PMID:29312406

Quantification of asymmetric microtubule nucleation at sub-cellular structures

PubMed Central

Zhu, Xiaodong; Kaverina, Irina

2012-01-01

Cell polarization is important for multiple physiological processes. In polarized cells, microtubules (MTs) are organized into a spatially polarized array. Generally, in non-differentiated cells, it is assumed that MTs are symmetrically nucleated exclusively from centrosome (microtubule organizing center, MTOC) and then reorganized into the asymmetric array. We have recently identified the Golgi complex as an additional MTOC that asymmetrically nucleates MTs toward one side of the cell. Methods used for alternative MTOC identification include microtubule re-growth after complete drug-induced depolymerization and tracking of growing microtubules using fluorescence labeled MT +TIP binding proteins in living cells. These approaches can be used for quantification of MT nucleation sites at diverse sub-cellular structures. PMID:21773933

3D imaging of a rice pollen grain using transmission X-ray microscopy.

PubMed

Wang, Shengxiang; Wang, Dajiang; Wu, Qiao; Gao, Kun; Wang, Zhili; Wu, Ziyu

2015-07-01

For the first time, the three-dimensional (3D) ultrastructure of an intact rice pollen cell has been obtained using a full-field transmission hard X-ray microscope operated in Zernike phase contrast mode. After reconstruction and segmentation from a series of projection images, complete 3D structural information of a 35 µm rice pollen grain is presented at a resolution of ∼100 nm. The reconstruction allows a clear differentiation of various subcellular structures within the rice pollen grain, including aperture, lipid body, mitochondrion, nucleus and vacuole. Furthermore, quantitative information was obtained about the distribution of cytoplasmic organelles and the volume percentage of each kind of organelle. These results demonstrate that transmission X-ray microscopy can be quite powerful for non-destructive investigation of 3D structures of whole eukaryotic cells.

Mating changes the subcellular distribution and the functionality of estrogen receptors in the rat oviduct

PubMed Central

2009-01-01

Background Mating changes the mode of action of 17beta-estradiol (E2) to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER) are required. This change was designated as intracellular path shifting (IPS). Methods Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta) in oviductal epithelial cells of rats on day 1 of cycle (C1) or pregnancy (P1) using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb) and calbindin 9 kDa (s100g) in rats on C1 or P1 treated with selective agonists for ESR1 (PPT) or ESR2 (DPN). The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats. Results Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c-fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats. Conclusion Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct. PMID:19948032

Mapping the subcellular localization of Fe3O4@TiO2 nanoparticles by X-ray Fluorescence Microscopy.

PubMed

Yuan, Y; Chen, S; Gleber, S C; Lai, B; Brister, K; Flachenecker, C; Wanzer, B; Paunesku, T; Vogt, S; Woloschak, G E

The targeted delivery of Fe 3 O 4 @TiO2 nanoparticles to cancer cells is an important step in their development as nanomedicines. We have synthesized nanoparticles that can bind the Epidermal Growth Factor Receptor, a cell surface protein that is overexpressed in many epithelial type cancers. In order to study the subcellular distribution of these nanoparticles, we have utilized the sub-micron resolution of X-ray Fluorescence Microscopy to map the locationof Fe 3 O4@TiO 2 NPs and other trace metal elements within HeLa cervical cancer cells. Here we demonstrate how the higher resolution of the newly installed Bionanoprobe at the Advanced Photon Source at Argonne National Laboratory can greatly improve our ability to distinguish intracellular nanoparticles and their spatial relationship with subcellular compartments.

The importance of immunohistochemical analyses in evaluating the phenotype of Kv channel knockout mice.

PubMed

Menegola, Milena; Clark, Eliana; Trimmer, James S

2012-06-01

To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

Different Subcellular Localization of ALCAM Molecules in Neuroblastoma: Association with Relapse

PubMed Central

Corrias, Maria Valeria; Gambini, Claudio; Gregorio, Andrea; Croce, Michela; Barisione, Gaia; Cossu, Claudia; Rossello, Armando; Ferrini, Silvano; Fabbi, Marina

2010-01-01

Background: The Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD), involved in nervous system development, has been linked to tumor progression and metastasis in several tumors. No information is available on ALCAM expression in neuroblastoma, a childhood neoplasia originating from the sympathetic nervous system. Methods: ALCAM expression was analysed by immunofluorescence and immunohistochemistry on differentiated neuroblastoma cell lines and on archival specimens of stroma-poor, not MYCN amplified, resectable neuroblastoma tumors, respectively. Results: ALCAM is variously expressed in neuroblastoma cell lines, is shed by metalloproteases and is cleaved by ADAM17/TACE in vitro. ALCAM is expressed in neuroblastoma primary tumors with diverse patterns of subcellular localization and is highly expressed in the neuropil area in a subgroup of cases. Tumor specimens showing high expression of ALCAM at the membrane of the neuroblast body or low levels in the neuropil area are associated with relapse (P = 0.044 and P < 0.0001, respectively). In vitro differentiated neuroblastoma cells show strong ALCAM expression on neurites, suggesting that ALCAM expression in the neuropil is related to a differentiated phenotype. Conclusions: Assessment of ALCAM localization by immunohistochemistry may help to identify patients who, in the absence of negative prognostic factors, are at risk of relapse and require a more careful follow-up. PMID:20208136

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duenas, Maria Emilia; Klein, Adam T.; Alexander, Liza E.

Metabolism in plants is compartmentalized among different tissues, cells and subcellular organelles. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption ionization (MALDI) has recently advanced to allow for the visualization of metabolites at single-cell resolution. Here we applied 5- and 10 μm high spatial resolution MALDI-MSI to the asymmetric Kranz anatomy of Zea mays (maize) leaves to study the differential localization of two major anionic lipids in thylakoid membranes, sulfoquinovosyldiacylglycerols (SQDG) and phosphatidylglycerols (PG). The quantification and localization of SQDG and PG molecular species, among mesophyll (M) and bundle sheath (BS) cells, are compared across the leaf developmental gradient frommore » four maize genotypes (the inbreds B73 and Mo17, and the reciprocal hybrids B73 × Mo17 and Mo17 × B73). SQDG species are uniformly distributed in both photosynthetic cell types, regardless of leaf development or genotype; however, PG shows photosynthetic cell-specific differential localization depending on the genotype and the fatty acyl chain constituent. Overall, 16:1-containing PGs primarily contribute to the thylakoid membranes of M cells, whereas BS chloroplasts are mostly composed of 16:0-containing PGs. Furthermore, PG 32:0 shows genotype-specific differences in cellular distribution, with preferential localization in BS cells for B73, but more uniform distribution between BS and M cells in Mo17. Maternal inheritance is exhibited within the hybrids, such that the localization of PG 32:0 in B73 × Mo17 is similar to the distribution in the B73 parental inbred, whereas that of Mo17 × B73 resembles the Mo17 parent. As a result, this study demonstrates the power of MALDI-MSI to reveal unprecedented insights on metabolic outcomes in multicellular organisms at single-cell resolution.« less

Modelling spatially regulated beta-catenin dynamics and invasion in intestinal crypts.

PubMed

Murray, Philip J; Kang, Jun-Won; Mirams, Gary R; Shin, Sung-Young; Byrne, Helen M; Maini, Philip K; Cho, Kwang-Hyun

2010-08-04

Experimental data (e.g., genetic lineage and cell population studies) on intestinal crypts reveal that regulatory features of crypt behavior, such as control via morphogen gradients, are remarkably well conserved among numerous organisms (e.g., from mouse and rat to human) and throughout the different regions of the small and large intestines. In this article, we construct a partial differential equation model of a single colonic crypt that describes the spatial distribution of Wnt pathway proteins along the crypt axis. The novelty of our continuum model is that it is based upon assumptions that can be directly related to processes at the cellular and subcellular scales. We use the model to predict how the distributions of Wnt pathway proteins are affected by mutations. The model is then extended to investigate how mutant cell populations can invade neighboring crypts. The model simulations suggest that cell crowding caused by increased proliferation and decreased cell loss may be sufficient for a mutant cell population to colonize a neighboring healthy crypt. 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Differential cellular and subcellular distribution of glutamate transporters in the cat retina.

PubMed

Fyk-Kolodziej, Bozena; Qin, Pu; Dzhagaryan, Arturik; Pourcho, Roberta G

2004-01-01

Retrieval of glutamate from extracellular sites in the retina involves at least five excitatory amino acid transporters. Immunocytochemical analysis of the cat retina indicates that each of these transporters exhibits a selective distribution which may reflect its specific function. The uptake of glutamate into Muller cells or astrocytes appears to depend upon GLAST and EAAT4, respectively. Staining for EAAT4 was also seen in the pigment epithelium. The remaining transporters are neuronal with GLT-1alpha localized to a number of cone bipolar, amacrine, and ganglion cells and GLT-1v in cone photoreceptors and several populations of bipolar cells. The EAAC1 transporter was found in horizontal, amacrine, and ganglion cells. Staining for EAAT5 was seen in the axon terminals of both rod and cone photoreceptors as well as in numerous amacrine and ganglion cells. Although some of the glutamate transporter molecules are positioned for presynaptic or postsynaptic uptake at glutamatergic synapses, others with localizations more distant from such contacts may serve in modulatory roles or provide protection against excitoxic or oxidative damage.

The neuroregenerative mechanism mediated by the Hsp90-binding immunophilin FKBP52 resembles the early steps of neuronal differentiation

PubMed Central

Quintá, HR; Galigniana, MD

2012-01-01

BACKGROUND AND PURPOSE The immunosuppressive macrolide FK506 (tacrolimus) shows neuroregenerative action by a mechanism that appears to involve the Hsp90-binding immunophilin FKBP52. This study analyses some aspects of the early steps of neuronal differentiation and neuroregeneration. EXPERIMENTAL APPROACH Undifferentiated murine neuroblastoma cells and hippocampal neurones isolated from embryonic day-17 rat embryos were induced to differentiate with FK506. Subcellular relocalization of FKBP52, Hsp90 and its co-chaperone p23 was analysed by indirect immunofluorescence confocal microscopy and by Western blots of axonal fractions isolated from cells grown on a porous transwell cell culture chamber. Neuroregeneration was evaluated using a scratch-wound assay. KEY RESULTS In undifferentiated cells, FKBP52, Hsp90 and p23 are located in the cell nucleus, forming an annular structure that disassembles when the differentiation process is triggered by FK506. This was observed in the N2a cell line and in hippocampal neurones. More importantly, the annular structure of chaperones is reassembled after damaging the neurones, whereas FK506 prompts their rapid regeneration, a process linked to the subcellular redistribution of the heterocomplex. CONCLUSIONS AND IMPLICATIONS There is a direct relationship between the disassembly of the chaperone complex and the progression of neuronal differentiation upon stimulation with the immunophilin ligand FK506. Both neuronal differentiation and neuroregeneration appear to be mechanistically linked, so the elucidation of one mechanism may lead to unravel the properties of the other. This study also implies that the discovery of FK506 derivatives, devoid of immunosuppressive action, would be therapeutically significant for neurotrophic use. PMID:22091865

Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

NASA Astrophysics Data System (ADS)

Chandra, Subhash

2004-06-01

Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13C and 15N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13C15N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39K, 23Na and 40Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

Identification, classification and differential expression of oleosin genes in tung tree (Vernicia fordii)

USDA-ARS?s Scientific Manuscript database

Triacylglycerols (TAG) are the major molecules of energy storage in eukaryotes. TAG are packed in subcellular structures called oil bodies or lipid droplets. Oleosins (OLE) are the major proteins in plant oil bodies. Multiple isoforms of OLE are present in plants such as tung tree (Vernicia fordii),...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raza, Haider; John, Annie

Tea polyphenols have been reported to be potent antioxidants and beneficial in oxidative stress related diseases. Prooxidant effects of tea polyphenols have also been reported in cell culture systems. In the present study, we have studied oxidative stress in the subcellular compartments of PC12 cells after treatment with different concentrations of the green tea polyphenol, epigallocatechin-3-gallate (EGCG). We have demonstrated that EGCG has differentially affected the production of reactive oxygen species (ROS), glutathione (GSH) metabolism and cytochrome P450 2E1 activity in the different subcellular compartments in PC12 cells. Our results have shown that although the cell survival was not inhibitedmore » by EGCG, there was, however, an increased DNA breakdown and activation of apoptotic markers, caspase 3 and poly- (ADP-ribose) polymerase (PARP) at higher concentrations of EGCG treatment. Our results suggest that the differential effects of EGCG might be related to the alterations in oxidative stress, GSH pools and CYP2E1 activity in different cellular compartments. These results may have implications in determining the chemopreventive therapeutic use of tea polyphenols in vivo.« less

Single-cell mechanics--An experimental-computational method for quantifying the membrane-cytoskeleton elasticity of cells.

PubMed

Tartibi, M; Liu, Y X; Liu, G-Y; Komvopoulos, K

2015-11-01

The membrane-cytoskeleton system plays a major role in cell adhesion, growth, migration, and differentiation. F-actin filaments, cross-linkers, binding proteins that bundle F-actin filaments to form the actin cytoskeleton, and integrins that connect the actin cytoskeleton network to the cell plasma membrane and extracellular matrix are major cytoskeleton constituents. Thus, the cell cytoskeleton is a complex composite that can assume different shapes. Atomic force microscopy (AFM)-based techniques have been used to measure cytoskeleton material properties without much attention to cell shape. A recently developed surface chemical patterning method for long-term single-cell culture was used to seed individual cells on circular patterns. A continuum-based cell model, which uses as input the force-displacement response obtained with a modified AFM setup and relates the membrane-cytoskeleton elastic behavior to the cell geometry, while treating all other subcellular components suspended in the cytoplasmic liquid (gel) as an incompressible fluid, is presented and validated by experimental results. The developed analytical-experimental methodology establishes a framework for quantifying the membrane-cytoskeleton elasticity of live cells. This capability may have immense implications in cell biology, particularly in studies seeking to establish correlations between membrane-cytoskeleton elasticity and cell disease, mortality, differentiation, and migration, and provide insight into cell infiltration through nonwoven fibrous scaffolds. The present method can be further extended to analyze membrane-cytoskeleton viscoelasticity, examine the role of other subcellular components (e.g., nucleus envelope) in cell elasticity, and elucidate the effects of mechanical stimuli on cell differentiation and motility. This is the first study to decouple the membrane-cytoskeleton elasticity from cell stiffness and introduce an effective approach for measuring the elastic modulus. The novelty of this study is the development of new technology for quantifying the elastic stiffness of the membrane-cytoskeleton system of cells. This capability could have immense implications in cell biology, particularly in establishing correlations between various cell diseases, mortality, and differentiation with membrane-cytoskeleton elasticity, examining through-tissue cell migration, and understanding cell infiltration in porous scaffolds. The present method can be further extended to analyze membrane-cytoskeleton viscous behavior, identify the contribution of other subcellular components (e.g., nucleus envelope) to load sharing, and elucidate mechanotransduction effects due to repetitive compressive loading and unloading on cell differentiation and motility. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The eukaryotic RNA exosome: same scaffold but variable catalytic subunits.

PubMed

Lykke-Andersen, Søren; Tomecki, Rafal; Jensen, Torben Heick; Dziembowski, Andrzej

2011-01-01

The RNA exosome is a versatile ribonucleolytic protein complex that participates in a multitude of cellular RNA processing and degradation events. It consists of an invariable nine-subunit core that associates with a variety of enzymatically active subunits and co-factors. These contribute to or even provide the catalytic activity and substrate specificity of the complex. The S. cerevisiae exosome has been intensively studied since its discovery in 1997 and thus serves as the archetype of eukaryotic exosomes. Notably, its catalytic potential, derived exclusively from associated subunits, differs between the nuclear and cytoplasmic versions of the complex. The same holds true for other eukaryotes, however, recent discoveries from various laboratories including our own have revealed that there are variations on this theme. Here, we review the latest findings concerning catalytic subunits of eukaryotic exosomes, and we discuss the apparent need for differential composition and subcellular distribution of exosome variants.

Subcellular localization and cytoplasmic complex status of endogenous Keap1.

PubMed

Watai, Yoriko; Kobayashi, Akira; Nagase, Hiroko; Mizukami, Mio; McEvoy, Justina; Singer, Jeffrey D; Itoh, Ken; Yamamoto, Masayuki

2007-10-01

Keap1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin-3-based ubiquitin ligase, and a regulator of Nrf2 activity through the interaction with Nrf2 Neh2 domain. However, the mechanism(s) of Nrf2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous Keap1 molecule. Here, we report the generation of a new monoclonal antibody for the detection of endogenous Keap1 molecules. Immunocytochemical analysis of mouse embryonic fibroblasts with the antibody revealed that under normal, unstressed condition, Keap1 is localized primarily in the cytoplasm with minimal amount in the nucleus and endoplasmic reticulum. This subcellular localization profile of Keap1 appears unchanged after treatment of cells with diethyl maleate, an electrophile, and/or Leptomycin B, a nuclear export inhibitor. Subcellular fractionation analysis of mouse liver cells showed similar results. No substantial change in the subcellular distribution profile could be observed in cells isolated from butylated hydroxyanisole-treated mice. Analyses of sucrose density gradient centrifugation of mouse liver cells indicated that Keap1 appears to form multiprotein complexes in the cytoplasm. These results demonstrate that endogenous Keap1 remains mostly in the cytoplasm, and electrophiles promote nuclear accumulation of Nrf2 without altering the subcellular localization of Keap1.

Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum).

PubMed

Gao, Wei; Nan, Tiegui; Tan, Guiyu; Zhao, Hongwei; Tan, Weiming; Meng, Fanyun; Li, Zhaohu; Li, Qing X; Wang, Baomin

2015-01-01

The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1(Ser137) involvement in spindle formation and REC8 cleavage.

PubMed

Du, Juan; Cao, Yan; Wang, Qian; Zhang, Nana; Liu, Xiaoyu; Chen, Dandan; Liu, Xiaoyun; Xu, Qunyuan; Ma, Wei

2015-01-01

Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1(Ser137) and pPlk1(Thr210)) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1(Ser137) with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1(Thr210) was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1(Ser137) was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1(Thr210) was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1(Ser137) accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1(Ser137) and pPlk1(Thr210), as well as the subcellular distribution of pPlk1(Thr210), were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1(Ser137) is the action executor, in mouse oocytes during meiotic division.

Analysis of Subcellular Prefoldin 1 Redistribution During Rabies Virus Infection

PubMed Central

Zhang, Jinyang; Han, Qinqin; Song, Yuzhu; Chen, Qiang; Xia, Xueshan

2015-01-01

Background: Rabies virus (RABV) is one of the old deadly zoonotic viruses. It attacks the central nervous system and causes acute encephalitis in humans and animals. Host factors are known to be essential for virus infection and replication in cells. The identification of the key host factors required for RABV infection may provide important information on RABV replication and may provide new potential targets for RABV drug discovery. Objectives: This study aimed to investigate the change in the subcellular distribution and expression of the host protein Prefoldin subunit 1 (PFDN1) in RABV-infected cells and the viral expression of plasmids in the transfected cells. Materials and Methods: Mouse Neuro-2a (N2a) cells were infected by RABV or transfected with the plasmids of the nucleoprotein (N) and/or phosphoprotein (P) gene of RABV. The subcellular distribution of PFDN1 was analyzed by confocal microscopy, and the transcription levels of PFDN1 in the N and/or P gene of the RABV-transfected or RABV-infected N2a cells were assessed via real-time quantitative polymerase chain reaction. Results: Confocal microscopy showed that PFDN1 was colocalized with the N protein of RABV in the infected N2a cells and was mainly recruited to the characteristic Negri-Body-Like (NBL) structures in the cytoplasm, as well as the cotransfection of the N and P genes of RABV. The transcription of PFDN1 in the RABV-infected N2a cells was upregulated, whereas the transfection of the N and/or P genes did not result in the upregulation of PFDN1. Conclusions: The results of this work demonstrated that the subcellular distribution of PFDN1 was altered in the RABV-infected N2a cells and colocalized with the N protein of RABV in the NBL structures. PMID:26421138

Localization of a portion of the liver isoform of fatty-acid-binding protein (L-FABP) to peroxisomes

PubMed Central

Antonenkov, Vasily D.; Sormunen, Raija T.; Ohlmeier, Steffen; Amery, Leen; Fransen, Marc; Mannaerts, Guy P.; Hiltunen, J. Kalervo

2005-01-01

The liver isoform of fatty-acid-binding protein (L-FABP) facilitates the cellular uptake, transport and metabolism of fatty acids and is also involved in the regulation of gene expressions and cell differentiation. Consistent with these functions, L-FABP is predominantly present in the cytoplasm and to a lesser extent in the nucleus; however, a significant portion of this protein has also been detected in fractions containing different organelles. More recent observations, notably on L-FABP-deficient mice, indicated a possible direct involvement of L-FABP in the peroxisomal oxidation of long-chain fatty acids. In order to clarify the links between L-FABP and peroxisomal lipid metabolism, we reinvestigated the subcellular distribution of the protein. Analytical subcellular fractionation by a method preserving the intactness of isolated peroxisomes, two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with MS analysis, and immunoelectron microscopy of liver sections demonstrate the presence of L-FABP in the matrix of peroxisomes as a soluble protein. Peroxisomal L-FABP was highly inducible by clofibrate. The induction of L-FABP was accompanied by a marked increase in the binding capacity of peroxisomal matrix proteins for oleic acid and cis-parinaric acid. The peroxisomal β-oxidation of palmitoyl-CoA and acyl-CoA thioesterase activity were stimulated by L-FABP, indicating that the protein modulates the function of peroxisomal lipid-metabolizing enzymes. The possible role of intraperoxisomal L-FABP in lipid metabolism is discussed. PMID:16262600

Localization of a portion of the liver isoform of fatty-acid-binding protein (L-FABP) to peroxisomes.

PubMed

Antonenkov, Vasily D; Sormunen, Raija T; Ohlmeier, Steffen; Amery, Leen; Fransen, Marc; Mannaerts, Guy P; Hiltunen, J Kalervo

2006-03-01

The liver isoform of fatty-acid-binding protein (L-FABP) facilitates the cellular uptake, transport and metabolism of fatty acids and is also involved in the regulation of gene expressions and cell differentiation. Consistent with these functions, L-FABP is predominantly present in the cytoplasm and to a lesser extent in the nucleus; however, a significant portion of this protein has also been detected in fractions containing different organelles. More recent observations, notably on L-FABP-deficient mice, indicated a possible direct involvement of L-FABP in the peroxisomal oxidation of long-chain fatty acids. In order to clarify the links between L-FABP and peroxisomal lipid metabolism, we reinvestigated the subcellular distribution of the protein. Analytical subcellular fractionation by a method preserving the intactness of isolated peroxisomes, two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with MS analysis, and immunoelectron microscopy of liver sections demonstrate the presence of L-FABP in the matrix of peroxisomes as a soluble protein. Peroxisomal L-FABP was highly inducible by clofibrate. The induction of L-FABP was accompanied by a marked increase in the binding capacity of peroxisomal matrix proteins for oleic acid and cis-parinaric acid. The peroxisomal beta-oxidation of palmitoyl-CoA and acyl-CoA thioesterase activity were stimulated by L-FABP, indicating that the protein modulates the function of peroxisomal lipid-metabolizing enzymes. The possible role of intraperoxisomal L-FABP in lipid metabolism is discussed.

Site of Fluoride Accumulation in Navel Orange Leaves 1

PubMed Central

Chang, Chong W.; Thompson, C. Ray

1966-01-01

Fluoride-polluted navel orange leaves, Citrus sinensis (Linn.) Osbeck, were fractionated into the subcellular components in hexane/carbon tetrachloride mixtures having various densities. Fluoride was determined at each fraction. Analyses were also made for the subcellular distribution of chlorophyll, nitrogen, and DNA to assess the extent of cross-contamination of each component. The fraction containing cell wall, nuclei, and partly broken cells apparently contained a major amount of fluoride. However, if allowance was made for the cross-contamination of chloroplasts and chloroplast fragments, the fraction of chloroplasts was found to be the site of the highest fluoride accumulation. When each particulate component was washed with water after drying, the combined washings contained more than 50% of the total fluoride of the isolated fractions. The usual method of subcellular fractionation with aqueous solvent shifted the major site of fluoride accumulation from the fraction of chloroplasts to that of the supernatant. PMID:5908632

Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration.

PubMed

Dwyer, Amy R; Mouchemore, Kellie A; Steer, James H; Sunderland, Andrew J; Sampaio, Natalia G; Greenland, Eloise L; Joyce, David A; Pixley, Fiona J

2016-07-01

A major role of colony-stimulating factor-1 is to stimulate the differentiation of mononuclear phagocytic lineage cells into adherent, motile, mature macrophages. The colony-stimulating factor-1 receptor transduces colony-stimulating factor-1 signaling, and we have shown previously that phosphatidylinositol 3-kinase p110δ is a critical mediator of colony-stimulating factor-1-stimulated motility through the colony-stimulating factor-1 receptor pY721 motif. Src family kinases are also implicated in the regulation of macrophage motility and in colony-stimulating factor-1 receptor signaling, although functional redundancy of the multiple SFKs expressed in macrophages makes it challenging to delineate their specific functions. We report a comprehensive analysis of individual Src family kinase expression in macrophage cell lines and primary macrophages and demonstrate colony-stimulating factor-1-induced changes in Src family kinase subcellular localization, which provides clues to their distinct and redundant functions in macrophages. Moreover, expression of individual Src family kinases is both species specific and dependent on colony-stimulating factor-1-induced macrophage differentiation. Hck associated with the activated colony-stimulating factor-1 receptor, whereas Lyn associated with the receptor in a constitutive manner. Consistent with this, inhibitor studies revealed that Src family kinases were important for both colony-stimulating factor-1 receptor activation and colony-stimulating factor-1-induced macrophage spreading, motility, and invasion. Distinct colony-stimulating factor-1-induced changes in the subcellular localization of individual SFKs suggest specific roles for these Src family kinases in the macrophage response to colony-stimulating factor-1. © Society for Leukocyte Biology.

Yes-Associated Protein (YAP) Promotes the Nuclear Import of p73

NASA Astrophysics Data System (ADS)

Zhang, Heng; Wu, Shengnan

2011-01-01

p73 has been identified as a structural and functional homolog of the tumor suppressor p53. However, mechanisms that regulate the localization of p73 have not been fully clarified. The Yes-associated protein (YAP) is a transcriptional coactivator. As a transcriptional coactivator, YAP needs to bind transcription factors to stimulate gene expression. p73 is a reported YAP target transcription factors and YAP has been shown to positively regulate p73 in promoting apoptosis. Previous studies show that p73 interacts with YAP through its PPPY motif, and increases p73 transactivation of apoptotic genes. In this study, we focused on YAP's regulation of the localization of p73. After transient transfection into Rat pheochromocytoma (PC12) cells and Human embryonic kidney 293T cells with GFP-YAP and/or YFP-p73, and incubated for 24 hours expression. p73 was fused to YFP to allow the examination of its subcellular localization. When expressed alone, YFP-p73 was distributed throughout the cell. When coexpressed with YAP, nuclear accumulation of YFP-p73 became evident. We quantitated the effect of YAP on the redistribution of YFP-p73 by counting cells with nuclear-only YFP signal. We found that YAP can influence the subcellular distribution of p73. Altogether, coexpression with YAP affected the subcellular distribution of the p73 protein. Our studies attribute a central role to YAP in regulating p73 accumulation and YAP, at least in part, might promote the nuclear import of p73.

Selenium alleviates chromium toxicity by preventing oxidative stress in cabbage (Brassica campestris L. ssp. Pekinensis) leaves.

PubMed

Qing, Xuejiao; Zhao, Xiaohu; Hu, Chengxiao; Wang, Peng; Zhang, Ying; Zhang, Xuan; Wang, Pengcheng; Shi, Hanzhi; Jia, Fen; Qu, Chanjuan

2015-04-01

The beneficial role of selenium (Se) in alleviation of chromium (Cr)-induced oxidative stress is well established. However, little is known about the underlying mechanism. The impacts of exogenous Se (0.1mg/L) on Cr(1mg/L)-induced oxidative stress and antioxidant systems in leaves of cabbage (Brassica campestris L. ssp. Pekinensis) were investigated by using cellular and biochemical approaches. The results showed that supplementation of the medium with Se was effective in reducing Cr-induced increased levels of lipid peroxides and superoxide free radicals (O(-)2(·)), as well as increasing activities of superoxide dismutase (SOD) and peroxidase (POD). Meanwhile, 1mg/L Cr induced loss of plasma membrane integrity, growth inhibition, as well as ultrastructural changes of leaves were significantly reversed due to Se supplementation in the medium. In addition, Se application significantly altered the subcellular distribution of Cr which transported from mitochondria, nucleus and the cell-wall material to the soluble fraction and chloroplasts. However, Se application did no significant alteration of Cr effects on osmotic adjustment accumulating products. The study suggested that Se is able to protect leaves of cabbage against Cr toxicity by alleviation of Cr induced oxidative stress, and re-distribution of Cr in the subcellular of the leaf. Furthermore, free radicals, lipid peroxides, activity of SOD and POD, and subcellular distribution of Cr can be considered the efficient biomarkers to indicate the efficiency of Se to detoxification Cr. Copyright © 2015 Elsevier Inc. All rights reserved.

Analysis of Hippo and TGFβ signaling in polarizing epithelial cells and mouse embryos.

PubMed

Narimatsu, Masahiro; Labibi, Batool; Wrana, Jeffrey L; Attisano, Liliana

2016-01-01

The Hippo signaling pathway is involved in numerous biological events ranging from early development to organogenesis and when disrupted, impacts various human diseases including cancer. The Hippo pathway also interacts with and controls the activity of other signaling pathways such as the TGFβ/Smad pathway, in which Hippo pathway activity influences the subcellular localization of Smad transcription factors. Here, we describe techniques for examining crosstalk between Hippo and TGFβ signaling in polarizing mammary epithelial cells. In addition, we provide detailed methods for analyzing the subcellular localization of the Hippo pathway effectors, Taz and Yap using both in vitro cultured epithelial cells and in vivo in pregastrulation mouse embryos. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

RNA-binding Protein Quaking Stabilizes Sirt2 mRNA during Oligodendroglial Differentiation*

PubMed Central

Thangaraj, Merlin P.; Furber, Kendra L.; Gan, Jotham K.; Ji, Shaoping; Sobchishin, Larhonda; Doucette, J. Ronald; Nazarali, Adil J.

2017-01-01

Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, plays a critical role in OL differentiation by promoting both arborization and downstream expression of myelin-specific genes. However, the mechanisms involved in regulating SIRT2 expression during OL development are largely unknown. The RNA-binding protein quaking (QKI) plays an important role in myelination by post-transcriptionally regulating the expression of several myelin specific genes. In quaking viable (qkv/qkv) mutant mice, SIRT2 protein is severely reduced; however, it is not known whether these genes interact to regulate OL differentiation. Here, we report for the first time that QKI directly binds to Sirt2 mRNA via a common quaking response element (QRE) located in the 3′ untranslated region (UTR) to control SIRT2 expression in OL lineage cells. This interaction is associated with increased stability and longer half-lives of Sirt2.1 and Sirt2.2 transcripts leading to increased accumulation of Sirt2 transcripts. Consistent with this, overexpression of qkI promoted the expression of Sirt2 mRNA and protein. However, overexpression of the nuclear isoform qkI-5 promoted the expression of Sirt2 mRNA, but not SIRT2 protein, and delayed OL differentiation. These results suggest that the balance in the subcellular distribution and temporal expression of QKI isoforms control the availability of Sirt2 mRNA for translation. Collectively, our study demonstrates that QKI directly plays a crucial role in the post-transcriptional regulation and expression of Sirt2 to facilitate OL differentiation. PMID:28188285

Characterization of Aquaporin 4 Protein Expression and Localization in Tissues of the Dogfish (Squalus acanthias)

PubMed Central

Cutler, Christopher P.; Harmon, Sheena; Walsh, Jonathon; Burch, Kia

2012-01-01

The role of aquaporin water channels such as aquaporin 4 (Aqp4) in elasmobranchs such as the dogfish Squalus acanthias is completely unknown. This investigation set out to determine the expression and cellular and sub-cellular localization of Aqp4 protein in dogfish tissues. Two polyclonal antibodies were generated (AQP4/1 and AQP4/2) and these showed somewhat different characteristics in Western blotting and immunohistochemistry. Western blots using the AQP4/1 antibody showed two bands (35.5 and 49.5 kDa) in most tissues in a similar fashion to mammals. Liver had an additional band of 57 kDa and rectal gland two further faint bands of 37.5 and 38.5 kDa. However, unlike in mammals, Aqp4 protein was ubiquitously expressed in all tissues including gill and liver. The AQP4/2 antibody appeared much less specific in Western blots. Both antibodies were used in immunohistochemistry and showed similar cellular localizations, although the AQP4/2 antibody had a more restricted sub-cellular distribution compared to AQP4/1 and therefore appeared to be more specific for Aqp4. In kidney a sub-set of tubules were stained which may represent intermediate tubule segments (In-III–In-VI). AQP4/1 and AQP4/2 antibodies localized to the same tubules segments in serial sections although the intensity and sub-cellular distribution were different. AQP4/2 showed a basal or basolateral membrane distribution whereas AQP4/1 was often distributed throughout the whole cell including the nuclear region. In rectal gland and cardiac stomach Aqp4 was localized to secretory tubules but again AQP/1 and AQP/2 exhibited different sub-cellular distributions. In gill, both antibodies stained large cells in the primary filament and secondary lamellae. Again AQP4/1 antibody stained most or all the cell including the nucleus, whereas AQP4/2 had a plasma membrane or plasma membrane and cytoplasmic distribution. Two types of large mitochondrial rich transport cells are known to exist in elasmobranchs, that express either Na, K-ATPase, or V-type ATPase ion transporters. Using Na, K-ATPase, and V-type ATPase antibodies, Aqp4 was colocalized with these proteins using the AQP4/1 antibody. Results show Aqp4 is expressed in both (and all) branchial Na, K-ATPase, and V-type ATPase expressing cells. PMID:22363294

Characterization of Aquaporin 4 Protein Expression and Localization in Tissues of the Dogfish (Squalus acanthias).

PubMed

Cutler, Christopher P; Harmon, Sheena; Walsh, Jonathon; Burch, Kia

2012-01-01

The role of aquaporin water channels such as aquaporin 4 (Aqp4) in elasmobranchs such as the dogfish Squalus acanthias is completely unknown. This investigation set out to determine the expression and cellular and sub-cellular localization of Aqp4 protein in dogfish tissues. Two polyclonal antibodies were generated (AQP4/1 and AQP4/2) and these showed somewhat different characteristics in Western blotting and immunohistochemistry. Western blots using the AQP4/1 antibody showed two bands (35.5 and 49.5 kDa) in most tissues in a similar fashion to mammals. Liver had an additional band of 57 kDa and rectal gland two further faint bands of 37.5 and 38.5 kDa. However, unlike in mammals, Aqp4 protein was ubiquitously expressed in all tissues including gill and liver. The AQP4/2 antibody appeared much less specific in Western blots. Both antibodies were used in immunohistochemistry and showed similar cellular localizations, although the AQP4/2 antibody had a more restricted sub-cellular distribution compared to AQP4/1 and therefore appeared to be more specific for Aqp4. In kidney a sub-set of tubules were stained which may represent intermediate tubule segments (In-III-In-VI). AQP4/1 and AQP4/2 antibodies localized to the same tubules segments in serial sections although the intensity and sub-cellular distribution were different. AQP4/2 showed a basal or basolateral membrane distribution whereas AQP4/1 was often distributed throughout the whole cell including the nuclear region. In rectal gland and cardiac stomach Aqp4 was localized to secretory tubules but again AQP/1 and AQP/2 exhibited different sub-cellular distributions. In gill, both antibodies stained large cells in the primary filament and secondary lamellae. Again AQP4/1 antibody stained most or all the cell including the nucleus, whereas AQP4/2 had a plasma membrane or plasma membrane and cytoplasmic distribution. Two types of large mitochondrial rich transport cells are known to exist in elasmobranchs, that express either Na, K-ATPase, or V-type ATPase ion transporters. Using Na, K-ATPase, and V-type ATPase antibodies, Aqp4 was colocalized with these proteins using the AQP4/1 antibody. Results show Aqp4 is expressed in both (and all) branchial Na, K-ATPase, and V-type ATPase expressing cells.

Subcellular Distribution of Glutathione Precursors in Arabidopsis thaliana

PubMed Central

Koffler, Barbara Eva; Maier, Romana; Zechmann, Bernd

2011-01-01

Abstract Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) – the first enzyme of glutathione synthesis – causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells. PMID:22050910

Rho-associated protein kinase regulates subcellular localisation of Angiomotin and Hippo-signalling during preimplantation mouse embryo development.

PubMed

Mihajlović, Aleksandar I; Bruce, Alexander W

2016-09-01

The differential activity of the Hippo-signalling pathway between the outer- and inner-cell populations of the developing preimplantation mouse embryo directs appropriate formation of trophectoderm and inner cell mass (ICM) lineages. Such distinct signalling activity is under control of intracellular polarization, whereby Hippo-signalling is either supressed in polarized outer cells or activated in apolar inner cells. The central role of apical-basolateral polarization to such differential Hippo-signalling regulation prompted us to reinvestigate the role of potential upstream molecular regulators affecting apical-basolateral polarity. This study reports that the chemical inhibition of Rho-associated kinase (Rock) is associated with failure to form morphologically distinct blastocysts, indicative of compromised trophectoderm differentiation, and defects in the localization of both apical and basolateral polarity factors associated with malformation of tight junctions. Moreover, Rock-inhibition mediates mislocalization of the Hippo-signalling activator Angiomotin (Amot), to the basolateral regions of outer cells and is concomitant with aberrant activation of the pathway. The Rock-inhibition phenotype is mediated by Amot, as RNAi-based Amot knockdown totally rescues the normal suppression of Hippo-signalling in outer cells. In conclusion, Rock, via regulating appropriate apical-basolateral polarization in outer cells, regulates the appropriate activity of the Hippo-signalling pathway, by ensuring correct subcellular localization of Amot protein in outer cells. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Systematic Analysis of Arabidopsis Organelles and a Protein Localization Database for Facilitating Fluorescent Tagging of Full-Length Arabidopsis Proteins1[W

PubMed Central

Li, Shijun; Ehrhardt, David W.; Rhee, Seung Y.

2006-01-01

Cells are organized into a complex network of subcellular compartments that are specialized for various biological functions. Subcellular location is an important attribute of protein function. To facilitate systematic elucidation of protein subcellular location, we analyzed experimentally verified protein localization data of 1,300 Arabidopsis (Arabidopsis thaliana) proteins. The 1,300 experimentally verified proteins are distributed among 40 different compartments, with most of the proteins localized to four compartments: mitochondria (36%), nucleus (28%), plastid (17%), and cytosol (13.3%). About 19% of the proteins are found in multiple compartments, in which a high proportion (36.4%) is localized to both cytosol and nucleus. Characterization of the overrepresented Gene Ontology molecular functions and biological processes suggests that the Golgi apparatus and peroxisome may play more diverse functions but are involved in more specialized processes than other compartments. To support systematic empirical determination of protein subcellular localization using a technology called fluorescent tagging of full-length proteins, we developed a database and Web application to provide preselected green fluorescent protein insertion position and primer sequences for all Arabidopsis proteins to study their subcellular localization and to store experimentally verified protein localization images, videos, and their annotations of proteins generated using the fluorescent tagging of full-length proteins technology. The database can be searched, browsed, and downloaded using a Web browser at http://aztec.stanford.edu/gfp/. The software can also be downloaded from the same Web site for local installation. PMID:16617091

Capillary electrophoretic analysis reveals subcellular binding between individual mitochondria and cytoskeleton

PubMed Central

Kostal, Vratislav; Arriaga, Edgar A.

2011-01-01

Interactions between the cytoskeleton and mitochondria are essential for normal cellular function. An assessment of such interactions is commonly based on bulk analysis of mitochondrial and cytoskeletal markers present in a given sample, which assumes complete binding between these two organelle types. Such measurements are biased because they rarely account for non-bound ‘free’ subcellular species. Here we report on the use of capillary electrophoresis with dual laser induced fluorescence detection (CE-LIF) to identify, classify, count and quantify properties of individual binding events of mitochondria and cytoskeleton. Mitochondria were fluorescently labeled with DsRed2 while F-actin, a major cytoskeletal component, was fluorescently labeled with Alexa488-phalloidin. In a typical subcellular fraction of L6 myoblasts, 79% of mitochondrial events did not have detectable levels of F-actin, while the rest had on average ~2 zeptomole F-actin, which theoretically represents a ~ 2.5-μm long network of actin filaments per event. Trypsin treatment of L6 subcellular fractions prior to analysis decreased the fraction of mitochondrial events with detectable levels of F-actin, which is expected from digestion of cytoskeletal proteins on the surface of mitochondria. The electrophoretic mobility distributions of the individual events were also used to further distinguish between cytoskeleton-bound from cytoskeleton-free mitochondrial events. The CE-LIF approach described here could be further developed to explore cytoskeleton interactions with other subcellular structures, the effects of cytoskeleton destabilizing drugs, and the progression of viral infections. PMID:21309532

PhosphoregDB: The tissue and sub-cellular distribution of mammalian protein kinases and phosphatases

PubMed Central

Forrest, Alistair RR; Taylor, Darrin F; Fink, J Lynn; Gongora, M Milena; Flegg, Cameron; Teasdale, Rohan D; Suzuki, Harukazu; Kanamori, Mutsumi; Kai, Chikatoshi; Hayashizaki, Yoshihide; Grimmond, Sean M

2006-01-01

Background Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse. Results The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases. Conclusion Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered. PMID:16504016

Subcellular distribution and potential detoxification mechanisms of mercury in the liver of the Javan mongoose (Herpestes javanicus) in Amamioshima Island, Japan.

PubMed

Horai, Sawako; Furukawa, Tatsuhiko; Ando, Tetsuo; Akiba, Suminori; Takeda, Yasuo; Yamada, Katsushi; Kuno, Katsuji; Abe, Shintaro; Watanabe, Izumi

2008-06-01

In a previous study, we showed that Hg accumulated to high levels in the liver of the Javan mongoose (Herpestes javanicus), a terrestrial mammal that lives on Amamioshima Island, Japan. This suggests a sophisticated mechanism of hepatic Hg detoxication. Assay of the subcellular localization of Hg and the expression of protective enzymes provides important clues for elucidating the mechanism of Hg detoxication. In the present study, the concentrations of 11 elements (Mg, Cr, Mn, Fe, Cu, Zn, Se, Rb, Cd, total Hg [T-Hg] and organic Hg [O-Hg], and Pb) were determined in the liver and in five liver subcellular fractions (plasma membrane, mitochondria, nuclei, microsome, and cytosol) of this species. As the T-Hg level increased, T-Hg markedly distributed to the plasma membrane. The T-Hg levels in all subcellular fractions correlated with Se levels. Although the T-Hg level in the microsomal fraction was relatively low, the ratio of O-Hg to T-Hg was significantly lower in the microsomes than in the other fractions. Significant positive correlations were found between the level of glutathione-S-transferase-pi, a marker of oxidative stress, and the O-Hg and T-Hg levels, but the correlation was better with O-Hg than with T-Hg. Western blot analysis of thioredoxin reductase 2 (TrxR2), a protein involved in protecting cells from mitochondrial oxidative stress, showed that the level of TrxR2 correlated with that of T-Hg. High TrxR2 levels may be one mechanism by which the Javan mongoose attenuates the toxicity of the high Hg levels present in the liver.

Distribution and change patterns of free IAA, ABP 1 and PM H⁺-ATPase during ovary and ovule development of Nicotiana tabacum L.

PubMed

Chen, Dan; Deng, Yingtian; Zhao, Jie

2012-01-15

Auxin plays key roles in flower induction, embryogenesis, seed formation and seedling development, but little is known about whether auxin regulates the development of ovaries and ovules before pollination. In the present report, we measured the content of free indole-3-acetic (IAA) in ovaries of Nicotiana tabacum L., and localized free IAA, auxin binding protein 1 (ABP1) and plasma membrane (PM) H⁺-ATPase in the ovaries and ovules. The level of free IAA in the developmental ovaries increased gradually from the stages of ovular primordium to the functional megaspore, but slightly decreased when the embryo sacs formed. Immunoenzyme labeling clearly showed that both IAA and ABP1 were distributed in the ovules, the edge of the placenta, vascular tissues and the ovary wall, while PM H⁺-ATPase was mainly localized in the ovules. By using immunogold labeling, the subcellular distributions of IAA, ABP1 and PM H⁺-ATPase in the ovules were also shown. The results suggest that IAA, ABP1 and PM H⁺-ATPase may play roles in the ovary and ovule initiation, formation and differentiation. Crown Copyright © 2011. Published by Elsevier GmbH. All rights reserved.

High spatial resolution mass spectrometry imaging reveals the genetically programmed, developmental modification of the distribution of thylakoid membrane lipids among individual cells of maize leaf

DOE PAGES

Duenas, Maria Emilia; Klein, Adam T.; Alexander, Liza E.; ...

2016-11-17

Metabolism in plants is compartmentalized among different tissues, cells and subcellular organelles. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption ionization (MALDI) has recently advanced to allow for the visualization of metabolites at single-cell resolution. Here we applied 5- and 10 μm high spatial resolution MALDI-MSI to the asymmetric Kranz anatomy of Zea mays (maize) leaves to study the differential localization of two major anionic lipids in thylakoid membranes, sulfoquinovosyldiacylglycerols (SQDG) and phosphatidylglycerols (PG). The quantification and localization of SQDG and PG molecular species, among mesophyll (M) and bundle sheath (BS) cells, are compared across the leaf developmental gradient frommore » four maize genotypes (the inbreds B73 and Mo17, and the reciprocal hybrids B73 × Mo17 and Mo17 × B73). SQDG species are uniformly distributed in both photosynthetic cell types, regardless of leaf development or genotype; however, PG shows photosynthetic cell-specific differential localization depending on the genotype and the fatty acyl chain constituent. Overall, 16:1-containing PGs primarily contribute to the thylakoid membranes of M cells, whereas BS chloroplasts are mostly composed of 16:0-containing PGs. Furthermore, PG 32:0 shows genotype-specific differences in cellular distribution, with preferential localization in BS cells for B73, but more uniform distribution between BS and M cells in Mo17. Maternal inheritance is exhibited within the hybrids, such that the localization of PG 32:0 in B73 × Mo17 is similar to the distribution in the B73 parental inbred, whereas that of Mo17 × B73 resembles the Mo17 parent. As a result, this study demonstrates the power of MALDI-MSI to reveal unprecedented insights on metabolic outcomes in multicellular organisms at single-cell resolution.« less

Subcellular distribution of trace elements in the liver of sea turtles.

PubMed

Anan, Yasumi; Kunito, Takashi; Sakai, Haruya; Tanabe, Shinsuke

2002-01-01

Subcellular distribution of Cu, Zn, Se, Rb, Mo, Ag, Cd and Pb was determined in the liver of green turtles (Chelonia mydas) and hawksbill turtles (Eretmochelys imbricata) from Yaeyama Islands, Japan. Also, hepatic cytosol from sea turtles was applied on a Sephadex G-75 column and elution profiles of trace elements were examined. Copper, Zn, Se, Rb, Ag and Cd were largely present in cytosol in the liver of both species, indicating that cytosol was the significant site for the accumulation of these elements in sea turtles. In contrast, Mo and Pb were accumulated specifically in nuclear and mitochondrial fraction and microsomal fraction, respectively. Gel filtration analysis showed that Cu, Zn, Ag and Cd were bound to metallothionein (MT) in the cytosol of sea turtles. To our knowledge, this is the first report on the association of trace elements with MT in sea turtles.

Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

PubMed

Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

2009-01-01

Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

Calibrated mitotic oscillator drives motile ciliogenesis.

PubMed

Al Jord, Adel; Shihavuddin, Asm; Servignat d'Aout, Raphaël; Faucourt, Marion; Genovesio, Auguste; Karaiskou, Anthi; Sobczak-Thépot, Joëlle; Spassky, Nathalie; Meunier, Alice

2017-11-10

Cell division and differentiation depend on massive and rapid organelle remodeling. The mitotic oscillator, centered on the cyclin-dependent kinase 1-anaphase-promoting complex/cyclosome (CDK1-APC/C) axis, spatiotemporally coordinates this reorganization in dividing cells. Here we discovered that nondividing cells could also implement this mitotic clocklike regulatory circuit to orchestrate subcellular reorganization associated with differentiation. We probed centriole amplification in differentiating mouse-brain multiciliated cells. These postmitotic progenitors fine-tuned mitotic oscillator activity to drive the orderly progression of centriole production, maturation, and motile ciliation while avoiding the mitosis commitment threshold. Insufficient CDK1 activity hindered differentiation, whereas excessive activity accelerated differentiation yet drove postmitotic progenitors into mitosis. Thus, postmitotic cells can redeploy and calibrate the mitotic oscillator to uncouple cytoplasmic from nuclear dynamics for organelle remodeling associated with differentiation. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

The subcellular distribution of aquaporin 5 in the cochlea reveals a water shunt at the perilymph-endolymph barrier.

PubMed

Hirt, B; Penkova, Z H; Eckhard, A; Liu, W; Rask-Andersen, H; Müller, M; Löwenheim, H

2010-07-28

Aquaporins are membrane water channel proteins that have also been identified in the cochlea. Auditory function critically depends on the homeostasis of the cochlear fluids perilymph and endolymph. In particular, the ion and water regulation of the endolymph is essential for sensory transduction. Within the cochlear duct the lateral wall epithelium has been proposed to secrete endolymph by an aquaporin-mediated flow of water across its epithelial tight junction barrier. This study identifies interspecies differences in the cellular distribution of aquaporin 5 (AQP5) in the cochlear lateral wall of mice, rats, gerbils and guinea pigs. In addition the cellular expression pattern of AQP5 is described in the human cochlea. Developmental changes in rats demonstrate longitudinal and radial gradients along the cochlear duct. During early postnatal development a pancochlear expression is detected. However a regression to the apical quadrant and limitation to outer sulcus cells (OSCs) is observed in the adult. This developmental loss of AQP5 expression in the basal cochlear segments coincides with a morphological loss of contact between OSCs and the endolymph. At the subcellular level, AQP5 exhibits polarized expression in the apical plasma membrane of the OSCs. Complementary, the basolateral membrane in the root processes of the OSCs exhibits AQP4 expression. This differential localization of AQP5 and AQP4 in the apical and basolateral membranes of the same epithelial cell type suggests a direct aquaporin-mediated transcellular water shunt between the perilymph and endolymph in the OSCs of the cochlear lateral wall. In the human cochlea these findings may have pathophysiological implications attributed to a dysfunctional water regulation by AQP5 such as endolymphatic hydrops (i.e. in Meniere's disease) or sensorineural hearing loss (i.e. in Sjögren's syndrome). Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Neutrophils Express Distinct RNA Receptors in a Non-canonical Way*

PubMed Central

Berger, Michael; Hsieh, Chin-Yuan; Bakele, Martina; Marcos, Veronica; Rieber, Nikolaus; Kormann, Michael; Mays, Lauren; Hofer, Laura; Neth, Olaf; Vitkov, Ljubomir; Krautgartner, Wolf Dietrich; von Schweinitz, Dietrich; Kappler, Roland; Hector, Andreas; Weber, Alexander; Hartl, Dominik

2012-01-01

RNAs are capable of modulating immune responses by binding to specific receptors. Neutrophils represent the major fraction of circulating immune cells, but receptors and mechanisms by which neutrophils sense RNA are poorly defined. Here, we analyzed the mRNA and protein expression patterns and the subcellular localization of the RNA receptors RIG-I, MDA-5, TLR3, TLR7, and TLR8 in primary neutrophils and immortalized neutrophil-like differentiated HL-60 cells. Our results demonstrate that both neutrophils and differentiated HL-60 cells express RIG-I, MDA-5, and TLR8 at the mRNA and protein levels, whereas TLR3 and TLR7 are not expressed at the protein level. Subcellular fractionation, flow cytometry, confocal laser scanning microscopy, and immuno-transmission electron microscopy provided evidence that, besides the cytoplasm, RIG-I and MDA-5 are stored in secretory vesicles of neutrophils and showed that RIG-I and its ligand, 3p-RNA, co-localize at the cell surface without triggering neutrophil activation. In summary, this study demonstrates that neutrophils express a distinct pattern of RNA recognition receptors in a non-canonical way, which could have essential implications for future RNA-based therapeutics. PMID:22532562

Bidirectional regulation of neurite elaboration by alternatively spliced metabotropic glutamate receptor 5 (mGluR5) isoforms.

PubMed

Mion, S; Corti, C; Neki, A; Shigemoto, R; Corsi, M; Fumagalli, G; Ferraguti, F

2001-06-01

Alternative splicing in the mGluR5 gene generates two different receptor isoforms, of which expression is developmentally regulated. However, little is known about the functional significance of mGluR5 splice variants. We have examined the functional coupling, subcellular targeting, and effect on neuronal differentiation of epitope-tagged mGluR5 isoforms by expression in neuroblastoma NG108-15 cells. We found that both mGluR5 splice variants give rise to comparable [Ca2+]i transients and have similar pharmacological profile. Tagged receptors were shown by immunofluorescence to be inserted in the plasma membrane. In undifferentiated cells the subcellular localization of the two mGluR5 isoforms was partially segregated, whereas in differentiated cells the labeling largely redistributed to the newly formed neurites. Interestingly, we demonstrate that mGluR5 splice variants dramatically influence the formation and maturation of neurites; mGluR5a hinders the acquisition of mature neuronal traits and mGluR5b fosters the elaboration and extension of neurites. These effects are partly inhibited by MPEP. Copyright 2001 Academic Press.

Dictyostelium cell death: early emergence and demise of highly polarized paddle cells.

PubMed

Levraud, Jean-Pierre; Adam, Myriam; Luciani, Marie-Françoise; de Chastellier, Chantal; Blanton, Richard L; Golstein, Pierre

2003-03-31

Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of "paddle" cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells.

Dictyostelium cell death

PubMed Central

Levraud, Jean-Pierre; Adam, Myriam; Luciani, Marie-Françoise; de Chastellier, Chantal; Blanton, Richard L.; Golstein, Pierre

2003-01-01

Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of “paddle” cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells. PMID:12654899

Divergent N-Terminal Sequences Target an Inducible Testis Deubiquitinating Enzyme to Distinct Subcellular Structures

PubMed Central

Lin, Haijiang; Keriel, Anne; Morales, Carlos R.; Bedard, Nathalie; Zhao, Qing; Hingamp, Pascal; Lefrançois, Stephane; Combaret, Lydie; Wing, Simon S.

2000-01-01

Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-γ-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action. PMID:10938131

Zea mays Taxilin Protein Negatively Regulates Opaque-2 Transcriptional Activity by Causing a Change in Its Sub-Cellular Distribution

PubMed Central

Zhang, Nan; Qiao, Zhenyi; Liang, Zheng; Mei, Bing; Xu, Zhengkai; Song, Rentao

2012-01-01

Zea mays (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity. PMID:22937104

Effects of cooking and subcellular distribution on the bioaccessibility of trace elements in two marine fish species.

PubMed

He, Mei; Ke, Cai-Huan; Wang, Wen-Xiong

2010-03-24

In current human health risk assessment, the maximum acceptable concentrations of contaminants in food are mostly based on the total concentrations. However, the total concentration of contaminants may not always reflect the available amount. Bioaccessibility determination is thus required to improve the risk assessment of contaminants. This study used an in vitro digestion model to assess the bioaccessibility of several trace elements (As, Cd, Cu, Fe, Se, and Zn) in the muscles of two farmed marine fish species (seabass Lateolabrax japonicus and red seabream Pagrosomus major ) of different body sizes. The total concentrations and subcellular distributions of these trace elements in fish muscles were also determined. Bioaccessibility of these trace elements was generally high (>45%), and the lowest bioaccessibility was observed for Fe. Cooking processes, including boiling, steaming, frying, and grilling, generally decreased the bioaccessibility of these trace elements, especially for Cu and Zn. The influences of frying and grilling were greater than those of boiling and steaming. The relationship of bioaccessibility and total concentration varied with the elements. A positive correlation was found for As and Cu and a negative correlation for Fe, whereas no correlation was found for Cd, Se, and Zn. A significant positive relationship was demonstrated between the bioaccessibility and the elemental partitioning in the heat stable protein fraction and in the trophically available fraction, and a negative correlation was observed between the bioaccessibility and the elemental partitioning in metal-rich granule fraction. Subcellular distribution may thus affect the bioaccessibility of metals and should be considered in the risk assessment for seafood safety.

Effect of titanium dioxide nanoparticles on the accumulation and distribution of arsenate in Daphnia magna in the presence of an algal food.

PubMed

Luo, Zhuanxi; Li, Mengting; Wang, Zhenhong; Li, Jinli; Guo, Jianhua; Rosenfeldt, Ricki R; Seitz, Frank; Yan, Changzhou

2018-05-15

The impact of titanium dioxide nanoparticles (nano-TiO 2 ) on the bioavailability of metals in aquatic filter-feeding organisms has rarely been investigated, especially in the presence of algae as a food source. In this study, we quantified the accumulation and subcellular distribution of arsenate (As V ) in Daphnia magna in the presence of nano-TiO 2 and a green alga (Scenedesmus obliquus) food source. Results showed that S. obliquus significantly increased the accumulation of total arsenic (As) and titanium (Ti) in D. magna. The presence of this food source increased As in metal-sensitive fractions (MSF) and as biologically detoxified metals (BDM), while it decreased Ti levels in MSF but increased levels as BDM. The difference in the subcellular distribution of As and Ti demonstrates the dissociation of As from nano-TiO 2 during digestion at subcellular partitioning irrespective of food availability. In turn, the presence of algae was shown to increase metal-based toxicity in D. magna due to the transfer of As from BMD to MSF. Furthermore, S. obliquus significantly increased the concentration of As and Ti in soluble fractions, indicating that As and nano-TiO 2 ingested by D. magna could be transferred more readily to their predators in the presence of S. obliquus. Our study shows the potential of algae to increase the toxicity and biomagnification of As V . Furthermore, it highlights food as an important factor in the toxicity assessment of nanomaterials and co-existing pollutants.

Differential distribution of the sodium‐activated potassium channels slick and slack in mouse brain

PubMed Central

Knaus, Hans‐Günther; Schwarzer, Christoph

2015-01-01

ABSTRACT The sodium‐activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are high‐conductance potassium channels of the Slo family. In neurons, Slick and Slack channels are involved in the generation of slow afterhyperpolarization, in the regulation of firing patterns, and in setting and stabilizing the resting membrane potential. The distribution and subcellular localization of Slick and Slack channels in the mouse brain have not yet been established in detail. The present study addresses this issue through in situ hybridization and immunohistochemistry. Both channels were widely distributed and exhibited distinct distribution patterns. However, in some brain regions, their expression overlapped. Intense Slick channel immunoreactivity was observed in processes, varicosities, and neuronal cell bodies of the olfactory bulb, granular zones of cortical regions, hippocampus, amygdala, lateral septal nuclei, certain hypothalamic and midbrain nuclei, and several regions of the brainstem. The Slack channel showed primarily a diffuse immunostaining pattern, and labeling of cell somata and processes was observed only occasionally. The highest Slack channel expression was detected in the olfactory bulb, lateral septal nuclei, basal ganglia, and distinct areas of the midbrain, brainstem, and cerebellar cortex. In addition, comparing our data obtained from mouse brain with a previously published study on rat brain revealed some differences in the expression and distribution of Slick and Slack channels in these species. J. Comp. Neurol. 524:2093–2116, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:26587966

Subcellular boron and fluorine distributions with SIMS ion microscopy in BNCT and cancer research

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subhash Chandra

2008-05-30

The development of a secondary ion mass spectrometry (SIMS) based technique of Ion Microscopy in boron neutron capture therapy (BNCT) was the main goal of this project, so that one can study the subcellular location of boron-10 atoms and their partitioning between the normal and cancerous tissue. This information is fundamental for the screening of boronated drugs appropriate for neutron capture therapy of cancer. Our studies at Cornell concentrated mainly on studies of glioblastoma multiforme (GBM). The early years of the grant were dedicated to the development of cryogenic methods and correlative microscopic approaches so that a reliable subcellular analysismore » of boron-10 atoms can be made with SIMS. In later years SIMS was applied to animal models and human tissues of GBM for studying the efficacy of potential boronated agents in BNCT. Under this grant the SIMS program at Cornell attained a new level of excellence and collaborative SIMS studies were published with leading BNCT researchers in the U.S.« less

Absorption Kinetics and Subcellular Fractionation of Zinc in Winter Wheat in Response to Nitrogen Supply.

PubMed

Nie, Zhaojun; Zhao, Peng; Wang, Jia; Li, Jinfeng; Liu, Hongen

2017-01-01

Nitrogen (N) is critical for zinc (Zn) absorption into plant roots; this in turn allows for Zn accumulation and biofortification of grain in winter wheat ( Triticum aestivum L.), an important food crop. However, little is known about root morphology and subcellular Zn distribution in response to N treatment at different levels of Zn supply. In this study, two nutrient solution culture experiments were conducted to examine Zn accumulation, Zn absorption kinetics, root morphology, and Zn subcellular distribution in wheat seedlings pre-cultured with different N concentrations. The results showed positive correlations between N and Zn concentrations, and N and Zn accumulation, respectively. The findings suggested that an increase in N supply enhanced root absorption and the root-to-shoot transport of Zn. Nitrogen combined with the high Zn (Zn 10 ) treatment increased the Zn concentration and consequently its accumulation in both shoots and roots. The maximum influx rate ( V max ), root length, surface area, and volume of 14-d-old seedlings, and root growth from 7 to 14 d in the medium N (N 7.5 ) treatment were higher, but the Michaelis constant ( K m ) and minimum equilibrium concentrations ( C min ) in this treatment were lower than those in the low (N 0.05 ) and high (N 15 ) N treatments, when Zn was supplied at a high level (Zn 10 ). Meanwhile, there were no pronounced differences in the above root traits between the N 0.05 Zn 0 and N 7.5 Zn 10 treatments. An increase in N supply decreased Zn in cell walls and cell organelles, while it increased Zn in the root soluble fraction. In leaves, an increase in N supply significantly decreased Zn in cell walls and the soluble fraction, while it increased Zn in cell organelles under Zn deficiency, but increased Zn distribution in the soluble fraction under medium and high Zn treatments. Therefore, a combination of medium N and high Zn treatments enhanced Zn absorption, apparently by enhancing Zn membrane transport and stimulating root development in winter wheat. An increase in N supply was beneficial in terms of achieving a balanced distribution of Zn subcellular fractions, thus enhancing Zn translocation to shoots, while maintaining normal metabolism.

Absorption Kinetics and Subcellular Fractionation of Zinc in Winter Wheat in Response to Nitrogen Supply

PubMed Central

Nie, Zhaojun; Zhao, Peng; Wang, Jia; Li, Jinfeng; Liu, Hongen

2017-01-01

Nitrogen (N) is critical for zinc (Zn) absorption into plant roots; this in turn allows for Zn accumulation and biofortification of grain in winter wheat (Triticum aestivum L.), an important food crop. However, little is known about root morphology and subcellular Zn distribution in response to N treatment at different levels of Zn supply. In this study, two nutrient solution culture experiments were conducted to examine Zn accumulation, Zn absorption kinetics, root morphology, and Zn subcellular distribution in wheat seedlings pre-cultured with different N concentrations. The results showed positive correlations between N and Zn concentrations, and N and Zn accumulation, respectively. The findings suggested that an increase in N supply enhanced root absorption and the root-to-shoot transport of Zn. Nitrogen combined with the high Zn (Zn10) treatment increased the Zn concentration and consequently its accumulation in both shoots and roots. The maximum influx rate (Vmax), root length, surface area, and volume of 14-d-old seedlings, and root growth from 7 to 14 d in the medium N (N7.5) treatment were higher, but the Michaelis constant (Km) and minimum equilibrium concentrations (Cmin) in this treatment were lower than those in the low (N0.05) and high (N15) N treatments, when Zn was supplied at a high level (Zn10). Meanwhile, there were no pronounced differences in the above root traits between the N0.05Zn0 and N7.5Zn10 treatments. An increase in N supply decreased Zn in cell walls and cell organelles, while it increased Zn in the root soluble fraction. In leaves, an increase in N supply significantly decreased Zn in cell walls and the soluble fraction, while it increased Zn in cell organelles under Zn deficiency, but increased Zn distribution in the soluble fraction under medium and high Zn treatments. Therefore, a combination of medium N and high Zn treatments enhanced Zn absorption, apparently by enhancing Zn membrane transport and stimulating root development in winter wheat. An increase in N supply was beneficial in terms of achieving a balanced distribution of Zn subcellular fractions, thus enhancing Zn translocation to shoots, while maintaining normal metabolism. PMID:28868060

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clarke, J.T.; Cook, H.W.; Spence, M.W.

1985-03-01

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22 h in the presence of D-(1-/sup 3/H)galactose or (/sup 3/H)GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipid-sialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellularmore » membrane fractions studied was recovered from plasma membrane and only 10-15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous (/sup 3/H)GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.« less

Spatially patterned matrix elasticity directs stem cell fate

NASA Astrophysics Data System (ADS)

Yang, Chun; DelRio, Frank W.; Ma, Hao; Killaars, Anouk R.; Basta, Lena P.; Kyburz, Kyle A.; Anseth, Kristi S.

2016-08-01

There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness.

Subcellular Localized Chemical Imaging of Benthic Algal Nutritional Content via HgCdTe Array FT-IR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wetzel, D.; Murdock, J; Dodds, W

2008-01-01

Algae respond rapidly and uniquely to changes in nutrient availability by adjusting pigment, storage product, and organelle content and quality. Cellular and subcellular variability of the relative abundance of macromolecular pools (e.g. protein, lipid, carbohydrate, and phosphodiesters) within the benthic (bottom dwelling) alga Cladophora glomerata (a common nuisance species in fresh and saline waters) was revealed by FT-IR microspectroscopic imaging. Nutrient heterogeneity was compared at the filament, cellular, and subcellular level, and localized nutrient uptake kinetics were studied by detecting the gradual incorporation of isotopically labeled nitrogen (N) (as K15NO3) from surrounding water into cellular proteins. Nutritional content differed substantiallymore » among filament cells, with differences driven by protein and lipid abundance. Whole cell imaging showed high subcellular macromolecular variability in all cells, including adjacent cells on a filament that developed clonally. N uptake was also very heterogeneous, both within and among cells, and did not appear to coincide with subcellular protein distribution. Despite high intercellular variability, some patterns emerged. Cells acquired more 15N the further they were away from the filament attachment point, and 15N incorporation was more closely correlated with phosphodiester content than protein, lipid, or carbohydrate content. Benthic algae are subject to substantial environmental heterogeneity induced by microscale hydrodynamic factors and spatial variability in nutrient availability. Species specific responses to nutrient heterogeneity are central to understanding this key component of aquatic ecosystems. FT-IR microspectroscopy, modified for benthic algae, allows determination of algal physiological responses at scales not available using current techniques.« less

Changes in subcellular distribution and antioxidant compounds involved in Pb accumulation and detoxification in Neyraudia reynaudiana.

PubMed

Zhou, Chuifan; Huang, Meiying; Li, Ying; Luo, Jiewen; Cai, Li Ping

2016-11-01

The effects of increasing concentrations of lead (Pb) on Pb accumulation, subcellular distribution, ultrastructure, photosynthetic characteristics, antioxidative enzyme activity, malondialdehyde content, and phytochelatin contents were investigated in Neyraudia reynaudiana seedlings after a 21-day exposure. A Pb analysis at the subcellular level showed that the majority of Pb in the roots was associated with the cell wall fraction, followed by the soluble fraction. In contrast, the majority of the Pb in the leaves was located in the soluble fraction based on transmission electron microscopy and energy dispersive X-ray analyses. Furthermore, high Pb concentrations adversely affected N. reynaudiana cellular structure. The changes in enzyme activity suggested that the antioxidant system plays an important role in eliminating or alleviating Pb toxicity, both in the roots and leaves of N. reynaudiana. Additionally, the phytochelatin contents in the roots and leaves differed significantly between Pb-spiked treatments and control plants. Our results provide strong evidence that cell walls restrict Pb uptake into the protoplasm and establish an important protective barrier. Subsequent vacuolar compartmentalization in leaves could isolate Pb from other substances in the cell and minimize Pb toxicity in other organelles over time. These results also demonstrated that the levels of antioxidant enzymes and phytochelatin in leaves and roots are correlated with Pb toxicity. These detoxification mechanisms promote Pb tolerance in N. reynaudiana.

My career as an immunoglycobiologist.

PubMed

Marcus, Donald M

2013-01-01

The research program of my laboratory included three major topics: the structures and immunology of human carbohydrate blood group and glycosphingolipid antigens; the tissue distribution, subcellular localization and biosynthesis of glycosphingolipids; and the structural basis of the binding of carbohydrates by antibodies and lectins.

Inhibition of partially purified K+/H+-ATPase from guinea-pig isolated and enriched parietal cells by substituted benzimidazoles.

PubMed Central

Beil, W.; Sewing, K. F.

1984-01-01

The cellular and subcellular distributions of adenosinetriphosphatases (ATPases) were examined in guinea-pig gastric mucosal cells. All cell types displayed Mg2+-ATPase and bicarbonate (HCO3-)-stimulated ATPase activity. K+-ATPase was located only in fractions derived from parietal cells. Differential and density-gradient centrifugation of material prepared from parietal cells revealed that K+-ATPase activity was located in a tubulo-vesicular membrane fraction. Enzyme activity was ten fold greater in this fraction than in a crude parietal cell homogenate. The substituted benzimidazoles, omeprazole and picoprazole, inhibited K+-ATPase (IC50 1.8 +/- 0.5 mumol l-1 and 3.1 +/- 0.4 mumol l-1, respectively). Detailed kinetic analysis indicated that these compounds were non-competitive and reversible inhibitors of the enzyme. In contrast cimetidine and verapamil were without effect on the enzyme. The relevance of the inhibition of K+-ATPase to the antisecretory activity of the benzimidazoles, in experimental animals and man, is discussed. PMID:6146367

Ultrastructural demonstration of Cx43 gap junctions in induced pluripotent stem cells from human cord blood.

PubMed

Beckmann, Anja; Schubert, Madline; Hainz, Nadine; Haase, Alexandra; Martin, Ulrich; Tschernig, Thomas; Meier, Carola

2016-11-01

Gap junction proteins are essential for direct intercellular communication but also influence cellular differentiation and migration. The expression of various connexin gap junction proteins has been demonstrated in embryonic stem cells, with Cx43 being the most intensely studied. As Cx43 is the most prominent gap junction protein in the heart, cardiomyocyte-differentiated stem cells have been studied intensely. To date, however, little is known about the expression and the subcellular distribution of Cx43 in undifferentiated stem cells or about the structural arrangement of channels. We, therefore, here investigate expression of Cx43 in undifferentiated human cord-blood-derived induced pluripotent stem cells (hCBiPS2). For this purpose, we carried out quantitative real-time PCR and immunohistochemistry. For analysis of Cx43 ultrastructure and protein assembly, we performed freeze-fracture replica immunogold labeling (FRIL). Cx43 expression was detected at mRNA and protein level in hCBIPS2 cells. For the first time, ultrastructural data are presented on gap junction morphology in induced pluripotent stem (iPS) cells from cord blood: Our FRIL and electron microscopical analysis revealed the occurrence of gap junction plaques in undifferentiated iPS cells. In addition, these gap junctions were shown to contain the gap junction protein Cx43.

The miR-29 family: genomics, cell biology, and relevance to renal and cardiovascular injury.

PubMed

Kriegel, Alison J; Liu, Yong; Fang, Yi; Ding, Xiaoqiang; Liang, Mingyu

2012-02-27

The human miR-29 family of microRNAs has three mature members, miR-29a, miR-29b, and miR-29c. miR-29s are encoded by two gene clusters. Binding sites for several transcriptional factors have been identified in the promoter regions of miR-29 genes. The miR-29 family members share a common seed region sequence and are predicted to target largely overlapping sets of genes. However, the miR-29 family members exhibit differential regulation in several cases and different subcellular distribution, suggesting their functional relevance may not be identical. miR-29s directly target at least 16 extracellular matrix genes, providing a dramatic example of a single microRNA targeting a large group of functionally related genes. Strong antifibrotic effects of miR-29s have been demonstrated in heart, kidney, and other organs. miR-29s have also been shown to be proapoptotic and involved in the regulation of cell differentiation. It remains to be explored how various cellular effects of miR-29s determine functional relevance of miR-29s to specific diseases and how the miR-29 family members may function cooperatively or separately.

Activated Cdc42 kinase regulates Dock localization in male germ cells during Drosophila spermatogenesis.

PubMed

Abdallah, Abbas M; Zhou, Xin; Kim, Christine; Shah, Kushani K; Hogden, Christopher; Schoenherr, Jessica A; Clemens, James C; Chang, Henry C

2013-06-15

Deregulation of the non-receptor tyrosine kinase ACK1 (Activated Cdc42-associated kinase) correlates with poor prognosis in cancers and has been implicated in promoting metastasis. To further understand its in vivo function, we have characterized the developmental defects of a null mutation in Drosophila Ack, which bears a high degree of sequence similarity to mammalian ACK1 but lacks a CRIB domain. We show that Ack, while not essential for viability, is critical for sperm formation. This function depends on Ack tyrosine kinase activity and is required cell autonomously in differentiating male germ cells at or after the spermatocyte stage. Ack associates predominantly with endocytic clathrin sites in spermatocytes, but disruption of Ack function has no apparent effect on clathrin localization and receptor-mediated internalization of Boss (Bride of sevenless) protein in eye discs. Instead, Ack is required for the subcellular distribution of Dock (dreadlocks), the Drosophila homolog of the SH2- and SH3-containing adaptor protein Nck. Moreover, Dock forms a complex with Ack, and the localization of Dock in male germ cells depends on its SH2 domain. Together, our results suggest that Ack-dependent tyrosine phosphorylation recruits Dock to promote sperm differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

Saccharomyces cerevisiae Bat1 and Bat2 Aminotransferases Have Functionally Diverged from the Ancestral-Like Kluyveromyces lactis Orthologous Enzyme

PubMed Central

Colón, Maritrini; Hernández, Fabiola; López, Karla; Quezada, Héctor; González, James; López, Geovani; Aranda, Cristina; González, Alicia

2011-01-01

Background Gene duplication is a key evolutionary mechanism providing material for the generation of genes with new or modified functions. The fate of duplicated gene copies has been amply discussed and several models have been put forward to account for duplicate conservation. The specialization model considers that duplication of a bifunctional ancestral gene could result in the preservation of both copies through subfunctionalization, resulting in the distribution of the two ancestral functions between the gene duplicates. Here we investigate whether the presumed bifunctional character displayed by the single branched chain amino acid aminotransferase present in K. lactis has been distributed in the two paralogous genes present in S. cerevisiae, and whether this conservation has impacted S. cerevisiae metabolism. Principal Findings Our results show that the KlBat1 orthologous BCAT is a bifunctional enzyme, which participates in the biosynthesis and catabolism of branched chain aminoacids (BCAAs). This dual role has been distributed in S. cerevisiae Bat1 and Bat2 paralogous proteins, supporting the specialization model posed to explain the evolution of gene duplications. BAT1 is highly expressed under biosynthetic conditions, while BAT2 expression is highest under catabolic conditions. Bat1 and Bat2 differential relocalization has favored their physiological function, since biosynthetic precursors are generated in the mitochondria (Bat1), while catabolic substrates are accumulated in the cytosol (Bat2). Under respiratory conditions, in the presence of ammonium and BCAAs the bat1Δ bat2Δ double mutant shows impaired growth, indicating that Bat1 and Bat2 could play redundant roles. In K. lactis wild type growth is independent of BCAA degradation, since a Klbat1Δ mutant grows under this condition. Conclusions Our study shows that BAT1 and BAT2 differential expression and subcellular relocalization has resulted in the distribution of the biosynthetic and catabolic roles of the ancestral BCAT in two isozymes improving BCAAs metabolism and constituting an adaptation to facultative metabolism. PMID:21267457

Understanding Acyl Chain and Glycerolipid Metabolism in Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohlrogge, John B.

2013-11-05

Progress is reported in these areas: acyl-editing in initial eukaryotic lipid assembly in soybean seeds; identification and characterization of two Arabidopsis thaliana lysophosphatidyl acyltransferases with preference for lysophosphatidylethanolamine; and characterization and subcellular distribution of lysolipid acyl transferase activity of pea leaves.

Three-dimensional Organization of Layered Apical Cytoskeletal Networks Associated with Mouse Airway Tissue Development

NASA Astrophysics Data System (ADS)

Tateishi, Kazuhiro; Nishida, Tomoki; Inoue, Kanako; Tsukita, Sachiko

2017-03-01

The cytoskeleton is an essential cellular component that enables various sophisticated functions of epithelial cells by forming specialized subcellular compartments. However, the functional and structural roles of cytoskeletons in subcellular compartmentalization are still not fully understood. Here we identified a novel network structure consisting of actin filaments, intermediate filaments, and microtubules directly beneath the apical membrane in mouse airway multiciliated cells and in cultured epithelial cells. Three-dimensional imaging by ultra-high voltage electron microscopy and immunofluorescence revealed that the morphological features of each network depended on the cell type and were spatiotemporally integrated in association with tissue development. Detailed analyses using Odf2 mutant mice, which lack ciliary basal feet and apical microtubules, suggested a novel contribution of the intermediate filaments to coordinated ciliary beating. These findings provide a new perspective for viewing epithelial cell differentiation and tissue morphogenesis through the structure and function of apical cytoskeletal networks.

Endocytosis and interaction of poly (amidoamine) dendrimers with Caco-2 cells.

PubMed

Kitchens, Kelly M; Foraker, Amy B; Kolhatkar, Rohit B; Swaan, Peter W; Ghandehari, Hamidreza

2007-11-01

To investigate the internalization and subcellular trafficking of fluorescently labeled poly (amidoamine) (PAMAM) dendrimers in intestinal cell monolayers. PAMAM dendrimers with positive or negative surface charge were conjugated to fluorescein isothiocyanate (FITC) and visualized for colocalization with endocytosis markers using confocal microscopy. Effect of concentration, generation and charge on the morphology of microvilli was observed using transmission electron microscopy. Both cationic and anionic PAMAM dendrimers internalized within 20 min, and differentially colocalized with endocytosis markers clathrin, EEA-1, and LAMP-1. Transmission electron microscopy analysis showed a concentration-, generation- and surface charge-dependent effect on microvilli morphology. These studies provide visual evidence that endocytic mechanism(s) contribute to the internalization and subcellular trafficking of PAMAM dendrimers across the intestinal cells, and that appropriate selection of PAMAM dendrimers based on surface charge, concentration and generation number allows the application of these polymers for oral drug delivery.

Multi-scale Imaging of Cellular and Sub-cellular Structures using Scanning Probe Recognition Microscopy.

NASA Astrophysics Data System (ADS)

Chen, Q.; Rice, A. F.

2005-03-01

Scanning Probe Recognition Microscopy is a new scanning probe capability under development within our group to reliably return to and directly interact with a specific nanobiological feature of interest. In previous work, we have successfully recognized and classified tubular versus globular biological objects from experimental atomic force microscope images using a method based on normalized central moments [ref. 1]. In this paper we extend this work to include recognition schemes appropriate for cellular and sub-cellular structures. Globular cells containing tubular actin filaments are under investigation. Thus there are differences in external/internal shapes and scales. Continuous Wavelet Transform with a differential Gaussian mother wavelet is employed for multi- scale analysis. [ref. 1] Q. Chen, V. Ayres and L. Udpa, ``Biological Investigation Using Scanning Probe Recognition Microscopy,'' Proceedings 3rd IEEE Conference on Nanotechnology, vol. 2, p 863-865 (2003).

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

NASA Astrophysics Data System (ADS)

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.; England, Grant T.; Kim, Philseok; Kolle, Mathias; Ferrante, Thomas; Zarzar, Lauren D.; Strong, Elizabeth; Aizenberg, Joanna

2017-03-01

Mechanical forces in the cell's natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging from the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques.

Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the Echinococcus granulosus Preadult Stage.

PubMed

Lorenzatto, Karina R; Kim, Kyunggon; Ntai, Ioanna; Paludo, Gabriela P; Camargo de Lima, Jeferson; Thomas, Paul M; Kelleher, Neil L; Ferreira, Henrique B

2015-11-06

Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC-MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentially acetylated, including histones. Bottom up analysis increased the overall number of identified proteins in nuclear and cytosolic fractions to 154 and 112, respectively. Overall, our results provided the first description of the low mass proteome of E. granulosus subcellular fractions and highlighted proteoforms with CTMs and PTMS whose characterization may lead to another level of understanding about molecular mechanisms controlling parasitic flatworm biology.

Arbuscular Mycorrhizal Colonization Alters Subcellular Distribution and Chemical Forms of Cadmium in Medicago sativa L. and Resists Cadmium Toxicity

PubMed Central

Gao, Yanzheng

2012-01-01

Some plants can tolerate and even detoxify soils contaminated with heavy metals. This detoxification ability may depend on what chemical forms of metals are taken up by plants and how the plants distribute the toxins in their tissues. This, in turn, may have an important impact on phytoremediation. We investigated the impact of arbuscular mycorrhizal (AM) fungus, Glomus intraradices, on the subcellular distribution and chemical forms of cadmium (Cd) in alfalfa (Medicago sativa L.) that were grown in Cd-added soils. The fungus significantly colonized alfalfa roots by day 25 after planting. Colonization of alfalfa by G. intraradices in soils contaminated with Cd ranged from 17% to 69% after 25–60 days and then decreased to 43%. The biomass of plant shoots with AM fungi showed significant 1.7-fold increases compared to no AM fungi addition under the treatment of 20 mg·kg−1 Cd. Concentrations of Cd in the shoots of alfalfa under 0.5, 5, and 20 mg·kg−1 Cd without AM fungal inoculation are 1.87, 2.92, and 2.38 times higher, respectively, than those of fungi-inoculated plants. Fungal inoculation increased Cd (37.2–80.5%) in the cell walls of roots and shoots and decreased in membranes after 80 days of incubation compared to untreated plants. The proportion of the inactive forms of Cd in roots was higher in fungi-treated plants than in controls. Furthermore, although fungi-treated plants had less overall Cd in subcellular fragments in shoots, they had more inactive Cd in shoots than did control plants. These results provide a basis for further research on plant-microbe symbioses in soils contaminated with heavy metals, which may potentially help us develop management regimes for phytoremediation. PMID:23139811

Shenmai injection enhances the cytotoxicity of chemotherapeutic drugs against colorectal cancers via improving their subcellular distribution

PubMed Central

Liu, Wen-yue; Zhang, Jing-wei; Yao, Xue-quan; Jiang, Chao; He, Ji-chao; Ni, Pin; Liu, Jia-li; Chen, Qian-ying; Li, Qing-ran; Zang, Xiao-jie; Yao, Lan; Liu, Ya-zhong; Wang, Mu-lan; Shen, Pei-qiang; Wang, Guang-ji; Zhou, Fang

2017-01-01

Shenmai injection (SMI) is a Chinese patent-protected injection, which was mainly made of Red Ginseng and Radix Ophiopogonis and widely used for treating coronary heart disease and tumors by boosting Qi and nourishing Yin. In this study we examined whether SMI could produce direct synergetic effects on the cytoxicity of adriamycin (ADR) and paclitaxel (PTX) in colorectal cancers in vivo and in vitro, and explored the underlying pharmacokinetic mechanisms. BALB/c nude mice with LoVo colon cancer xenografts were intraperitoneally injected with ADR (2 mg·kg−1·3d−1) or PTX (7.5 mg·kg−1·3d−1) with or without SMI (0.01 mL·g−1·d−1) for 13 d. Co-administration of SMI significantly enhanced the chemotherapeutic efficacy of ADR and PTX, whereas administration of SMI alone at the given dosage did not produce visible anti-cancer effects, The chemosensitizing action of SMI was associated with increased concentrations of ADR and PTX in the plasma and tumors. In Caco-2 and LoVo cells in vitro, co-treatment with SMI (2 μL/mL) significantly enhanced the cytotoxicity of ADR and PTX, and resulted in some favorable pharmacokinetic changes in the subcellular distribution of ADR and PTX. In addition, SMI-induced intracellular accumulation of ADR was closely correlated with the increased expression levels of P-glycoprotein in 4 colon cancer cell lines (r2=+0.8558). SMI enhances the anti-cancer effects of ADR and PTX in colon cancers in vivo and in vitro by improving the subcellular distributions of ADR and PTX. PMID:27867186

Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome*

PubMed Central

Jadot, Michel; Boonen, Marielle; Thirion, Jaqueline; Wang, Nan; Xing, Jinchuan; Zhao, Caifeng; Tannous, Abla; Qian, Meiqian; Zheng, Haiyan; Everett, John K.; Moore, Dirk F.; Sleat, David E.; Lobel, Peter

2017-01-01

Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease gene. PMID:27923875

Acute Liver Injury Induces Nucleocytoplasmic Redistribution of Hepatic Methionine Metabolism Enzymes

PubMed Central

Delgado, Miguel; Garrido, Francisco; Pérez-Miguelsanz, Juliana; Pacheco, María; Partearroyo, Teresa; Pérez-Sala, Dolores

2014-01-01

Abstract Aims: The discovery of methionine metabolism enzymes in the cell nucleus, together with their association with key nuclear processes, suggested a putative relationship between alterations in their subcellular distribution and disease. Results: Using the rat model of d-galactosamine intoxication, severe changes in hepatic steady-state mRNA levels were found; the largest decreases corresponded to enzymes exhibiting the highest expression in normal tissue. Cytoplasmic protein levels, activities, and metabolite concentrations suffered more moderate changes following a similar trend. Interestingly, galactosamine treatment induced hepatic nuclear accumulation of methionine adenosyltransferase (MAT) α1 and S-adenosylhomocysteine hydrolase tetramers, their active assemblies. In fact, galactosamine-treated livers showed enhanced nuclear MAT activity. Acetaminophen (APAP) intoxication mimicked most galactosamine effects on hepatic MATα1, including accumulation of nuclear tetramers. H35 cells that overexpress tagged-MATα1 reproduced the subcellular distribution observed in liver, and the changes induced by galactosamine and APAP that were also observed upon glutathione depletion by buthionine sulfoximine. The H35 nuclear accumulation of tagged-MATα1 induced by these agents correlated with decreased glutathione reduced form/glutathione oxidized form ratios and was prevented by N-acetylcysteine (NAC) and glutathione ethyl ester. However, the changes in epigenetic modifications associated with tagged-MATα1 nuclear accumulation were only prevented by NAC in galactosamine-treated cells. Innovation: Cytoplasmic and nuclear changes in proteins that regulate the methylation index follow opposite trends in acute liver injury, their nuclear accumulation showing potential as disease marker. Conclusion: Altogether these results demonstrate galactosamine- and APAP-induced nuclear accumulation of methionine metabolism enzymes as active oligomers and unveil the implication of redox-dependent mechanisms in the control of MATα1 subcellular distribution. Antioxid. Redox Signal. 20, 2541–2554. PMID:24124652

Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome.

PubMed

Jadot, Michel; Boonen, Marielle; Thirion, Jaqueline; Wang, Nan; Xing, Jinchuan; Zhao, Caifeng; Tannous, Abla; Qian, Meiqian; Zheng, Haiyan; Everett, John K; Moore, Dirk F; Sleat, David E; Lobel, Peter

2017-02-01

Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease gene. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of DA-6 and EDTA alone or in combination on uptake, subcellular distribution and chemical form of Pb in Lolium perenne.

PubMed

He, Shanying; Wu, Qiuling; He, Zhenli

2013-11-01

The effects of growth-promoting hormone diethyl aminoethyl hexanoate (DA-6) and EDTA, either alone or in combination applied to original soil or lead (Pb) spiked soil on Pb phytoextraction, subcellular distribution and chemical forms in Lolium perenne were studied. EDTA addition alone significantly reduced plant biomass though it increased Pb accumulation (P<0.05). Foliar spray of DA-6 alone increased both plant biomass and Pb accumulation (P<0.05), with 10μM DA-6 being the most effective. DA-6 combined with EDTA compensated the adverse effect of the latter on plant growth, and resulted in a synergistic effect on Pb uptake and translocation, with the maximum accumulation occurring in the EDTA+10μM DA-6 treatment. At the subcellular level, about 35-66% of Pb was distributed in cell wall and 21-42% in soluble fraction, with a minority present in cellular organelles fraction. EDTA addition alone increased the proportion of Pb in soluble and cellular organelles fraction, while DA-6 detoxified Pb in plant by storing additional Pb in cell wall, and 10μM DA-6 was the most effective. Of the total Pb in plant shoot, 27-52% was NaCl extractable, 22-47% HAc extractable, followed by other fractions. Contrary to EDTA, DA-6 significantly decreased Pb migration in plant. These results suggest that Pb fixation by pectates and proteins in cell wall and compartmentalization by vacuole might be responsible for Pb detoxification in plant, and the combined use of EDTA and 10μM DA-6 appears to be optimal for improving the remediation efficiency of L. perenne for Pb contaminated soil. Copyright © 2013 Elsevier Ltd. All rights reserved.

Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43.

PubMed

Tomatis, Vanesa M; Trenchi, Alejandra; Gomez, Guillermo A; Daniotti, Jose L

2010-11-30

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.

Uptake and subcellular distribution of triclosan in typical hydrophytes under hydroponic conditions.

PubMed

He, Yupeng; Nie, Enguang; Li, Chengming; Ye, Qingfu; Wang, Haiyan

2017-01-01

The increasing discharge of pharmaceuticals and personal care products (PPCPs) into the environment has generated serious public concern. The recent awareness of the environmental impact of this emerging class of pollutants and their potential adverse effects on human health have been documented in many reports. However, information regarding uptake and intracellular distribution of PPCPs in hydrophytes under hydroponic conditions, and potential human exposure is very limited. A laboratory experiment was conducted using 14 C-labeled triclosan (TCS) to investigate uptake and distribution of TCS in six aquatic plants (water spinach, purple perilla, cress, penny grass, cane shoot, and rice), and the subcellular distribution of 14 C-TCS was determined in these plants. The results showed that the uptake and removal rate of TCS from nutrient solution by hydrophytes followed the order of cress (96%) > water spinach (94%) > penny grass (87%) > cane shoot (84%) > purple perilla (78%) > rice (63%) at the end of incubation period (192 h). The range of 14 C-TCS content in the roots was 94.3%-99.0% of the added 14 C-TCS, and the concentrations in roots were 2-3 orders of magnitude greater than those in shoots. Furthermore, the subcellular fraction-concentration factor (3.6 × 10 2 -2.6 × 10 3  mL g -1 ), concentration (0.58-4.47 μg g -1 ), and percentage (30%-61%) of 14 C-TCS in organelles were found predominantly greater than those in cell walls and/or cytoplasm. These results indicate that for these plants, the roots are the primary storage for TCS, and within plant cells organelles are the major domains for TCS accumulation. These findings provide a better understanding of translocation and accumulation of TCS in aquatic plants at the cellular level, which is valuable for environmental and human health assessments of TCS. Copyright © 2016 Elsevier Ltd. All rights reserved.

The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

PubMed

Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

1995-07-15

The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process.

Subcellular distribution and chemical forms of cadmium in the edible seaweed, Porphyra yezoensis.

PubMed

Zhao, Yanfang; Wu, Jifa; Shang, Derong; Ning, Jinsong; Zhai, Yuxiu; Sheng, Xiaofeng; Ding, Haiyan

2015-02-01

The subcellular distribution and chemical forms of Cd were investigated in the edible seaweed, Porphyra yezoensis. The seaweed was exposed to different Cd concentrations (0.01, 0.05, 0.1, 0.5, 1.0 and 5.0mgl(-1)) for up to 96h. In both the controls (no Cd added) and treatment groups, 41.2-79.2% of Cd was localised in the cell wall, and the proportion of Cd in the cell wall increased with increasing concentrations of Cd and exposure time. In the control groups, 74.8% of Cd was extracted by 1M NaCl, followed by 2% acetic acid, HAC (18.9%). In the treatment groups, most Cd was extracted by 2% HAC. The proportion of Cd extracted by 2% HAC increased with exposure to increasing concentrations of Cd and over time. Cell wall deposition and forming of precipitates with phosphate may be a key strategy to reduce Cd toxicity in P. yezoensis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cytoplasmic YY1 Is Associated with Increased Smooth Muscle-Specific Gene Expression

PubMed Central

Favot, Laure; Hall, Susan M.; Haworth, Sheila G.; Kemp, Paul R.

2005-01-01

Immediately after birth the adluminal vascular SMCs of the pulmonary elastic arteries undergo transient actin cytoskeletal remodeling as well as cellular de-differentiation and proliferation. Vascular smooth muscle phenotype is regulated by serum response factor, which is itself regulated in part by the negative regulator YY1. We therefore studied the subcellular localization of YY1 in arteries of normal newborn piglets and piglets affected by neonatal pulmonary hypertension. We found that YY1 localization changed during development and that expression of γ-smooth muscle actin correlated with expression of cytoplasmic rather than nuclear YY1. Analysis of the regulation of YY1 localization in vitro demonstrated that polymerized γ-actin sequestered EGFP-YY1 in the cytoplasm and that YY1 activation of c-myc promoter activity was inhibited by LIM kinase, which increases actin polymerization. Consistent with these data siRNA-mediated down-regulation of YY1 in C2C12 cells increased SM22-α expression and inhibited cell proliferation. Thus, actin polymerization controls subcellular YY1 localization, which contributes to vascular SMC proliferation and differentiation in normal pulmonary artery development. In the absence of actin depolymerization, YY1 does not relocate to the nucleus, and this lack of relocation may contribute to the pathobiology of pulmonary hypertension. PMID:16314465

Distribution and Characterization of Antigens Found in Subcellular Fractions of African Trypanosomes.

DTIC Science & Technology

1979-08-01

flagellate, Tritrichomonas foetus . The specific activities for enzymes in the original homogenate, cumulative percentage distributions in the various...with another protozoan T. foetus (Lloyd, Lindmark and Muller in press). The lack of latency for this trypanosomal ATPase indicates the enzyme to occupy...flagellate protozoan Tritrichomonas foetus . J. Gen. Microbiol. (in press). . Lowry, 0. H., Rosebrough, N. D., Farr, A. L. and Randall, R. J. (1951) Protein 9

Intracellular delivery and trafficking dynamics of a lymphoma-targeting antibody-polymer conjugate.

PubMed

Berguig, Geoffrey Y; Convertine, Anthony J; Shi, Julie; Palanca-Wessels, Maria Corinna; Duvall, Craig L; Pun, Suzie H; Press, Oliver W; Stayton, Patrick S

2012-12-03

Ratiometric fluorescence and cellular fractionation studies were employed to characterize the intracellular trafficking dynamics of antibody-poly(propylacrylic acid) (PPAA) conjugates in CD22+ RAMOS-AW cells. The HD39 monoclonal antibody (mAb) directs CD22-dependent, receptor-mediated uptake in human B-cell lymphoma cells, where it is rapidly trafficked to the lysosomal compartment. To characterize the intracellular-release dynamics of the polymer-mAb conjugates, HD39-streptavidin (HD39/SA) was dual-labeled with pH-insensitive Alexa Fluor 488 and pH-sensitive pHrodo fluorophores. The subcellular pH distribution of the HD39/SA-polymer conjugates was quantified as a function of time by live-cell fluorescence microscopy, and the average intracellular pH value experienced by the conjugates was also characterized as a function of time by flow cytometry. PPAA was shown to alter the intracellular trafficking kinetics strongly relative to HD39/SA alone or HD39/SA conjugates with a control polymer, poly(methacryclic acid) (PMAA). Subcellular trafficking studies revealed that after 6 h, only 11% of the HD39/SA-PPAA conjugates had been trafficked to acidic lysosomal compartments with values at or below pH 5.6. In contrast, the average intracellular pH of HD39/SA alone dropped from 6.7 ± 0.2 at 1 h to 5.6 ± 0.5 after 3 h and 4.7 ± 0.6 after 6 h. Conjugation of the control polymer PMAA to HD39/SA showed an average pH drop similar to that of HD39/SA. Subcellular fractionation studies with tritium-labeled HD39/SA demonstrated that after 6 h, 89% of HD39/SA was associated with endosomes (Rab5+) and lysosomes (Lamp2+), while 45% of HD39/SA-PPAA was translocated to the cytosol (lactate dehydrogenase+). These results demonstrate the endosomal-releasing properties of PPAA with antibody-polymer conjugates and detail their intracellular trafficking dynamics and subcellular compartmental distributions over time.

Subcellular Distribution and Chemical Forms of Pb in Corn: Strategies Underlying Tolerance in Pb Stress.

PubMed

Sun, Jianling; Luo, Liqiang

2018-06-22

Studying the accumulation position and forms of heavy metals (HMs) in organisms and cells is helpful to understand the transport process and detoxification mechanism. As typical HMs, lead (Pb) subcellular content, localization, and speciation of corn subcellular fractions were studied by a series of technologies, including transmission electron microscopy, inductively coupled plasma mass spectrometry, and X-ray absorption near edge structure. The results revealed that the electrodense granules of Pb were localized in the cell wall, intercellular space, and plasma membranes. About 71% Pb was localized at the cell wall and soluble fraction. In cell walls, the total amount of pyromorphite and Pb carbonate was about 80% and the remaining was Pb stearate. In the nuclear and chloroplast fraction, which demonstrated significant changes, major speciations were Pb sulfide (72%), basic Pb carbonate (16%), and Pb stearate (12%). Pb is blocked by cell walls as pyromorphite and Pb carbonate sediments and compartmentalized by vacuoles, which both play an inportant role in cell detoxification. Besides, sulfur-containing compounds form inside the cells.

Modulation of integrin-linked kinase nucleo-cytoplasmic shuttling by ILKAP and CRM1.

PubMed

Nakrieko, Kerry-Ann; Vespa, Alisa; Mason, David; Irvine, Timothy S; D'Souza, Sudhir J A; Dagnino, Lina

2008-07-15

Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.

Differential distribution of the sodium-activated potassium channels slick and slack in mouse brain.

PubMed

Rizzi, Sandra; Knaus, Hans-Günther; Schwarzer, Christoph

2016-07-01

The sodium-activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are high-conductance potassium channels of the Slo family. In neurons, Slick and Slack channels are involved in the generation of slow afterhyperpolarization, in the regulation of firing patterns, and in setting and stabilizing the resting membrane potential. The distribution and subcellular localization of Slick and Slack channels in the mouse brain have not yet been established in detail. The present study addresses this issue through in situ hybridization and immunohistochemistry. Both channels were widely distributed and exhibited distinct distribution patterns. However, in some brain regions, their expression overlapped. Intense Slick channel immunoreactivity was observed in processes, varicosities, and neuronal cell bodies of the olfactory bulb, granular zones of cortical regions, hippocampus, amygdala, lateral septal nuclei, certain hypothalamic and midbrain nuclei, and several regions of the brainstem. The Slack channel showed primarily a diffuse immunostaining pattern, and labeling of cell somata and processes was observed only occasionally. The highest Slack channel expression was detected in the olfactory bulb, lateral septal nuclei, basal ganglia, and distinct areas of the midbrain, brainstem, and cerebellar cortex. In addition, comparing our data obtained from mouse brain with a previously published study on rat brain revealed some differences in the expression and distribution of Slick and Slack channels in these species. J. Comp. Neurol. 524:2093-2116, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Live imaging of companion cells and sieve elements in Arabidopsis leaves.

PubMed

Cayla, Thibaud; Batailler, Brigitte; Le Hir, Rozenn; Revers, Frédéric; Anstead, James A; Thompson, Gary A; Grandjean, Olivier; Dinant, Sylvie

2015-01-01

The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

Live Imaging of Companion Cells and Sieve Elements in Arabidopsis Leaves

PubMed Central

Cayla, Thibaud; Batailler, Brigitte; Le Hir, Rozenn; Revers, Frédéric; Anstead, James A.; Thompson, Gary A.; Grandjean, Olivier; Dinant, Sylvie

2015-01-01

The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo. PMID:25714357

Subcellular distribution of a new fluorinated, biocompatible, non-ionic telomeric carrier: a study in cultured B16 melanoma and rat skin fibroblasts.

PubMed

Chehade, F; Maurizis, J C; Pucci, B; Pavia, A A; Ollier, M; Veyre, A; Escaig, F; Jeanguillaume, C; Dennebouy, R; Slodzian, G; Hindié, E

1996-05-01

Tris-hydroxymethyl-amino-methane telomers bearing a fluorinated end have recently been proposed as potential drug carriers. Using ion microscopy, we have investigated the cell uptake and subcellular distribution of a perfluorinated telomere, called F-TAC, in two cell lines, malignant murine B16 melanoma and normal rat skin fibroblasts. Single layer cell cultures on gold plates were incubated with F-TAC at different concentrations. Ion microscopy using mass spectrometry enabled the detection of Fluorine 19 atoms entering into F-TAC constitution. This microanalytical study showed an elective cytoplasmic localization of the molecule, wherein the distribution is relatively homogeneous. Within same culture and incubation conditions, intercellular variations in F-TAC content were very low. In the malignant line, the intracellular concentration remains practically identical when increasing F-TAC concentration in the culture medium above 0.2 mg/ml, indicating that the uptake phenomenon is saturable. In conclusion, the F-TAC telomer easily crosses the plasma membrane, however, it has difficulties in crossing the nuclear membrane. It is likely that intracellular penetration is essentially due to rapid endocytosis of the telomer.

Relationship between aquaporin-5 expression and saliva flow in streptozotocin-induced diabetic mice?

PubMed

Soyfoo, M S; Bolaky, N; Depoortere, I; Delporte, C

2012-07-01

To investigate the expression and distribution of AQP5 in submandibular acinar cells from sham- and streptozotocin (STZ)-treated mice in relation to the salivary flow. Mice were sham or STZ injected. Distribution of AQP5 subcellular expression in submandibular glands was determined by immunohistochemistry. AQP5 labelling indices (LI), reflecting AQP5 subcellular distribution, were determined in acinar cells. Western blotting was performed to determine the expression of AQP5 in submandibular glands. Blood glycaemia and osmolality and saliva flow rates were also determined. AQP5 immunoreactivity was primarily located at the apical and apical-basolateral membranes of submandibular gland acinar cells from sham- and STZ-treated mice. No significant differences in AQP5 protein levels were observed between sham- and STZ-treated mice. Compared to sham-treated mice, STZ-treated mice had significant increased glycaemia, while no significant differences in blood osmolality were observed. Saliva flow rate was significantly decreased in STZ-treated mice as compared to sham-treated mice. In STZ-treated mice, significant reduction in salivary flow rate was observed without any concomitant modification in AQP5 expression and localization. © 2011 John Wiley & Sons A/S.

Estrogen and Hydroxysteroid Sulfotransterases in Guinea Pig Adrenal Cortex: Cellular and Subcellular Distributions

DTIC Science & Technology

1993-06-01

hydroxysteroid substrate specificities (32 and 33 kilodaltons, respectively) were previously purified from guinea pig adrenal cortex and characterized. Western...labeling with these antisera revealed that the sulfortransferases were expressed only within the ACTH- responsive layers of the guinea pig adrenal cortex

Evidence for membrane-bound carbonic anhydrase in the air bladder of bowfin (Amia calva), a primitive air-breathing fish.

PubMed

Gervais, M R; Tufts, B L

1998-07-01

The purpose of this study was to examine the subcellular distribution and isoenzyme characteristics of carbonic anhydrase from the gills and respiratory air bladder of bowfin Amia calva, a primitive air-breathing fish. Separation of subcellular fractions by differential centrifugation revealed that the vast majority of carbonic anhydrase from the gills of bowfin originated from the cytoplasmic fraction. Washing of the gill microsomal pellet also indicated that the carbonic anhydrase originally associated with this pellet was largely due to contamination from the cytoplasmic fraction. Experiments with a carbonic anhydrase inhibitor, sulphanilamide, and the plasma carbonic anhydrase inhibitor from this species confirmed that the bowfin gill probably contains only one carbonic anhydrase isoenzyme which had properties resembling those of CA II. In contrast to the situation in the gills, a relatively large percentage (27%) of the total air bladder carbonic anhydrase was associated with the microsomal fraction. Washing of the air bladder microsomal pellet removed little of the carbonic anhydrase activity, indicating that most of the carbonic anhydrase in the microsomal fraction was associated with the membranes. Like the mammalian pulmonary CA IV isoenzyme, microsomal carbonic anhydrase from the bowfin air bladder was less sensitive to the bowfin plasma carbonic anhydrase inhibitor, sodium dodecylsulphate (SDS) and sulphanilamide than was cytoplasmic carbonic anhydrase from the air bladder. Microsomal carbonic anhydrase from the bowfin air bladder also resembled CA IV in that it appears to be anchored to the membrane via a phosphatidylinositol-glycan linkage which could be cleaved by phosphatidylinositol-specific phospholipase C. Taken together, these results suggest that a membrane-bound carbonic anhydrase isoenzyme resembling mammalian CA IV in terms of inhibition characteristics and membrane attachment is present in the air-breathing organ of one of the most primitive air-breathing vertebrates.

CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN11[OPEN

PubMed Central

Wilkop, Thomas E.; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

2015-01-01

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

CESA TRAFFICKING INHIBITOR inhibits cellulose deposition and interferes with the trafficking of cellulose synthase complexes and their associated proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1.

PubMed

Worden, Natasha; Wilkop, Thomas E; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

2015-02-01

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. © 2015 American Society of Plant Biologists. All Rights Reserved.

Protein localization as a principal feature of the etiology and comorbidity of genetic diseases

PubMed Central

Park, Solip; Yang, Jae-Seong; Shin, Young-Eun; Park, Juyong; Jang, Sung Key; Kim, Sanguk

2011-01-01

Proteins targeting the same subcellular localization tend to participate in mutual protein–protein interactions (PPIs) and are often functionally associated. Here, we investigated the relationship between disease-associated proteins and their subcellular localizations, based on the assumption that protein pairs associated with phenotypically similar diseases are more likely to be connected via subcellular localization. The spatial constraints from subcellular localization significantly strengthened the disease associations of the proteins connected by subcellular localizations. In particular, certain disease types were more prevalent in specific subcellular localizations. We analyzed the enrichment of disease phenotypes within subcellular localizations, and found that there exists a significant correlation between disease classes and subcellular localizations. Furthermore, we found that two diseases displayed high comorbidity when disease-associated proteins were connected via subcellular localization. We newly explained 7584 disease pairs by using the context of protein subcellular localization, which had not been identified using shared genes or PPIs only. Our result establishes a direct correlation between protein subcellular localization and disease association, and helps to understand the mechanism of human disease progression. PMID:21613983

Distribution and Characterization of Antigens Found in Subcellular Fractions of African Trypanosomes.

DTIC Science & Technology

1982-08-01

CLASSIFICATION AUTHORITY 3. DISTRIBUTION /AVAILABILITY OF REPORT 2b. DECLASSIFICATION / DOWNGRADING SCHEDULE 4. PERFORMING ORGANIZATION REPORT NUMBER(S...S. MONITORING ORGANIZATION REPORT NUMBER(S) 6a. NAME OF PERFORMING ORGANIZATION 6b OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATION University of...was passed through the column using a peristaltic pump adjusted to flow rate of 8.0 ml/h. To allow full binding of sugar residues to lectin the eluent

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions.

PubMed

Perez White, Bethany E; Ventrella, Rosa; Kaplan, Nihal; Cable, Calvin J; Thomas, Paul M; Getsios, Spiro

2017-01-01

EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions. © 2017. Published by The Company of Biologists Ltd.

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions

PubMed Central

Perez White, Bethany E.; Ventrella, Rosa; Kaplan, Nihal; Cable, Calvin J.; Thomas, Paul M.

2017-01-01

ABSTRACT EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions. PMID:27815408

The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

PubMed Central

Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

1995-01-01

The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7626033

Studying dyadic structure-function relationships: a review of current modeling approaches and new insights into Ca2+ (mis)handling.

PubMed

Maleckar, Mary M; Edwards, Andrew G; Louch, William E; Lines, Glenn T

2017-01-01

Excitation-contraction coupling in cardiac myocytes requires calcium influx through L-type calcium channels in the sarcolemma, which gates calcium release through sarcoplasmic reticulum ryanodine receptors in a process known as calcium-induced calcium release, producing a myoplasmic calcium transient and enabling cardiomyocyte contraction. The spatio-temporal dynamics of calcium release, buffering, and reuptake into the sarcoplasmic reticulum play a central role in excitation-contraction coupling in both normal and diseased cardiac myocytes. However, further quantitative understanding of these cells' calcium machinery and the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease requires accurate knowledge of cardiac ultrastructure, protein distribution and subcellular function. As current imaging techniques are limited in spatial resolution, limiting insight into changes in calcium handling, computational models of excitation-contraction coupling have been increasingly employed to probe these structure-function relationships. This review will focus on the development of structural models of cardiac calcium dynamics at the subcellular level, orienting the reader broadly towards the development of models of subcellular calcium handling in cardiomyocytes. Specific focus will be given to progress in recent years in terms of multi-scale modeling employing resolved spatial models of subcellular calcium machinery. A review of the state-of-the-art will be followed by a review of emergent insights into calcium-dependent etiologies in heart disease and, finally, we will offer a perspective on future directions for related computational modeling and simulation efforts.

Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

PubMed Central

Nikiforov, Andrey; Dölle, Christian; Niere, Marc; Ziegler, Mathias

2011-01-01

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule. PMID:21504897

A novel mechanism of E2F1 regulation via nucleocytoplasmic shuttling: determinants of nuclear import and export.

PubMed

Ivanova, Iordanka A; Vespa, Alisa; Dagnino, Lina

2007-09-01

E2F1 is a transcription factor central for cell survival, proliferation, and repair following genomic insult. Depending on the cell type and conditions, E2F1 can induce apoptosis in transformed cells, behaving as a tumour suppressor, or impart growth advantages favouring tumour formation. The pleiotropic functions of E2F1 are a likely consequence of its ability to transcriptionally control a wide variety of target genes, and require tight regulation of its activity at multiple levels. Although sequestration of proteins to particular cellular compartments is a well-established regulatory mechanism, virtually nothing is known about its contribution to modulation of E2F1 target gene expression. We have examined the subcellular trafficking of E2F1 and, contrary to the widely held notion that this factor is constitutively nuclear, we now demonstrate that it is subjected to continuous nucleocytoplasmic shuttling. We have also defined two nuclear localization domains and a nuclear export region, which mediates CRM1-dependent transit out of the nucleus. The predominant subcellular location of E2F1 is likely determined by the balance between the activity of nuclear import and export domains, and can be modulated by differentiation stimuli in epidermal cells. Thus, we have identified a hitherto unrecognized mechanism to control E2F1 function through modulation of its subcellular localization.

Plasmodium berghei MAPK1 Displays Differential and Dynamic Subcellular Localizations during Liver Stage Development

PubMed Central

Wierk, Jannika Katharina; Langbehn, Annette; Kamper, Maria; Richter, Stefanie; Burda, Paul-Christian; Heussler, Volker Theo; Deschermeier, Christina

2013-01-01

Mitogen-activated protein kinases (MAPKs) regulate key signaling events in eukaryotic cells. In the genomes of protozoan Plasmodium parasites, the causative agents of malaria, two genes encoding kinases with significant homology to other eukaryotic MAPKs have been identified (mapk1, mapk2). In this work, we show that both genes are transcribed during Plasmodium berghei liver stage development, and analyze expression and subcellular localization of the PbMAPK1 protein in liver stage parasites. Live cell imaging of transgenic parasites expressing GFP-tagged PbMAPK1 revealed a nuclear localization of PbMAPK1 in the early schizont stage mediated by nuclear localization signals in the C-terminal domain. In contrast, a distinct localization of PbMAPK1 in comma/ring-shaped structures in proximity to the parasite’s nuclei and the invaginating parasite membrane was observed during the cytomere stage of parasite development as well as in immature blood stage schizonts. The PbMAPK1 localization was found to be independent of integrity of a motif putatively involved in ATP binding, integrity of the putative activation motif and the presence of a predicted coiled-coil domain in the C-terminal domain. Although PbMAPK1 knock out parasites showed normal liver stage development, the kinase may still fulfill a dual function in both schizogony and merogony of liver stage parasites regulated by its dynamic and stage-dependent subcellular localization. PMID:23544094

Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells.

PubMed

Roudeau, Stéphane; Carmona, Asuncion; Perrin, Laura; Ortega, Richard

2014-11-01

X-ray chemical element imaging has the potential to enable fundamental breakthroughs in the understanding of biological systems because chemical element interactions with organelles can be studied at the sub-cellular level. What is the distribution of trace metals in cells? Do some elements accumulate within sub-cellular organelles? What are the chemical species of the elements in these organelles? These are some of the fundamental questions that can be addressed by use of X-ray chemical element imaging with synchrotron radiation beams. For precise location of the distribution of the elements, identification of cellular organelles is required; this can be achieved, after appropriate labelling, by use of fluorescence microscopy. As will be discussed, this approach imposes some limitations on sample preparation. For example, standard immunolabelling procedures strongly modify the distribution of the elements in cells as a result of the chemical fixation and permeabilization steps. Organelle location can, however, be performed, by use of a variety of specific fluorescent dyes or fluorescent proteins, on living cells before cryogenic fixation, enabling preservation of element distribution. This article reviews the methods used for fluorescent organelle labelling and X-ray chemical element imaging and speciation of single cells. Selected cases from our work and from other research groups are presented to illustrate the potential of the combination of the two techniques.

Plant subcellular proteomics: Application for exploring optimal cell function in soybean.

PubMed

Wang, Xin; Komatsu, Setsuko

2016-06-30

Plants have evolved complicated responses to developmental changes and stressful environmental conditions. Subcellular proteomics has the potential to elucidate localized cellular responses and investigate communications among subcellular compartments during plant development and in response to biotic and abiotic stresses. Soybean, which is a valuable legume crop rich in protein and vegetable oil, can grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. To date, numerous proteomic studies have been performed in soybean to examine the specific protein profiles of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum. In this review, methods for the purification and purity assessment of subcellular organelles from soybean are summarized. In addition, the findings from subcellular proteomic analyses of soybean during development and under stresses, particularly flooding stress, are presented and the proteins regulated among subcellular compartments are discussed. Continued advances in subcellular proteomics are expected to greatly contribute to the understanding of the responses and interactions that occur within and among subcellular compartments during development and under stressful environmental conditions. Subcellular proteomics has the potential to investigate the cellular events and interactions among subcellular compartments in response to development and stresses in plants. Soybean could grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. Numerous proteomics of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum was carried out to investigate the respecting proteins and their functions in soybean during development or under stresses. In this review, methods of subcellular-organelle enrichment and purity assessment are summarized. In addition, previous findings of subcellular proteomics are presented, and functional proteins regulated among different subcellular are discussed. Subcellular proteomics contributes greatly to uncovering responses and interactions among subcellular compartments during development and under stressful environmental conditions in soybean. Copyright © 2016 Elsevier B.V. All rights reserved.

Oligodendroglial Maturation Is Dependent on Intracellular Protein Shuttling

PubMed Central

Göttle, Peter; Sabo, Jennifer K.; Heinen, André; Venables, Gene; Torres, Klintsy; Tzekova, Nevena; Parras, Carlos M.; Kremer, David; Hartung, Hans-Peter; Cate, Holly S.

2015-01-01

Multiple sclerosis is an autoimmune disease of the CNS resulting in degeneration of myelin sheaths and loss of oligodendrocytes, which means that protection and electrical insulation of axons and rapid signal propagation are impaired, leading to axonal damage and permanent disabilities. Partial replacement of lost oligodendrocytes and remyelination can occur as a result of activation and recruitment of resident oligodendroglial precursor cells. However, the overall remyelination capacity remains inefficient because precursor cells often fail to generate new oligodendrocytes. Increasing evidence points to the existence of several molecular inhibitors that act on these cells and interfere with their cellular maturation. The p57kip2 gene encodes one such potent inhibitor of oligodendroglial differentiation and this study sheds light on the underlying mode of action. We found that subcellular distribution of the p57kip2 protein changed during differentiation of rat, mouse, and human oligodendroglial cells both in vivo and in vitro. Nuclear export of p57kip2 was correlated with promoted myelin expression, higher morphological phenotypes, and enhanced myelination in vitro. In contrast, nuclear accumulation of p57kip2 resulted in blocked oligodendroglial differentiation. Experimental evidence suggests that the inhibitory role of p57kip2 depends on specific interactions with binding proteins such as LIMK-1, CDK2, Mash1, and Hes5 either by controlling their site of action or their activity. Because functional restoration in demyelinating diseases critically depends on the successful generation of oligodendroglial cells, a therapeutic need that is currently unmet, the regulatory mechanism described here might be of particular interest for identifying suitable drug targets and devising novel therapeutic approaches. PMID:25609610

Bioindication of mercury, arsenic and uranium in the apple snail Pomacea canaliculata (Caenogastropoda, Ampullariidae): Bioconcentration and depuration in tissues and symbiotic corpuscles.

PubMed

Campoy-Diaz, Alejandra D; Arribére, María A; Guevara, Sergio Ribeiro; Vega, Israel A

2018-04-01

Pomacea canaliculata is a mollusk potentially useful as a biomonitor species of freshwater quality. This work explores the ability of snail tissues and symbiotic corpuscles to bioconcentrate and depurate mercury, arsenic, and uranium. Adult snails cultured in metal-free reconstituted water were exposed for eight weeks (bioaccumulation phase) to water with Hg (2 μgL -1 ), As (10 μgL -1 ), and U (30 μgL -1 ) and then returned to the reconstituted water for other additional eight weeks (depuration phase). Elemental concentrations in digestive gland, kidney, symbiotic corpuscles and particulate excreta were determined by neutron activation analysis. The glandular symbiotic occupancy was measured by morphometric analysis. After exposure, the kidney showed the highest concentration of Hg, while the digestive gland accumulated mainly As and U. The subcellular distribution in symbiotic corpuscles was ∼71%, ∼48%, and ∼11% for U, Hg, and As, respectively. Tissue depuration between weeks 8 and 16 was variable amongst elements. At week 16, the tissue depuration of U was the highest (digestive gland = 92%; kidney = 80%), while it was lower for Hg (digestive gland = 51%; kidney = 53%). At week 16, arsenic showed a differential pattern of tissue depuration (digestive gland = 23%; kidney = 88%). The symbiotic detoxification of the three elements in excreta was fast between weeks 8 and 10 and it was slower after on. At the end of the depuration, each element distributed differentially in digestive gland and symbiotic corpuscles. Our findings show that symbiotic corpuscles, digestive gland and kidney P. canaliculata are sensitive places for biomonitoring of Hg, As and U. Copyright © 2017 Elsevier Ltd. All rights reserved.

Local depletion of glycogen with supramaximal exercise in human skeletal muscle fibres

PubMed Central

Ørtenblad, Niels; Andersson, Erik; Plomgaard, Peter; Holmberg, Hans‐Christer; Nielsen, Joachim

2016-01-01

Key points Glycogen is stored in local spatially distinct compartments within skeletal muscle fibres and is the main energy source during supramaximal exercise.Using quantitative electron microscopy, we show that supramaximal exercise induces a differential depletion of glycogen from these compartments and also demonstrate how this varies with fibre types.Repeated exercise alters this compartmentalized glycogen depletion.The results obtained in the present study help us understand the muscle metabolic dynamics of whole body repeated supramaximal exercise, and suggest that the muscle has a compartmentalized local adaptation to repeated exercise, which affects glycogen depletion. Abstract Skeletal muscle glycogen is heterogeneously distributed in three separated compartments (intramyofibrillar, intermyofibrillar and subsarcolemmal). Although only constituting 3–13% of the total glycogen volume, the availability of intramyofibrillar glycogen is of particular importance to muscle function. The present study aimed to investigate the depletion of these three subcellular glycogen compartments during repeated supramaximal exercise in elite athletes. Ten elite cross‐country skiers (aged 25 ± 4 years, V˙O2 max : 65 ± 4 ml kg−1 min−1; mean ± SD) performed four ∼4 min supramaximal sprint time trials (STT 1–4) with 45 min of recovery. The subcellular glycogen volumes in musculus triceps brachii were quantified from electron microscopy images before and after both STT 1 and 4. During STT 1, the depletion of intramyofibrillar glycogen was higher in type 1 fibres [−52%; (−89:−15%)] than type 2 fibres [−15% (−52:22%)] (P = 0.02), whereas the depletion of intermyofibrillar glycogen [main effect: −19% (−33:0%), P = 0.006] and subsarcolemmal glycogen [main effect: −35% (−66:0%), P = 0.03] was similar between fibre types. By contrast, only intermyofibrillar glycogen volume was significantly reduced during STT 4, in both fibre types [main effect: −31% (−50:−11%), P = 0.002]. Furthermore, for each of the subcellular compartments, the depletion of glycogen during STT 1 was associated with the volumes of glycogen before STT 1. In conclusion, the depletion of spatially distinct glycogen compartments differs during supramaximal exercise. Furthermore, the depletion changes with repeated exercise and is fibre type‐dependent. PMID:27689320

Local depletion of glycogen with supramaximal exercise in human skeletal muscle fibres.

PubMed

Gejl, Kasper D; Ørtenblad, Niels; Andersson, Erik; Plomgaard, Peter; Holmberg, Hans-Christer; Nielsen, Joachim

2017-05-01

Glycogen is stored in local spatially distinct compartments within skeletal muscle fibres and is the main energy source during supramaximal exercise. Using quantitative electron microscopy, we show that supramaximal exercise induces a differential depletion of glycogen from these compartments and also demonstrate how this varies with fibre types. Repeated exercise alters this compartmentalized glycogen depletion. The results obtained in the present study help us understand the muscle metabolic dynamics of whole body repeated supramaximal exercise, and suggest that the muscle has a compartmentalized local adaptation to repeated exercise, which affects glycogen depletion. Skeletal muscle glycogen is heterogeneously distributed in three separated compartments (intramyofibrillar, intermyofibrillar and subsarcolemmal). Although only constituting 3-13% of the total glycogen volume, the availability of intramyofibrillar glycogen is of particular importance to muscle function. The present study aimed to investigate the depletion of these three subcellular glycogen compartments during repeated supramaximal exercise in elite athletes. Ten elite cross-country skiers (aged 25 ± 4 years, V̇O2 max : 65 ± 4 ml kg -1  min -1 ; mean ± SD) performed four ∼4 min supramaximal sprint time trials (STT 1-4) with 45 min of recovery. The subcellular glycogen volumes in musculus triceps brachii were quantified from electron microscopy images before and after both STT 1 and 4. During STT 1, the depletion of intramyofibrillar glycogen was higher in type 1 fibres [-52%; (-89:-15%)] than type 2 fibres [-15% (-52:22%)] (P = 0.02), whereas the depletion of intermyofibrillar glycogen [main effect: -19% (-33:0%), P = 0.006] and subsarcolemmal glycogen [main effect: -35% (-66:0%), P = 0.03] was similar between fibre types. By contrast, only intermyofibrillar glycogen volume was significantly reduced during STT 4, in both fibre types [main effect: -31% (-50:-11%), P = 0.002]. Furthermore, for each of the subcellular compartments, the depletion of glycogen during STT 1 was associated with the volumes of glycogen before STT 1. In conclusion, the depletion of spatially distinct glycogen compartments differs during supramaximal exercise. Furthermore, the depletion changes with repeated exercise and is fibre type-dependent. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Detection of tumorigenesis in urinary bladder with optical coherence tomography: optical characterization of morphological changes

NASA Astrophysics Data System (ADS)

Xie, T.-Q.; Zeidel, M. L.; Pan, Yingtian

2002-12-01

Most transitional cell tumorigenesis involves three stages of subcellular morphological changes: hyperplasia, dysplasia and neoplasia. Previous studies demonstrated that owing to its high spatial resolution and intermediate penetration depth, current OCT technology including endoscopic OCT could delineate the urothelium, submucosa and the upper muscular layers of the bladder wall. In this paper, we will discuss the sensitivity and limitations of OCT in diagnosing and staging bladder cancer. Based on histomorphometric evaluations of nuclear morphology, we modeled the resultant backscattering changes and the characteristic changes in OCT image contrast. In the theoretical modeling, we assumed that nuclei were the primary sources of scattering and were uniformly distributed in the uroepithelium, and compared with the results of the corresponding prior OCT measurements. According to our theoretical modeling, normal bladder shows a thin, uniform and low scattering urothelium, so does an inflammatory lesion except thickening in the submucosa. Compared with a normal bladder, a hyperplastic lesion exhibits a thickened, low scattering urothelium whereas a neoplastic lesion shows a thickened urothelium with increased backscattering. These results support our previous animal study that OCT has the potential to differentiate inflammation, hyperplasia, and neoplasia by quantifying the changes in urothelial thickening and backscattering. The results also suggest that OCT might not have the sensitivity to differentiate the subtle morphological changes between hyperplasia and dysplasia based on minor backscattering differences.

Detection of tumorigenesis in urinary bladder with optical coherence tomography: optical characterization of morphological changes.

PubMed

Xie, T; Zeidel, M; Pan, Yingtian

2002-12-02

Most transitional cell tumorigenesis involves three stages of subcellular morphological changes: hyperplasia, dysplasia and neoplasia. Previous studies demonstrated that owing to its high spatial resolution and intermediate penetration depth, current OCT technology including endoscopic OCT could delineate the urothelium, submucosa and the upper muscular layers of the bladder wall. In this paper, we will discuss the sensitivity and limitations of OCT in diagnosing and staging bladder cancer. Based on histomorphometric evaluations of nuclear morphology, we modeled the resultant backscattering changes and the characteristic changes in OCT image contrast. In the theoretical modeling, we assumed that nuclei were the primary sources of scattering and were uniformly distributed in the uroepithelium, and compared with the results of the corresponding prior OCT measurements. According to our theoretical modeling, normal bladder shows a thin, uniform and low scattering urothelium, so does an inflammatory lesion except thickening in the submucosa. Compared with a normal bladder, a hyperplastic lesion exhibits a thickened, low scattering urothelium whereas a neoplastic lesion shows a thickened urothelium with increased backscattering. These results support our previous animal study that OCT has the potential to differentiate inflammation, hyperplasia, and neoplasia by quantifying the changes in urothelial thickening and backscattering. The results also suggest that OCT might not have the sensitivity to differentiate the subtle morphological changes between hyperplasia and dysplasia based on minor backscattering differences.

Population-level coordination of pigment response in individual cyanobacterial cells under altered nitrogen levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murton, Jaclyn; Nagarajan, Aparna; Nguyen, Amelia Y.

Cyanobacterial phycobilisome (PBS) pigment-protein complexes harvest light and transfer the energy to reaction centers. Previous ensemble studies have shown that cyanobacteria respond to changes in nutrient availability by modifying the structure of PBS complexes, but this process has not been visualized for individual pigments at the single-cell level due to spectral overlap. We characterized the response of four key photosynthetic pigments to nitrogen depletion and repletion at the subcellular level in individual, live Synechocystis sp. PCC 6803 cells using hyperspectral confocal fluorescence microscopy and multivariate image analysis. Our results revealed that PBS degradation and re-synthesis comprise a rapid response tomore » nitrogen fluctuations, with coordinated populations of cells undergoing pigment modifications. Chlorophyll fluorescence originating from photosystem I and II decreased during nitrogen starvation, but no alteration in subcellular chlorophyll localization was found. Lastly, we observed differential rod and core pigment responses to nitrogen deprivation, suggesting that PBS complexes undergo a stepwise degradation process.« less

A graphene-based physiometer array for the analysis of single biological cells

NASA Astrophysics Data System (ADS)

Paulus, Geraldine L. C.; Nelson, Justin T.; Lee, Katherine Y.; Wang, Qing Hua; Reuel, Nigel F.; Grassbaugh, Brittany R.; Kruss, Sebastian; Landry, Markita P.; Kang, Jeon Woong; Vander Ende, Emma; Zhang, Jingqing; Mu, Bin; Dasari, Ramachandra R.; Opel, Cary F.; Wittrup, K. Dane; Strano, Michael S.

2014-10-01

A significant advantage of a graphene biosensor is that it inherently represents a continuum of independent and aligned sensor-units. We demonstrate a nanoscale version of a micro-physiometer - a device that measures cellular metabolic activity from the local acidification rate. Graphene functions as a matrix of independent pH sensors enabling subcellular detection of proton excretion. Raman spectroscopy shows that aqueous protons p-dope graphene - in agreement with established doping trajectories, and that graphene displays two distinct pKa values (2.9 and 14.2), corresponding to dopants physi- and chemisorbing to graphene respectively. The graphene physiometer allows micron spatial resolution and can differentiate immunoglobulin (IgG)-producing human embryonic kidney (HEK) cells from non-IgG-producing control cells. Population-based analyses allow mapping of phenotypic diversity, variances in metabolic activity, and cellular adhesion. Finally we show this platform can be extended to the detection of other analytes, e.g. dopamine. This work motivates the application of graphene as a unique biosensor for (sub)cellular interrogation.

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

DOE PAGES

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.; ...

2017-03-13

Mechanical forces in the cell’s natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging frommore » the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques.« less

Kibra and Merlin Activate the Hippo Pathway Spatially Distinct from and Independent of Expanded.

PubMed

Su, Ting; Ludwig, Michael Z; Xu, Jiajie; Fehon, Richard G

2017-03-13

The Hippo pathway is emerging as a key evolutionarily conserved signaling mechanism that controls organ size. Three membrane-associated proteins, Kibra, Merlin, and Expanded, regulate pathway activity, but the precise molecular mechanism by which they function is still poorly understood. Here we provide evidence that Merlin and Kibra activate Hippo signaling in parallel to Expanded at a spatially distinct cellular domain, the medial apical cortex. Merlin and Kibra together recruit the adapter protein Salvador, which in turn recruits the core kinase Hippo. In addition, we show that Crumbs has a dual effect on Hippo signaling. Crumbs promotes the ability of Expanded to activate the pathway but also sequesters Kibra to downregulate Hippo signaling. Together, our findings elucidate the mechanism of Hippo pathway activation by Merlin and Kibra, identify a subcellular domain for Hippo pathway regulation, and demonstrate differential activity of upstream regulators in different subcellular domains. Copyright © 2017 Elsevier Inc. All rights reserved.

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

PubMed Central

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.; England, Grant T; Kim, Philseok; Kolle, Mathias; Ferrante, Thomas; Zarzar, Lauren D; Strong, Elizabeth; Aizenberg, Joanna

2017-01-01

Mechanical forces in the cell’s natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging from the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques. PMID:28287116

Population-level coordination of pigment response in individual cyanobacterial cells under altered nitrogen levels

DOE PAGES

Murton, Jaclyn; Nagarajan, Aparna; Nguyen, Amelia Y.; ...

2017-07-21

Cyanobacterial phycobilisome (PBS) pigment-protein complexes harvest light and transfer the energy to reaction centers. Previous ensemble studies have shown that cyanobacteria respond to changes in nutrient availability by modifying the structure of PBS complexes, but this process has not been visualized for individual pigments at the single-cell level due to spectral overlap. We characterized the response of four key photosynthetic pigments to nitrogen depletion and repletion at the subcellular level in individual, live Synechocystis sp. PCC 6803 cells using hyperspectral confocal fluorescence microscopy and multivariate image analysis. Our results revealed that PBS degradation and re-synthesis comprise a rapid response tomore » nitrogen fluctuations, with coordinated populations of cells undergoing pigment modifications. Chlorophyll fluorescence originating from photosystem I and II decreased during nitrogen starvation, but no alteration in subcellular chlorophyll localization was found. Lastly, we observed differential rod and core pigment responses to nitrogen deprivation, suggesting that PBS complexes undergo a stepwise degradation process.« less

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.

Mechanical forces in the cell’s natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging frommore » the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques.« less

Evaluating the efficacy of subcellular fractionation of blast cells using live cell labeling and 2D DIGE.

PubMed

Ho, Yin Ying; Penno, Megan; Perugini, Michelle; Lewis, Ian; Hoffmann, Peter

2012-01-01

Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel.

Nutrient-dependent phosphorylation channels lipid synthesis to regulate PPARα

PubMed Central

Jensen-Urstad, Anne P. L.; Song, Haowei; Lodhi, Irfan J.; Funai, Katsuhiko; Yin, Li; Coleman, Trey; Semenkovich, Clay F.

2013-01-01

Peroxisome proliferator-activated receptor (PPAR)α is a nuclear receptor that coordinates liver metabolism during fasting. Fatty acid synthase (FAS) is an enzyme that stores excess calories as fat during feeding, but it also activates hepatic PPARα by promoting synthesis of an endogenous ligand. Here we show that the mechanism underlying this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated. In mouse liver and cultured hepatoma cells, the ratio of cytoplasmic to membrane FAS-specific activity was increased with fasting, indicating higher cytoplasmic FAS activity under conditions associated with PPARα activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS. Cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPARα target gene expression. Rapamycin-induced inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a mediator of the feeding/insulin signal to induce lipogenesis, reduced FAS phosphorylation, increased cytoplasmic FAS enzyme activity, and increased PPARα target gene expression. Rapamycin-mediated induction of the same gene was abrogated with FAS knockdown. These findings suggest that hepatic FAS channels lipid synthesis through specific subcellular compartments that allow differential gene expression based on nutritional status. PMID:23585690

Elution of Labile Fluorescent Dye from Nanoparticles during Biological Use

PubMed Central

Tenuta, Tiziana; Monopoli, Marco P.; Kim, JongAh; Salvati, Anna; Dawson, Kenneth A.; Sandin, Peter; Lynch, Iseult

2011-01-01

Cells act as extremely efficient filters for elution of unbound fluorescent tags or impurities associated with nanoparticles, including those that cannot be removed by extensive cleaning. This has consequences for quantification of nanoparticle uptake and sub-cellular localization in vitro and in vivo as a result of the presence of significant amount of labile dye even following extensive cleaning by dialysis. Polyacrylamide gel electrophoresis (PAGE) can be used to monitor the elution of unbound fluorescent probes from nanoparticles, either commercially available or synthesized in-house, and to ensure their complete purification for biological studies, including cellular uptake and sub-cellular localisation. Very different fluorescence distribution within cells is observed after short dialysis times versus following extensive dialysis against a solvent in which the free dye is more soluble, due to the contribution from free dye. In the absence of an understanding of the presence of residual free dye in (most) labeled nanoparticle solutions, the total fluorescence intensity in cells following exposure to nanoparticle solutions could be mis-ascribed to the presence of nanoparticles through the cell, rather than correctly assigned to either a combination of free-dye and nanoparticle-bound dye, or even entirely to free dye depending on the exposure conditions (i.e. aggregation of the particles etc). Where all of the dye is nanoparticle-bound, the particles are highly localized in sub-cellular organelles, likely lysosomes, whereas in a system containing significant amounts of free dye, the fluorescence is distributed through the cell due to the free diffusion of the molecule dye across all cellular barriers and into the cytoplasm. PMID:21998668

Fine and distributed subcellular retinotopy of excitatory inputs to the dendritic tree of a collision-detecting neuron

PubMed Central

Zhu, Ying

2016-01-01

Individual neurons in several sensory systems receive synaptic inputs organized according to subcellular topographic maps, yet the fine structure of this topographic organization and its relation to dendritic morphology have not been studied in detail. Subcellular topography is expected to play a role in dendritic integration, particularly when dendrites are extended and active. The lobula giant movement detector (LGMD) neuron in the locust visual system is known to receive topographic excitatory inputs on part of its dendritic tree. The LGMD responds preferentially to objects approaching on a collision course and is thought to implement several interesting dendritic computations. To study the fine retinotopic mapping of visual inputs onto the excitatory dendrites of the LGMD, we designed a custom microscope allowing visual stimulation at the native sampling resolution of the locust compound eye while simultaneously performing two-photon calcium imaging on excitatory dendrites. We show that the LGMD receives a distributed, fine retinotopic projection from the eye facets and that adjacent facets activate overlapping portions of the same dendritic branches. We also demonstrate that adjacent retinal inputs most likely make independent synapses on the excitatory dendrites of the LGMD. Finally, we show that the fine topographic mapping can be studied using dynamic visual stimuli. Our results reveal the detailed structure of the dendritic input originating from individual facets on the eye and their relation to that of adjacent facets. The mapping of visual space onto the LGMD's dendrites is expected to have implications for dendritic computation. PMID:27009157

Bioavailability of biologically sequestered cadmium and the implications of metal detoxification

USGS Publications Warehouse

Wallace, W.G.; Lopez, G.R.

1997-01-01

The deposit-feeding oligochaete Limnodrilus hoffmeisteri possesses metallothionein-like proteins and metal-rich granules for storing and detoxifying cadmium (Cd). In this study we investigated the bioavailability of Cd sequestered within this oligochaete by conducting feeding experiments with 109Cd-labeled oligochaetes and the omnivorous grass shrimp Palaemonetes pugio. We also make predictions on Cd trophic transfer based on oligochaete subcellular Cd distributions and absorption efficiencies of Cd by shrimp Cytosol [including metallothionein-like proteins and other proteins) and a debris fraction (including metal-rich granules and tissue fragments) isolated from homogenized 109Cd-labeled oligochaetes were embedded in gelatin and fed to shrimp. The 109Cd absorption efficiencies of shrimp fed these subcellular fractions were 84.8 and 48.6%, respectively, and were significantly different (p < 0.001), indicating that 109Cd bound in these fractions was not equally available to a predator. Mass balance equations demonstrate that shrimp fed whole worms absorb 61.5% of the ingested 109Cd, an absorption efficiency similar to that obtained experimentally (57.1%). Furthermore, the majority of the absorbed 109Cd comes from the fraction containing metallothionein-like proteins (i.e. cytosol). 109Cd absorbed from the debris fraction probably comes from the digestion of tissue fragments, rather than metal-rich granules. The ecological significance of these findings is that prey detoxification mechanisms may mediate the bioreduction or bioaccumulation of toxic metals along fond chains by altering metal bioavailability. Another important finding is that trophic transfer of metal can be predicted based on the subcellular metal distribution of prey.

Alterations in the characteristic size distributions of subcellular scatterers at the onset of apoptosis: effect of Bcl-xL and Bax/Bak

NASA Astrophysics Data System (ADS)

Zheng, Jing-Yi; Boustany, Nada N.

2010-07-01

Optical scatter imaging is used to estimate organelle size distributions in immortalized baby mouse kidney cells treated with 0.4 μM staurosporine to induce apoptosis. The study comprises apoptosis competent iBMK cells (W2) expressing the proapoptotic proteins Bax/Bak, apoptosis resistant Bax/Bak null cells (D3), and W2 and D3 cells expressing yellow fluorescent protein (YFP) or YFP fused to the antiapoptotic protein Bcl-xL (YFP-Bcl-xL). YFP expression is diffuse within the transfected cells, while YFP-Bcl-xL is localized to the mitochondria. Our results show a significant increase in the mean subcellular particle size from approximately 1.1 to 1.4 μm in both Bax/Bak expressing and Bax/Bak null cells after 60 min of STS treatment compared to DMSO-treated control cells. This dynamic is blocked by overexpression of YFP-Bcl-xL in Bax/Bak expressing cells, but is less significantly inhibited by YFP-Bcl-xL in Bax/Bak null cells. Our data suggest that the increase in subcellular particle size at the onset of apoptosis is modulated by Bcl-xL in the presence of Bax/Bak, but it occurs upstream of the final commitment to programmed cell death. Mitochondrial localization of YFP-Bcl-xL and the finding that micron-sized particles give rise to the scattering signal further suggest that alterations in mitochondrial morphology may underlie the observed changes in light scattering.

Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms.

PubMed

Ariotti, Nicholas; Hall, Thomas E; Rae, James; Ferguson, Charles; McMahon, Kerrie-Ann; Martel, Nick; Webb, Robyn E; Webb, Richard I; Teasdale, Rohan D; Parton, Robert G

2015-11-23

Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines. Copyright © 2015 Elsevier Inc. All rights reserved.

Transmembrane topology, subcellular distribution and turnover of the gamma-aminobutyric acid/benzodizaepine receptor in chick brain cell cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Czajkowski, C.M.

1987-01-01

Experiments were performed utilizing trypsinization of the GABA/BZD-R in intact cells to determine (1) the subcellular distribution of membrane-associated GABA/BZD-Rs and (2) aspects of the transmembrane topology of the BZD-R. Additionally, R07-0213, a positively charged benzodiazepine, was used to distinguish between cell surface and intracellular BZD-Rs. Following trypsin treatment of intact cells a cleaved receptor fragment of M{sub r} = 24,000 (xRF24) is generated. It remains anchored in the plasma membrane and not only retains the ability to bind ({sup 3}H)flunitrazepan reversibly and irreversibly but also retains the ability to be modulated by GABA. xRF24 is not observed following trypsinizationmore » of saponin-treated cells or cell homogenates, indicating that it has a cytoplasmic domain as well as a cell surface domain, as expected for a transmembrane fragment of the BZD-R. By utilizing ({sup 3}H)flunitrazepam as an irreversible photoaffinity label, BZD-R turnover was also investigated.« less

A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions.

PubMed Central

Enrich, C; Bachs, O; Evans, W H

1988-01-01

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3214436

Molecular assembly and subcellular distribution of ATP-sensitive potassium channel proteins in rat hearts.

PubMed

Kuniyasu, Akihiko; Kaneko, Kazuyoshi; Kawahara, Kohichi; Nakayama, Hitoshi

2003-09-25

Cardiac ATP-sensitive K(+) (K(ATP)) channels are proposed to contribute to cardio-protection and ischemic preconditioning. Although mRNAs for all subunits of K(ATP) channels (Kir6.0 and sulfonylurea receptors SURs) were detected in hearts, subcellular localization of their proteins and the subunit combination are not well elucidated. We address these questions in rat hearts, using anti-peptide antibodies raised against each subunit. By immunoblot analysis, all of the subunits were detected in microsomal fractions including sarcolemmal membranes, while they were not detected in mitochondrial fractions at all. Immunoprecipitation and sucrose gradient sedimentation of the digitonin-solubilized microsomes indicated that Kir6.2 exclusively assembled with SUR2A. The molecular mass of the Kir6.2-SUR2A complex estimated by sucrose sedimentation was 1150 kDa, significantly larger than the calculated value for (Kir6.2)(4)-(SUR2A)(4), suggesting a potential formation of micellar complex with digitonin but no indication of hybrid channel formation under the conditions. These findings provide additional information on the structural and functional relationships of cardiac K(ATP) channel proteins involving subcellular localization and roles for cardioprotection and ischemic preconditioning.

Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by Tobamovirus Infection and Transient Expression of Viral Replication and Movement Proteins

PubMed Central

Sasaki, Nobumitsu; Takashima, Eita; Nyunoya, Hiroshi

2018-01-01

Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexvirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by tomato mosaic virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamovirus movement. PMID:29868075

Role of PIN-mediated auxin efflux in apical hook development of Arabidopsis thaliana.

PubMed

Zádníková, Petra; Petrásek, Jan; Marhavy, Peter; Raz, Vered; Vandenbussche, Filip; Ding, Zhaojun; Schwarzerová, Katerina; Morita, Miyo T; Tasaka, Masao; Hejátko, Jan; Van Der Straeten, Dominique; Friml, Jirí; Benková, Eva

2010-02-01

The apical hook of dark-grown Arabidopsis seedlings is a simple structure that develops soon after germination to protect the meristem tissues during emergence through the soil and that opens upon exposure to light. Differential growth at the apical hook proceeds in three sequential steps that are regulated by multiple hormones, principally auxin and ethylene. We show that the progress of the apical hook through these developmental phases depends on the dynamic, asymmetric distribution of auxin, which is regulated by auxin efflux carriers of the PIN family. Several PIN proteins exhibited specific, partially overlapping spatial and temporal expression patterns, and their subcellular localization suggested auxin fluxes during hook development. Genetic manipulation of individual PIN activities interfered with different stages of hook development, implying that specific combinations of PIN genes are required for progress of the apical hook through the developmental phases. Furthermore, ethylene might modulate apical hook development by prolonging the formation phase and strongly suppressing the maintenance phase. This ethylene effect is in part mediated by regulation of PIN-dependent auxin efflux and auxin signaling.

SIRT1 Activity Is Linked to Its Brain Region-Specific Phosphorylation and Is Impaired in Huntington’s Disease Mice

PubMed Central

Tulino, Raffaella; Benjamin, Agnesska C.; Jolinon, Nelly; Smith, Donna L.; Chini, Eduardo N.; Carnemolla, Alisia; Bates, Gillian P.

2016-01-01

Huntington’s disease (HD) is a neurodegenerative disorder for which there are no disease-modifying treatments. SIRT1 is a NAD+-dependent protein deacetylase that is implicated in maintaining neuronal health during development, differentiation and ageing. Previous studies suggested that the modulation of SIRT1 activity is neuroprotective in HD mouse models, however, the mechanisms controlling SIRT1 activity are unknown. We have identified a striatum-specific phosphorylation-dependent regulatory mechanism of SIRT1 induction under normal physiological conditions, which is impaired in HD. We demonstrate that SIRT1 activity is down-regulated in the brains of two complementary HD mouse models, which correlated with altered SIRT1 phosphorylation levels. This SIRT1 impairment could not be rescued by the ablation of DBC1, a negative regulator of SIRT1, but was linked to changes in the sub-cellular distribution of AMPK-α1, a positive regulator of SIRT1 function. This work provides insights into the regulation of SIRT1 activity with the potential for the development of novel therapeutic strategies. PMID:26815359

Analysis of doxorubicin distribution in MCF-7 cells treated with drug-loaded nanoparticles by combination of two fluorescence-based techniques, confocal spectral imaging and capillary electrophoresis.

PubMed

Gautier, Juliette; Munnier, Emilie; Soucé, Martin; Chourpa, Igor; Douziech Eyrolles, Laurence

2015-05-01

The intracellular distribution of the antiancer drug doxorubicin (DOX) was followed qualitatively by fluorescence confocal spectral imaging (FCSI) and quantitatively by capillary electrophoresis (CE). FCSI permits the localization of the major fluorescent species in cell compartments, with spectral shifts indicating the polarity of the respective environment. However, distinction between drug and metabolites by FCSI is difficult due to their similar fluorochromes, and direct quantification of their fluorescence is complicated by quantum yield variation between different subcellular environments. On the other hand, capillary electrophoresis with fluorescence detection (CE-LIF) is a quantitative method capable of separating doxorubicin and its metabolites. In this paper, we propose a method for determining drug and metabolite concentration in enriched nuclear and cytosolic fractions of cancer cells by CE-LIF, and we compare these data with those of FCSI. Significant differences in the subcellular distribution of DOX are observed between the drug administered as a molecular solution or as a suspension of drug-loaded iron oxide nanoparticles coated with polyethylene glycol. Comparative analysis of the CE-LIF vs FCSI data may lead to a tentative calibration of this latter method in terms of DOX fluorescence quantum yields in the nucleus and more or less polar regions of the cytosol.

Subcellular distributions of metals and metal induced stress: A field study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jenkins, K.D.; Howe, S.; Sanders, B.M.

This paper reports the results of a field study which took place around an exploratory well located in the Santa Barbara Channel. This study was designed to test for significant temporal and spatial differences in the concentrations of a number of drilling fluid-associated metals in both the sediments and biota. Temporal changes in the distribution of Ba, Cd, Cr, Cu, Hg, Ni, Pb, and Zn were examined in the sediments, and the bioaccumulation and subcellular distribution of these metals were examined in three benthic invertebrate species before and after drilling. Statistically significant increases in the accumulation of several of themore » metals were found in the surface sediments down current from the site after drilling with Ba showing the most pronounced increase. Statistically significant increases in the bioaccumulation of Ba were also observed in two of the three species examined, Cyclocardia ventricosa and Pactinaria californiensis. Within these organisms the majority of the Ba was localized in the granular pellets (>97%) and less than 0.1% accumulated in the cytosol. These data indicate that although bioaccumulation of Ba occurs in some species immediately down current from the well, most of it remains in an insoluble for, presumably as BaSO{sub 4}.« less

Uptake, transport and distribution of molybdenum in two oilseed rape (Brassica napus L.) cultivars under different nitrate/ammonium ratios*

PubMed Central

Qin, Shi-yu; Sun, Xue-cheng; Hu, Cheng-xiao; Tan, Qi-ling; Zhao, Xiao-hu

2017-01-01

Objectives: To investigate the effects of different nitrate sources on the uptake, transport, and distribution of molybdenum (Mo) between two oilseed rape (Brassica napus L.) cultivars, L0917 and ZS11. Methods: A hydroponic culture experiment was conducted with four nitrate/ammonium (NO3 −:NH4 +) ratios (14:1, 9:6, 7.5:7.5, and 1:14) at a constant nitrogen concentration of 15 mmol/L. We examined Mo concentrations in roots, shoots, xylem and phloem sap, and subcellular fractions of leaves to contrast Mo uptake, transport, and subcellular distribution between ZS11 and L0917. Results: Both the cultivars showed maximum biomass and Mo accumulation at the 7.5:7.5 ratio of NO3 −:NH4 + while those were decreased by the 14:1 and 1:14 treatments. However, the percentages of root Mo (14.8% and 15.0% for L0917 and ZS11, respectively) were low under the 7.5:7.5 treatment, suggesting that the equal NO3 −:NH4 + ratio promoted Mo transportation from root to shoot. The xylem sap Mo concentration and phloem sap Mo accumulation of L0917 were lower than those of ZS11 under the 1:14 treatment, which suggests that higher NO3 −:NH4 + ratio was more beneficial for L0917. On the contrary, a lower NO3 −:NH4 + ratio was more beneficial for ZS11 to transport and remobilize Mo. Furthermore, the Mo concentrations of both the cultivars’ leaf organelles were increased but the Mo accumulations of the cell wall and soluble fraction were reduced significantly under the 14:1 treatment, meaning that more Mo was accumulated in organelles under the highest NO3 −:NH4 + ratio. Conclusions: This investigation demonstrated that the capacities of Mo absorption, transportation and subcellular distribution play an important role in genotype-dependent differences in Mo accumulation under low or high NO3 −:NH4 + ratio conditions. PMID:28585427

PDZK1 binding and serine phosphorylation regulate subcellular trafficking of organic anion transport protein 1a1

PubMed Central

Choi, Jo H.; Murray, John W.

2011-01-01

Although perturbation of organic anion transport protein (oatp) cell surface expression can result in drug toxicity, little is known regarding mechanisms regulating its subcellular distribution. Many members of the oatp family, including oatp1a1, have a COOH-terminal PDZ consensus binding motif that interacts with PDZK1, while serines upstream of this site (S634 and S635) can be phosphorylated. Using oatp1a1 as a prototypical member of the oatp family, we prepared plasmids in which these serines were mutated to glutamic acid [E634E635 (oatp1a1EE), phosphomimetic] or alanine [A634A635 (oatp1a1AA), nonphosphorylatable]. Distribution of oatp1a1AA and oatp1a1EE was largely intracellular in transfected human embryonic kidney (HEK) 293T cells. Cotransfection with a plasmid encoding PDZK1 revealed that oatp1a1AA was now expressed largely on the cell surface, while oatp1a1EE remained intracellular. To quantify these changes, studies were performed in HuH7 cells stably transfected with these oatp1a1 plasmids. These cells endogenously express PDZK1. Surface biotinylation at 4°C followed by shift to 37°C showed that oatp1a1EE internalizes quickly compared with oatp1a1AA. To examine a physiological role for phosphorylation in oatp1a1 subcellular distribution, studies were performed in rat hepatocytes exposed to extracellular ATP, a condition that stimulates serine phosphorylation of oatp1a1 via activity of a purinergic receptor. Internalization of oatp1a1 under these conditions was rapid. Thus, although PDZK1 binding is required for optimal cell surface expression of oatp1a1, phosphorylation provides a mechanism for fast regulation of the distribution of oatp1a1 between the cell surface and intracellular vesicular pools. Identification of the proteins and motor molecules that mediate these trafficking events represents an important area for future study. PMID:21183661

Proteome-wide Subcellular Topologies of E. coli Polypeptides Database (STEPdb)*

PubMed Central

Orfanoudaki, Georgia; Economou, Anastassios

2014-01-01

Cell compartmentalization serves both the isolation and the specialization of cell functions. After synthesis in the cytoplasm, over a third of all proteins are targeted to other subcellular compartments. Knowing how proteins are distributed within the cell and how they interact is a prerequisite for understanding it as a whole. Surface and secreted proteins are important pathogenicity determinants. Here we present the STEP database (STEPdb) that contains a comprehensive characterization of subcellular localization and topology of the complete proteome of Escherichia coli. Two widely used E. coli proteomes (K-12 and BL21) are presented organized into thirteen subcellular classes. STEPdb exploits the wealth of genetic, proteomic, biochemical, and functional information on protein localization, secretion, and targeting in E. coli, one of the best understood model organisms. Subcellular annotations were derived from a combination of bioinformatics prediction, proteomic, biochemical, functional, topological data and extensive literature re-examination that were refined through manual curation. Strong experimental support for the location of 1553 out of 4303 proteins was based on 426 articles and some experimental indications for another 526. Annotations were provided for another 320 proteins based on firm bioinformatic predictions. STEPdb is the first database that contains an extensive set of peripheral IM proteins (PIM proteins) and includes their graphical visualization into complexes, cellular functions, and interactions. It also summarizes all currently known protein export machineries of E. coli K-12 and pairs them, where available, with the secretory proteins that use them. It catalogs the Sec- and TAT-utilizing secretomes and summarizes their topological features such as signal peptides and transmembrane regions, transmembrane topologies and orientations. It also catalogs physicochemical and structural features that influence topology such as abundance, solubility, disorder, heat resistance, and structural domain families. Finally, STEPdb incorporates prediction tools for topology (TMHMM, SignalP, and Phobius) and disorder (IUPred) and implements the BLAST2STEP that performs protein homology searches against the STEPdb. PMID:25210196

Demonstration of subcellular migration of CK2α localization from nucleus to sarco(endo)plasmic reticulum in mammalian cardiomyocytes under hyperglycemia.

PubMed

Bitirim, Ceylan Verda; Tuncay, Erkan; Turan, Belma

2018-06-01

The cellular control of glucose uptake and glycogen metabolism in mammalian tissues is in part mediated through the regulation of protein-serine/threonine kinases including CK2. Although it participates to several cellular signaling processes, however, its subcellular localization is not well-defined while some documents mentioned its localization change under pathological conditions. The activation/phosphorylation of some proteins including Zn 2+ -transporter ZIP7 in cardiomyocytes is controlled with CK2α, thereby, inducing changes in the level of intracellular free Zn 2+ ([Zn 2+ ] i ). In this regard, we aimed to examine cellular localization of CK2α in cardiomyocytes and its possible subcellular migration under hyperglycemia. Our confocal imaging together with biochemical analysis in isolated sarco(endo)plasmic reticulum [S(E)R] and nuclear fractions from hearts have shown that CK2α localized highly to S(E)R and Golgi and weakly to nuclear fractions in physiological condition. However, it can migrate from nuclear fractions to S(E)R under hyperglycemia. This migration can further underlie phosphorylation of a target protein ZIP7 as well as some endogenous kinases and phosphatases including PKA, CaMKII, and PP2A. We also have shown that CK2α activation is responsible for hyperglycemia-associated [Zn 2+ ] i increase in diabetic heart. Therefore, our present data demonstrated, for the first time, the physiological relevance of CK2α in cellular control of Zn 2+ -distribution via inducing ZIP7 phosphorylation and activation of these above endogenous actors in hyperglycemia/diabetes-associated cardiac dysfunction. Moreover, our present data also emphasized the multi-subcellular compartmental localizations of CK2α and a tightly regulation of these localizations in cardiomyocytes. Therefore, taken into consideration of all data, one can emphasize the important role of the subcellular localization of CK2α as a novel target-pathway for understanding of diabetic cardiomyopathy.

Hepatic Concentration and Distribution of Coenzyme A and Carnitine during a Streptococcus pneumoniae Infection in the Rat: Possible Implications on Fatty Acid Metabolism and Ketogenesis

DTIC Science & Technology

1981-01-09

subcellular distribution of carnitine and coenzyme A (CoA). Compared to fasted control ILJ rats, fasted-infected rats have a decreased ketogenic capacity...decreased ketogenic capacity that is associated with an accumulation of total hepatic carnitine and a decrease in total hepatic coenzyme A. The...cholesterol. IiA .Ii INTRODUCTION Rats infected with Streptococcus pneumoniae have a decreased hep-tic ketogenic capacity which is associated with an

Intracellular delivery and trafficking dynamics of a lymphoma-targeting antibody-polymer conjugate

PubMed Central

Berguig, Geoffrey Y.; Convertine, Anthony J.; Shi, Julie; Palanca-Wessels, Maria Corinna; Duvall, Craig L.; Pun, Suzie H.; Press, Oliver W.; Stayton, Patrick S.

2012-01-01

Ratiometric fluorescence and cellular fractionation studies were employed to characterize the intracellular trafficking dynamics of antibody-poly(propylacrylic acid) (PPAA) conjugates in CD22+ RAMOS-AW cells. The HD39 monoclonal antibody (mAb) directs CD22-dependent, receptor-mediated uptake in human B-cell lymphoma cells where it is rapidly trafficked to the lysosomal compartment. To characterize the intracellular-releasing dynamics of the polymer-mAb conjugates, HD39-streptavidin (HD39/SA) was dual-labeled with pH-insensitive Alex Fluor 488 and pH-sensitive pHrodo fluorophores. The subcellular pH-distribution of the HD39/SA-polymer conjugates were quantified as a function of time by live-cell fluorescence microscopy, and the average intracellular pH values experienced by the conjugates were also characterized as a function of time by flow cytometry. PPAA was shown to strongly alter the intracellular trafficking kinetics compared to HD39/SA alone or HD39/SA conjugates with a control polymer, poly(methacryclic acid) (PMAA). Subcellular trafficking studies revealed that after 6 hours only 11% of the HD39/SA-PPAA conjugates had been trafficked to acidic lysosomal compartments with values at or below pH 5.6. In contrast the average intracellular pH of HD39/SA alone dropped from pH 6.7 ± 0.2 at 1 hour to pH 5.6 ± 0.5 after 3 hours and pH 4.7 ± 0.6 after 6 hours. Conjugation of the control PMAA to HD39/SA showed an average pH drop similar to HD39/SA. Subcellular fractionation studies with tritium-labeled HD39/SA demonstrated that after 6 hours, 89% of HD39/SA was associated with endosomes (Rab5+) and lysosomes (Lamp2+), while 45% of HD39/SA-PPAA was translocated to the cytosol (lactate dehydrogenase+). These results demonstrate the endosomal-releasing properties of PPAA with antibody-polymer conjugates and detail their intracellular trafficking dynamics and subcellular compartmental distributions over time. PMID:23075320

WE-AB-204-12: Dosimetry at the Sub-Cellular Scale of Auger-Electron Emitter 99m-Tc in a Mouse Single Thyroid Follicle Model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taborda, A; Benabdallah, N; Desbree, A

2015-06-15

Purpose: To perform a dosimetry study at the sub-cellular scale of Auger-electron emitter 99m-Tc using a mouse single thyroid cellular model to investigate the contribution of the 99m-Tc Auger-electrons to the absorbed dose and possible link to the thyroid stunning in in vivo experiments in mice, recently reported in literature. Methods: The simulation of S-values for Auger-electron emitting radionuclides was performed using both the recent MCNP6 software and the Geant4-DNA extension of the Geant4 toolkit. The dosimetric calculations were validated through comparison with results from literature, using a simple model of a single cell consisting of two concentric spheres ofmore » unit density water and for six Auger-electron emitting radionuclides. Furthermore, the S-values were calculated using a single thyroid follicle model for uniformly distributed 123-I and 125-I radionuclides and compared with published S-values. After validation, the simulation of the S-values was performed for the 99m-Tc radionuclide within the several mouse thyroid follicle cellular compartments, considering the radiative and non-radiative transitions of the 99m-Tc radiation spectrum. Results: The calculated S-values using MCNP6 are in good agreement with the results from literature, validating its use for the 99m-Tc S-values calculations. The most significant absorbed dose corresponds to the case where the radionuclide is uniformly distributed in the follicular cell’s nucleus, with a S-value of 7.8 mGy/disintegration, due mainly to the absorbed Auger-electrons. The results show that, at a sub-cellular scale, the emitted X-rays and gamma particles do not contribute significantly to the absorbed dose. Conclusion: In this work, MCNP6 was validated for dosimetric studies at the sub-cellular scale. It was shown that the contribution of the Auger-electrons to the absorbed dose is important at this scale compared to the emitted photons’ contribution and can’t be neglected. The obtained S-values of Auger-electron emitting 99m-Tc radionuclide will be presented and discussed.« less

Epidermal Growth Factor Receptor mediated cellular and subcellular targeted delivery of Iron oxide core-Titanium dioxide shell nanoparticles

NASA Astrophysics Data System (ADS)

Yuan, Ye

TiO2 nanomaterials can carry a multitude of therapeutic and diagnostic agents and the semiconductor properties of TiO2 allow for the production of cytotoxic reactive oxygen species following photoactivation. However, the delivery of these nanomaterials to specific cancer cells and specific subcellular organelles within these cells can have a substantial impact on the efficacy and safety of TiO2 nanoparticle therapeutics. Targeting cell surface receptors that are overexpressed by cancer cells is one strategy to improve the specificity of nanoparticle delivery. Therefore we decided to target the Epidermal Growth Factor Receptor (EGFR) because ligand- binding induces rapid receptor endocytosis and ligand-bound EGFR can translocate to the nucleus of cancer cells. To create NPs that can bind EGFR, we identified a peptide derived from the B-loop of Epidermal Growth Factor (EGF) that has been shown to bind and activate EGFR and conjugated it to the surface of Fe3O4 core-TiO2 shell NPs to produce B-loop NCs. We then devised a pulldown assay to show that B-loop NCs, but not bare NPs or NCs carrying a scrambled B-loop peptide, can bind and extract EGFR from HeLa cell protein extracts. Interestingly, B-loop NCs can also pulldown importin-beta, a protein that can transport EGFR to the nucleus. Furthermore, we used flow cytometry and fluorescently labeled NPs to show that B-loop peptides can significantly improve the internalization of NPs by EGFR-expressing HeLa cells. We determined that B-loop NCs can bind EGFR on the membrane of HeLa cells and that these NCs can be transported to the nucleus, by using a combination of confocal microscopy and X-ray Fluorescence Microscopy (XFM) to indirectly and directly track the subcellular distribution of NCs. Finally, we demonstrate how the Bionanoprobe, a novel high-resolution XFM apparatus that can scan whole-mounted, frozen-hydrated cells at multiple angles can be used to verify the subcellular distribution of B-loop NCs.

HPASubC: A suite of tools for user subclassification of human protein atlas tissue images.

PubMed

Cornish, Toby C; Chakravarti, Aravinda; Kapoor, Ashish; Halushka, Marc K

2015-01-01

The human protein atlas (HPA) is a powerful proteomic tool for visualizing the distribution of protein expression across most human tissues and many common malignancies. The HPA includes immunohistochemically-stained images from tissue microarrays (TMAs) that cover 48 tissue types and 20 common malignancies. The TMA data are used to provide expression information at the tissue, cellular, and occasionally, subcellular level. The HPA also provides subcellular data from confocal immunofluorescence data on three cell lines. Despite the availability of localization data, many unique patterns of cellular and subcellular expression are not documented. To get at this more granular data, we have developed a suite of Python scripts, HPASubC, to aid in subcellular, and cell-type specific classification of HPA images. This method allows the user to download and optimize specific HPA TMA images for review. Then, using a playstation-style video game controller, a trained observer can rapidly step through 10's of 1000's of images to identify patterns of interest. We have successfully used this method to identify 703 endothelial cell (EC) and/or smooth muscle cell (SMCs) specific proteins discovered within 49,200 heart TMA images. This list will assist us in subdividing cardiac gene or protein array data into expression by one of the predominant cell types of the myocardium: Myocytes, SMCs or ECs. The opportunity to further characterize unique staining patterns across a range of human tissues and malignancies will accelerate our understanding of disease processes and point to novel markers for tissue evaluation in surgical pathology.

HPASubC: A suite of tools for user subclassification of human protein atlas tissue images

PubMed Central

Cornish, Toby C.; Chakravarti, Aravinda; Kapoor, Ashish; Halushka, Marc K.

2015-01-01

Background: The human protein atlas (HPA) is a powerful proteomic tool for visualizing the distribution of protein expression across most human tissues and many common malignancies. The HPA includes immunohistochemically-stained images from tissue microarrays (TMAs) that cover 48 tissue types and 20 common malignancies. The TMA data are used to provide expression information at the tissue, cellular, and occasionally, subcellular level. The HPA also provides subcellular data from confocal immunofluorescence data on three cell lines. Despite the availability of localization data, many unique patterns of cellular and subcellular expression are not documented. Materials and Methods: To get at this more granular data, we have developed a suite of Python scripts, HPASubC, to aid in subcellular, and cell-type specific classification of HPA images. This method allows the user to download and optimize specific HPA TMA images for review. Then, using a playstation-style video game controller, a trained observer can rapidly step through 10's of 1000's of images to identify patterns of interest. Results: We have successfully used this method to identify 703 endothelial cell (EC) and/or smooth muscle cell (SMCs) specific proteins discovered within 49,200 heart TMA images. This list will assist us in subdividing cardiac gene or protein array data into expression by one of the predominant cell types of the myocardium: Myocytes, SMCs or ECs. Conclusions: The opportunity to further characterize unique staining patterns across a range of human tissues and malignancies will accelerate our understanding of disease processes and point to novel markers for tissue evaluation in surgical pathology. PMID:26167380

Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Nonprotein Thiol Imaging and Quantification in Mitochondria.

PubMed

Wang, Shenggang; Yin, Huihui; Huang, Yue; Guan, Xiangming

2018-06-11

Cellular thiols are divided into two major categories: nonprotein thiols (NPSH) and protein thiols (PSH). Thiols are unevenly distributed inside the cell and compartmentalized in subcellular structures. Most of our knowledge on functions/dysfunctions of cellular/subcellular thiols is based on the quantification of cellular/subcellular thiols through homogenization of cellular/subcellular structures followed by a thiol quantification method. We would like to report a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide {7,7'-thiobis( N-rhodamine-benzo[c][1,2,5]oxadiazole-4-sulfonamide) (TBROS)} that can effectively image and quantify live cell NPSH in mitochondria through fluorescence intensity. Limited methods are available for imaging thiols in mitochondria in live cells especially in a quantitative manner. The thiol specificity of TBROS was demonstrated by its ability to react with thiols and inability to react with biologically relevant nucleophilic functional groups other than thiols. TBROS, with minimal fluorescence, formed strong fluorescent thiol adducts (λ ex = 550 nm, λ em = 580 nm) when reacting with NPSH confirming its fluorogenicity. TBROS failed to react with PSH from bovine serum albumin and cell homogenate proteins. The high mitochondrial thiol selectivity of TBROS was achieved by its mitochondria targeting structure and its higher reaction rate with NPSH at mitochondrial pH. Imaging of mitochondrial NPSH in live cells was confirmed by two colocalization methods and use of a thiol-depleting reagent. TBROS effectively imaged NPSH changes in a quantitative manner in mitochondria in live cells. The reagent will be a useful tool in exploring physiological and pathological roles of mitochondrial thiols.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rojas, Joselyn; Teran-Angel, Guillermo; Barbosa, Luisa

Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttlingmore » of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection. - Highlights: • The expression and subcellular distribution of Foxp3, is modulated by PMA and preS1/2. • PMA and preS1/2 increase Foxp3 expression on HepG2. • PMA and preS1/2 induce foxp3 enrichment at mitochondrial, microsomal and nuclear compartments. • Results suggest a non-canonical function of Foxp3 or a mitochondrial transcriptional activity.« less

Interplay of CREB and ATF2 in Ionizing Radiation-Induced Neuroendocrine Differentiation of Prostate Cancer Cells

DTIC Science & Technology

2011-06-01

h-actin. Analysis of ATF2 and CREB subcellular localization. LNCaP cells were fixed in ice-cold 3.7% formaldehyde for 20 min, followed by... phenol -free RPMI 1640 supplemented with 10% charcoal-dextran–treated FBS (CD-FBS) for 3 wk and similarly assayed for morphologic changes and the... phenol -free RPMI 1640 supplemented with 10% CD-FBS for the indicated times. Cell viability for IR- and docetaxel-treated cells was determined by a 3

Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

PubMed

Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

2015-07-01

The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be associated with ferritin cages or transferrin receptor endosomes. The iron content and its distribution in puncta were similar in all neuron types studied including primary dopaminergic neurons. In summary, quantitative measurements of steady state metal levels in single primary cultured neurons made possible by SRXRF analyses provide unique information on the relative levels of each metal in neuronal soma and processes, subcellular location of zinc loads, and have confirmed and extended the characterization of heretofore poorly understood cytoplasmic iron puncta.

[Effects of Different Modifier Concentrations on Lead-Zinc Tolerance, Subcellular Distribution and Chemical Forms for Four Kinds of Woody Plants].

PubMed

Chen, Yong-hua; Zhang, Fu-yun; Wu, Xiao-fu; Liang, Xi; Yuan, Si-wen

2015-10-01

Four kinds of lead-zinc tolerant woody plants: Nerium oleander, Koelreuteria paniculata, Paulownia and Boehmeria were used as materials to estimate their enrichment and transferable capacity of lead (Pb) and zinc (Zn) and analyze the subcellular distribution and chemical speciation of Zn and Ph in different parts of plants, under different modifier concentrations (CK group: 100% lead-zinc slag plus a small amount of phosphate fertilizer, improved one: 85% of lead-zinc slag ± 10% peat ± 5% bacterial manure plus a small amount of phosphate fertilizer, improved two: 75% lead-zinc slag ± 20% peat ± 5% bacterial manure ± a small amount of phosphate). Results showed that: (1) The content of Pb, Zn in matrix after planting four kinds of plants was lower than before, no significant difference between improved one and improved two of Nerium oleander and Boehmeria was found, but improved two was better than improved one of Paulownia, while improved one was better than improved two of Koelreuteria paniculata; Four plants had relatively low aboveground enrichment coefficient of Pb and Zn, but had a high transfer coefficient, showed that the appropriate modifier concentration was able to improve the Pb and Zn enrichment and transfer ability of plants. (2) In subcellular distribution, most of Pb and Zn were distributed in plant cell wall components and soluble components while the distribution in cell organelles such as mitochondria, chloroplasts and nucleus component were less. Compared with CK group, two improved group made soluble components of the cell walls of Pb fixation and retention of zinc role in the enhancement. (3) As for the chemical forms of Pb and Zn in plants, the main chemical forms of Pb were hydrochloric acid, sodium chloride and ethanol extractable forms, while other chemical form contents were few, the main chemical forms of Zn were different based on plant type. Compared with CK group, the proportion of the active Pb chemical form in different plant parts decreased in two improved groups, while the proportion of strong activity chemical forms increased; two improved groups led strong activity Zn chemical form of root increased, while strong activity Zn chemical form of aboveground decreased.

Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus

USDA-ARS?s Scientific Manuscript database

In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution of Turnip mosaic virus (TuMV). Brassica chinensis sap-inoculated with TuMV-infected radish tissue showed different symptom severity with three isolates. In order to investigate variation among Korean Tu...

Regulating Set-β's Subcellular Localization Toggles Its Function between Inhibiting and Promoting Axon Growth and Regeneration

PubMed Central

Wang, Yan; Morkin, Melina I.; Fernandez, Stephanie G.; Mlacker, Gregory M.; Shechter, Jesse M.; Liu, Xiongfei; Patel, Karan H.; Lapins, Allison; Yang, Steven; Dombrowski, Susan M.

2014-01-01

The failure of the CNS neurons to regenerate axons after injury or stroke is a major clinical problem. Transcriptional regulators like Set-β are well positioned to regulate intrinsic axon regeneration capacity, which declines developmentally in maturing CNS neurons. Set-β also functions at cellular membranes and its subcellular localization is disrupted in Alzheimer's disease, but many of its biological mechanisms have not been explored in neurons. We found that Set-β was upregulated postnatally in CNS neurons, and was primarily localized to the nucleus but was also detected in the cytoplasm and adjacent to the plasma membrane. Remarkably, nuclear Set-β suppressed, whereas Set-β localized to cytoplasmic membranes promoted neurite growth in rodent retinal ganglion cells and hippocampal neurons. Mimicking serine 9 phosphorylation, as found in Alzheimer's disease brains, delayed nuclear import and furthermore blocked the ability of nuclear Set-β to suppress neurite growth. We also present data on gene regulation and protein binding partner recruitment by Set-β in primary neurons, raising the hypothesis that nuclear Set-β may preferentially regulate gene expression whereas Set-β at cytoplasmic membranes may regulate unique cofactors, including PP2A, which we show also regulates axon growth in vitro. Finally, increasing recruitment of Set-β to cellular membranes promoted adult rat optic nerve axon regeneration after injury in vivo. Thus, Set-β differentially regulates axon growth and regeneration depending on subcellular localization and phosphorylation. PMID:24849368

Differential growth of pavement cells of Arabidopsis thaliana leaf epidermis as revealed by microbead labeling.

PubMed

Elsner, Joanna; Lipowczan, Marcin; Kwiatkowska, Dorota

2018-02-01

In numerous vascular plants, pavement cells of the leaf epidermis are shaped like a jigsaw-puzzle piece. Knowledge about the subcellular pattern of growth that accompanies morphogenesis of such a complex shape is crucial for studies of the role of the cytoskeleton, cell wall and phytohormones in plant cell development. Because the detailed growth pattern of the anticlinal and periclinal cell walls remains unknown, our aim was to measure pavement cell growth at a subcellular resolution. Using fluorescent microbeads applied to the surface of the adaxial leaf epidermis of Arabidopsis thaliana as landmarks for growth computation, we directly assessed the growth rates for the outer periclinal and anticlinal cell walls at a subcellular scale. We observed complementary tendencies in the growth pattern of the outer periclinal and anticlinal cell walls. Central portions of periclinal walls were characterized by relatively slow growth, while growth of the other wall portions was heterogeneous. Local growth of the periclinal walls accompanying lobe development after initiation was relatively fast and anisotropic, with maximal extension usually in the direction along the lobe axis. This growth pattern of the periclinal walls was complemented by the extension of the anticlinal walls, which was faster on the lobe sides than at the tips. Growth of the anticlinal and outer periclinal walls of leaf pavement cells is heterogeneous. The growth of the lobes resembles cell elongation via diffuse growth rather than tip growth. © 2018 Botanical Society of America.

An Automatic Segmentation Method Combining an Active Contour Model and a Classification Technique for Detecting Polycomb-group Proteinsin High-Throughput Microscopy Images.

PubMed

Gregoretti, Francesco; Cesarini, Elisa; Lanzuolo, Chiara; Oliva, Gennaro; Antonelli, Laura

2016-01-01

The large amount of data generated in biological experiments that rely on advanced microscopy can be handled only with automated image analysis. Most analyses require a reliable cell image segmentation eventually capable of detecting subcellular structures.We present an automatic segmentation method to detect Polycomb group (PcG) proteins areas isolated from nuclei regions in high-resolution fluorescent cell image stacks. It combines two segmentation algorithms that use an active contour model and a classification technique serving as a tool to better understand the subcellular three-dimensional distribution of PcG proteins in live cell image sequences. We obtained accurate results throughout several cell image datasets, coming from different cell types and corresponding to different fluorescent labels, without requiring elaborate adjustments to each dataset.

Subcellular Localization of Arabidopsis 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase1

PubMed Central

Leivar, Pablo; González, Víctor M.; Castel, Susanna; Trelease, Richard N.; López-Iglesias, Carmen; Arró, Montserrat; Boronat, Albert; Campos, Narciso; Ferrer, Albert; Fernàndez-Busquets, Xavier

2005-01-01

Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-μm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR-ER. Nevertheless, they represent a previously undescribed subcellular compartment likely capable of synthesizing mevalonate, which provides new evidence for multiorganelle compartmentalization of the isoprenoid biosynthetic pathways in plants. PMID:15618432

Subcellular fractionation by zonal centrifugation of glucose-repressed anaerobically grown Saccharomyces carlsbergensis

PubMed Central

Cartledge, T. G.; Lloyd, D.

1972-01-01

1. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucoserepressed Saccharomyces carlsbergensis, and the distributions of marker enzymes investigated after zonal centrifugation on sucrose gradients containing 2mm-MgCl2. 2. These homogenates contained no detectable cytochrome c oxidase, succinate–cytochrome c oxidoreductase, succinate–ferricyanide oxidoreductase, l(+)-lactate–cytochrome c oxidoreductase or catalase. Cytochromes a+a3 and c were not detected. 3. Zonal centrifugation of whole homogenates indicated complex density distributions of the sedimentable portions of NADH– and NADPH–cytochrome c oxidoreductases, adenosine triphosphatases (ATPases), adenosine pyrophosphatase (ADPase), pyrophosphatase and acid p-nitrophenyl phosphatase. Several different ATPases were distinguished on the basis of their sensitivities to oligomycin and ouabain. 4. Differential centrifugation of whole homogenates at 105g-min left 80–90% of the protein, dithionite-reducible cytochrome b, acid hydrolases and pyrophosphatase in a supernatant (S1) together with 65 and 56% of the NADH– and NADPH–cytochrome c oxidoreductases respectively, 25% of the ATPases and 71% of the adenosine monophosphatase. 5. Further analysis of supernatant S1 revealed the presence of a class of small particles containing NADPH–cytochrome c oxidoreductases and ATPases. 6. At least four different populations of large particles were distinguished. 7. Electron microscopy indicated that one of these corresponded to `promitochondria' as described by other workers. ImagesPLATE 1PLATE 2PLATE 3 PMID:4405573

Small glutamine-rich tetratricopeptide repeat-containing protein alpha is present in human ovaries but may not be differentially expressed in relation to polycystic ovary syndrome.

PubMed

Butler, Miriam S; Yang, Xing; Ricciardelli, Carmela; Liang, Xiaoyan; Norman, Robert J; Tilley, Wayne D; Hickey, Theresa E

2013-06-01

To evaluate the expression and function of small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), an androgen receptor (AR) molecular chaperone, in human ovarian tissues. Examine the effect of SGTA on AR subcellular localization in granulosa tumor cells (KGN) and SGTA expression in ovarian tissues. University-based research laboratory. Archived tissues from premenopausal women and granulosa cells from infertile women receiving assisted reproduction. None. AR subcellular localization and SGTA protein or mRNA levels. SGTA and AR proteins were expressed in the cytoplasm of KGN cells and exposure to androgen stimulated AR nuclear localization. SGTA protein knockdown increased AR nuclear localization at low (0-0.1 nmol/L) but not high (1-10 nmol/L) concentrations of androgen hormone. In ovarian tissues, SGTA was localized to the cytoplasm of granulosa cells at all stages of folliculogenesis and in thecal cells of antral follicles. SGTA protein levels were similar when comparing primordial and primary follicles within core biopsies (n = 40) from women with and without polycystic ovary syndrome (PCOS). Likewise, SGTA mRNA levels were not significantly different in granulosa cells from preovulatory follicles after hyperstimulation of women with and without PCOS. SGTA is present in human ovaries and has the potential to modulate AR signalling, but it may not be differentially expressed in PCOS. Copyright © 2013 American Society for Reproductive Medicine. All rights reserved.

Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

NASA Technical Reports Server (NTRS)

Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.; Evans, M. L. (Principal Investigator)

2003-01-01

Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

Selection for Cd Pollution-Safe Cultivars of Chinese Kale (Brassica alboglabra L. H. Bailey) and Biochemical Mechanisms of the Cultivar-Dependent Cd Accumulation Involving in Cd Subcellular Distribution.

PubMed

Guo, Jing-Jie; Tan, Xiao; Fu, Hui-Ling; Chen, Jing-Xin; Lin, Xiao-Xia; Ma, Yuan; Yang, Zhong-Yi

2018-02-28

Two pot experiments were conducted to compare and verify Cd accumulation capacities of different cultivars under Cd exposures (0.215, 0.543, and 0.925 mg kg -1 in Exp-1 and 0.143, 0.619, and 1.407 mg kg -1 in Exp-2) and Cd subcellular distributions between low- and high-Cd cultivars. Shoot Cd concentrations between the selected low- and high-Cd cultivars were 1.4-fold different and the results were reproducible. The proportions of Cd-in-cell-wall of shoots and roots were all higher in a typical low-Cd cultivar (DX102) than in a typical high-Cd cultivar (HJK), while those of Cd-in-chloroplast or Cd-in-trophoplast and Cd-in-membrane-and-organelle were opposite. The proportions of Cd-in-vacuoles-and-cytoplasm of roots in DX102 were always higher than in HJK under Cd stresses, while there was no clear pattern in those of shoots. These findings may help to reduce health risk of Cd from Chinese kale consumption and explained biochemical mechanisms of cultivar-dependent Cd accumulation among the species.

Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

PubMed Central

Zechmann, Bernd; Liou, Liang-Chun; Koffler, Barbara E; Horvat, Lucija; Tomašić, Ana; Fulgosi, Hrvoje; Zhang, Zhaojie

2011-01-01

Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H2O2) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress. PMID:22093747

Immunocytochemical analysis of the subcellular distribution of ferritin in Imperata cylindrica (L.) Raeuschel, an iron hyperaccumulator plant.

PubMed

de la Fuente, Vicenta; Rodríguez, Nuria; Amils, Ricardo

2012-05-01

Ferritin is of interest at the structural and functional level not only as storage for iron, a critical element, but also as a means to prevent cell damage produced by oxidative stress. The main objective of this work was to confirm by immunocytochemistry the presence and the subcellular distribution of the ferritin detected by Mösbauer spectroscopy in Imperata cylindrica, a plant which accumulates large amounts of iron. The localization of ferritin was performed in epidermal, parenchymal and vascular tissues of shoots and leaves of I. cylindrica. The highest density of immunolabeling in shoots appeared in the intracellular space of cell tissues, near the cell walls and in the cytoplasm. In leaves, ferritin was detected in the proximity of the dense network of the middle lamella of cell walls, following a similar path to that observed in shoots. Immunolabeling was also localized in chloroplasts. The abundance of immunogold labelling in mitochondria for I. cylindrica was rather low, probably because the study dealt with tissues from old plants. These results further expand the localization of ferritin in cell components other than chloroplasts and mitochondria in plants. Copyright © 2011 Elsevier GmbH. All rights reserved.

Drosophila Pelle phosphorylates Dichaete protein and influences its subcellular distribution in developing oocytes.

PubMed

Mutsuddi, Mousumi; Mukherjee, Ashim; Shen, Baohe; Manley, James L; Nambu, John R

2010-01-01

The Drosophila Dichaete gene encodes a member of the Sox family of high mobility group (HMG) domain proteins that have crucial gene regulatory functions in diverse developmental processes. The subcellular localization and transcriptional regulatory activities of Sox proteins can be regulated by several post-translational modifications. To identify genes that functionally interact with Dichaete, we undertook a genetic modifier screen based on a Dichaete gain-of-function phenotype in the adult eye. Mutations in several genes, including decapentaplegic, engrailed and pelle, behaved as dominant modifiers of this eye phenotype. Further analysis of pelle mutants revealed that loss of pelle function results in alterations in the distinctive cytoplasmic distribution of Dichaete protein within the developing oocyte, as well as defects in the elaboration of individual egg chambers. The death domain-containing region of the Pelle protein kinase was found to associate with both Dichaete and mouse Sox2 proteins, and Pelle can phosphorylate Dichaete protein in vitro. Overall, these findings reveal that maternal functions of pelle are essential for proper localization of Dichaete protein in the oocyte and normal egg chamber formation. Dichaete appears to be a novel phosphorylation substrate for Pelle and may function in a Pelle-dependent signaling pathway during oogenesis.

Muscle glycogen and cell function--Location, location, location.

PubMed

Ørtenblad, N; Nielsen, J

2015-12-01

The importance of glycogen, as a fuel during exercise, is a fundamental concept in exercise physiology. The use of electron microscopy has revealed that glycogen is not evenly distributed in skeletal muscle fibers, but rather localized in distinct pools. In this review, we present the available evidence regarding the subcellular localization of glycogen in skeletal muscle and discuss this from the perspective of skeletal muscle fiber function. The distribution of glycogen in the defined pools within the skeletal muscle varies depending on exercise intensity, fiber phenotype, training status, and immobilization. Furthermore, these defined pools may serve specific functions in the cell. Specifically, reduced levels of these pools of glycogen are associated with reduced SR Ca(2+) release, muscle relaxation rate, and membrane excitability. Collectively, the available literature strongly demonstrates that the subcellular localization of glycogen has to be considered to fully understand the role of glycogen metabolism and signaling in skeletal muscle function. Here, we propose that the effect of low muscle glycogen on excitation-contraction coupling may serve as a built-in mechanism, which links the energetic state of the muscle fiber to energy utilization. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[Study of gene mutation and pathogenetic mechanism for a family with Waardenburg syndrome].

PubMed

Chen, Hongsheng; Liao, Xinbin; Liu, Yalan; He, Chufeng; Zhang, Hua; Jiang, Lu; Feng, Yong; Mei, Lingyun

2017-08-10

To explore the pathogenetic mechanism of a family affected with Waardenburg syndrome. Clinical data of the family was collected. Potential mutation of the MITF, SOX10 and SNAI2 genes were screened. Plasmids for wild type (WT) and mutant MITF proteins were constructed to determine their exogenous expression and subcellular distribution by Western blotting and immunofluorescence assay, respectively. A heterozygous c.763C>T (p.R255X) mutation was detected in exon 8 of the MITF gene in the proband and all other patients from the family. No pathological mutation of the SOX10 and SNAI2 genes was detected. The DNA sequences of plasmids of MITF wild and mutant MITF R255X were confirmed. Both proteins were detected with the expected size. WT MITF protein only localized in the nucleus, whereas R255X protein showed aberrant localization in the nucleus as well as the cytoplasm. The c.763C>T mutation of the MITF gene probably underlies the disease in this family. The mutation can affect the subcellular distribution of MITF proteins in vitro, which may shed light on the molecular mechanism of Waardenburg syndrome caused by mutations of the MITF gene.

Enantiospecific toxicity, distribution and bioaccumulation of chiral antidepressant venlafaxine and its metabolite in loach (Misgurnus anguillicaudatus) co-exposed to microplastic and the drugs.

PubMed

Qu, Han; Ma, Ruixue; Wang, Bin; Yang, Jian; Duan, Lei; Yu, Gang

2018-04-22

In present study, we investigated the enantioselective behaviors of the chiral antidepressant venlafaxine and its metabolite O-desmethylvenlafaxine in loach Misgurnus anguillicaudatus (M. anguillicaudatus), as well as effects of microplastic on toxicity, distribution and metabolism through a 40-day co-exposure. The contents of SOD and MDA in loach liver elevated when the loach was exposed to venlafaxine and O-desmethylvenlafaxine. Moreover, co-exposure with microplastic might lead to more adverse effect against loach. The distribution of venlafaxine and O-desmethylvenlafaxine were both detected in loach tissues and liver subcellular. The concentrations of venlafaxine and O-desmethylvenlafaxine were lower in water in microplastic-present treatment. Whilst, more contaminants were accumulated in liver through the "vehicle" (microplastic). Enantioselective behavior of venlafaxine and O-desmethylvenlafaxine occurred with R-enantiomers being preferentially degraded. With microplastic present, the bioaccumulation factor (BAF) of venlafaxine and O-desmethylvenlafaxine in loach tissue amplified more than 10 times. In liver subcellular structure, microplastic may help to transport more compounds into subtle areas and postpone the contaminants metabolism in organisms. The present study for the first time gained an insight into the potential ecological effects and environmental behaviors of combined pollutions of chiral pharmaceuticals and microplastic, which could supply important information for environment risk assessment of concurrent organic pollutants and microplastic. Copyright © 2018 Elsevier B.V. All rights reserved.

Fundamental studies of adrenal retinoid-X-receptor: Protein isoform, tissue expression, subcellular distribution, and ligand availability.

PubMed

Cheng, Behling; Al-Shammari, Fatema H; Ghader, Isra'a A; Sequeira, Fatima; Thakkar, Jitendra; Mathew, Thazhumpal C

2017-07-01

Adrenal gland reportedly expresses many nuclear receptors that are known to heterodimerize with retinoid-X-receptor (RXR) for functions, but the information regarding the glandular RXR is not adequate. Studies of rat adrenal homogenate by Western blotting revealed three RXR proteins: RXRα (55kDa), RXRβ (47kDa) and RXR (56kDa). RXRγ was not detectable. After fractionation, RXRα was almost exclusively localized in the nuclear fraction. In comparison, substantial portions of RXRβ and RXR were found in both nuclear and post-nuclear particle fractions, suggesting genomic and non-genomic functions. Cells immunostained for RXRα were primarily localized in zona fasciculata (ZF) and medulla, although some stained cells were found in zona glomerulosa (ZG) and zona reticularis (ZR). In contrast, cells immunostained for RXRβ were concentrated principally in ZG, although some stained cells were seen in ZR, ZF, and medulla (in descending order, qualitatively). Analysis of adrenal lipid extracts by LC/MS did not detect 9-cis-retinoic acid (a potent RXR-ligand) but identified all-trans retinoic acid. Since C20 and C22 polyunsaturated fatty acids (PUFAs) can also activate RXR, subcellular availabilities of unesterified fatty acids were investigated by GC/MS. As results, arachidonic acid (C20:4), adrenic acid (C22:4), docosapentaenoic acid (C22:5), and cervonic acid (C22:6) were detected in the lipids extracted from each subcellular fraction. Thus, the RXR-agonizing PUFAs are available in all the main subcellular compartments considerably. The present findings not only shed light on the adrenal network of RXRs but also provide baseline information for further investigations of RXR heterodimers in the regulation of adrenal steroidogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

A graphene-based physiometer array for the analysis of single biological cells

PubMed Central

Paulus, Geraldine L. C.; Nelson, Justin T.; Lee, Katherine Y.; Wang, Qing Hua; Reuel, Nigel F.; Grassbaugh, Brittany R.; Kruss, Sebastian; Landry, Markita P.; Kang, Jeon Woong; Vander Ende, Emma; Zhang, Jingqing; Mu, Bin; Dasari, Ramachandra R.; Opel, Cary F.; Wittrup, K. Dane; Strano, Michael S.

2014-01-01

A significant advantage of a graphene biosensor is that it inherently represents a continuum of independent and aligned sensor-units. We demonstrate a nanoscale version of a micro-physiometer – a device that measures cellular metabolic activity from the local acidification rate. Graphene functions as a matrix of independent pH sensors enabling subcellular detection of proton excretion. Raman spectroscopy shows that aqueous protons p-dope graphene – in agreement with established doping trajectories, and that graphene displays two distinct pKa values (2.9 and 14.2), corresponding to dopants physi- and chemisorbing to graphene respectively. The graphene physiometer allows micron spatial resolution and can differentiate immunoglobulin (IgG)-producing human embryonic kidney (HEK) cells from non-IgG-producing control cells. Population-based analyses allow mapping of phenotypic diversity, variances in metabolic activity, and cellular adhesion. Finally we show this platform can be extended to the detection of other analytes, e.g. dopamine. This work motivates the application of graphene as a unique biosensor for (sub)cellular interrogation. PMID:25359450

Analyzing the spatial positioning of nuclei in polynuclear giant cells

NASA Astrophysics Data System (ADS)

Stange, Maike; Hintsche, Marius; Sachse, Kirsten; Gerhardt, Matthias; Valleriani, Angelo; Beta, Carsten

2017-11-01

How cells establish and maintain a well-defined size is a fundamental question of cell biology. Here we investigated to what extent the microtubule cytoskeleton can set a predefined cell size, independent of an enclosing cell membrane. We used electropulse-induced cell fusion to form giant multinuclear cells of the social amoeba Dictyostelium discoideum. Based on dual-color confocal imaging of cells that expressed fluorescent markers for the cell nucleus and the microtubules, we determined the subcellular distributions of nuclei and centrosomes in the giant cells. Our two- and three-dimensional imaging results showed that the positions of nuclei in giant cells do not fall onto a regular lattice. However, a comparison with model predictions for random positioning showed that the subcellular arrangement of nuclei maintains a low but still detectable degree of ordering. This can be explained by the steric requirements of the microtubule cytoskeleton, as confirmed by the effect of a microtubule degrading drug.

Trehalose Alters Subcellular Trafficking and the Metabolism of the Alzheimer-associated Amyloid Precursor Protein*

PubMed Central

Tien, Nguyen T.; Karaca, Ilker; Tamboli, Irfan Y.

2016-01-01

The disaccharide trehalose is commonly considered to stimulate autophagy. Cell treatment with trehalose could decrease cytosolic aggregates of potentially pathogenic proteins, including mutant huntingtin, α-synuclein, and phosphorylated tau that are associated with neurodegenerative diseases. Here, we demonstrate that trehalose also alters the metabolism of the Alzheimer disease-related amyloid precursor protein (APP). Cell treatment with trehalose decreased the degradation of full-length APP and its C-terminal fragments. Trehalose also reduced the secretion of the amyloid-β peptide. Biochemical and cell biological experiments revealed that trehalose alters the subcellular distribution and decreases the degradation of APP C-terminal fragments in endolysosomal compartments. Trehalose also led to strong accumulation of the autophagic marker proteins LC3-II and p62, and decreased the proteolytic activation of the lysosomal hydrolase cathepsin D. The combined data indicate that trehalose decreases the lysosomal metabolism of APP by altering its endocytic vesicular transport. PMID:26957541

INTRACELLULAR DISTRIBUTION OF CALCIUM IN DEVELOPING BREAST MUSCLE OF NORMAL AND DYSTROPHIC CHICKENS

PubMed Central

Cosmos, Ethel

1964-01-01

To follow the intracellular distribution of calcium in the breast muscles of developing chickens, Ca45 was injected into the albumen of predeveloped eggs. Since the embryos were grown in a radioactive medium, a complete exchange of the isotope for its non-radioactive counterpart in muscles was accomplished. Subcellular particulates of the muscle cells were separated by the method of differential centrifugation. Analysis of the separated fractions showed that in the muscles of the 13-day embryo, when the nuclear-myofibrillar ratio is high, 65 per cent of the muscle calcium is in the nuclei. With the increased synthesis of myofibrils, the nuclear-myofibrillar ratio decreases with a concomitant fall in radioactivity. Thus, calcium was not associated with the developing myofibrils. At the time of hatching, when myofibrils perform physiological work, the highest level of calcium is in the mitochondria. This suggests that the mitochondria play a key role in the physiological activities of calcium in the cell. The microsomal fraction reaches a maximal level of calcium when the adult composition of muscle is attained. Results of investigations on dystrophic muscles show changes in the calcium distribution of the fractions as early as the 3rd week of embryonic development, which are interpreted to indicate an alteration in the protein metabolism of the cell, or an early destruction of muscle tissue. Further, alterations in the calcium content of fractions which seem to regulate the movements of this ion in the cell are discussed. A new technique for homogenizing tissues from embryos of different ages is presented. PMID:14222812

Subcellular Redox Signaling.

PubMed

Zhu, Liping; Lu, Yankai; Zhang, Jiwei; Hu, Qinghua

2017-01-01

Oxidative and antioxidative system of cells and tissues maintains a balanced state under physiological conditions. A disruption in this balance of redox status has been associated with numerous pathological processes. Reactive oxygen species (ROS) as a major redox signaling generates in a spatiotemporally dependent manner. Subcellular organelles such as mitochondria, endoplasmic reticulum, plasma membrane and nuclei contribute to the production of ROS. In addition to downstream effects of ROS signaling regulated by average ROS changes in cytoplasm, whether subcelluar ROS mediate biological effect(s) has drawn greater attentions. With the advance in redox-sensitive probes targeted to different subcellular compartments, the investigation of subcellular ROS signaling and its associated cellular function has become feasible. In this review, we discuss the subcellular ROS signaling, with particular focus on mechanisms of subcellular ROS production and its downstream effects.

Regulation of Sterol Content in Membranes by Subcellular Compartmentation of Steryl-Esters Accumulating in a Sterol-Overproducing Tobacco Mutant.

PubMed Central

Gondet, L.; Bronner, R.; Benveniste, P.

1994-01-01

The study of sterol overproduction in tissues of LAB 1-4 mutant tobacco (Nicotiana tabacum L. cv Xanthi) (P. Maillot-Vernier, H. Schaller, P. Benveniste, G. Belliard [1989] Biochem Biophys Res Commun 165: 125-130) over several generations showed that the overproduction phenotype is stable in calli, with a 10-fold stimulation of sterol content when compared with wild-type calli. However, leaves of LAB 1-4 plants obtained after two steps of self-fertilization were characterized by a mere 3-fold stimulation, whereas calli obtained from these plants retained a typical sterol-overproducing mutant phenotype (i.e. a 10-fold increase of sterol content). These results suggest that the expression of the LAB 1-4 phenotype is dependent on the differentiation state of cells. Most of the sterols accumulating in the mutant tissues were present as steryl-esters, which were minor species in wild-type tissues. Subcellular fractionation showed that in both mutant and wild-type tissues, free sterols were associated mainly with microsomal membranes. In contrast, the bulk of steryl-esters present in mutant tissues was found in the soluble fraction of cells. Numerous lipid droplets were detected in the hyaloplasm of LAB 1-4 cells by cytochemical and cytological techniques. After isolation, these lipid granules were shown to contain steryl-esters. These results show that the overproduced sterols of mutant tissues accumulate as steryl-esters in hyaloplasmic bodies. The esterification process thus allows regulation of the amount of free sterols in membranes by subcellular compartmentation. PMID:12232218

New insights into globoids of protein storage vacuoles in wheat aleurone using synchrotron soft X-ray microscopy

PubMed Central

Regvar, Marjana; Eichert, Diane; Kaulich, Burkhard; Gianoncelli, Alessandra; Pongrac, Paula; Vogel-Mikuš, Katarina; Kreft, Ivan

2011-01-01

Mature developed seeds are physiologically and biochemically committed to store nutrients, principally as starch, protein, oils, and minerals. The composition and distribution of elements inside the aleurone cell layer reflect their biogenesis, structural characteristics, and physiological functions. It is therefore of primary importance to understand the mechanisms underlying metal ion accumulation, distribution, storage, and bioavailability in aleurone subcellular organelles for seed fortification purposes. Synchrotron radiation soft X-ray full-field imaging mode (FFIM) and low-energy X-ray fluorescence (LEXRF) spectromicroscopy were applied to characterize major structural features and the subcellular distribution of physiologically important elements (Zn, Fe, Na, Mg, Al, Si, and P). These direct imaging methods reveal the accumulation patterns between the apoplast and symplast, and highlight the importance of globoids with phytic acid mineral salts and walls as preferential storage structures. C, N, and O chemical topographies are directly linked to the structural backbone of plant substructures. Zn, Fe, Na, Mg, Al, and P were linked to globoid structures within protein storage vacuoles with variable levels of co-localization. Si distribution was atypical, being contained in the aleurone apoplast and symplast, supporting a physiological role for Si in addition to its structural function. These results reveal that the immobilization of metals within the observed endomembrane structures presents a structural and functional barrier and affects bioavailability. The combination of high spatial and chemical X-ray microscopy techniques highlights how in situ analysis can yield new insights into the complexity of the wheat aleurone layer, whose precise biochemical composition, morphology, and structural characteristics are still not unequivocally resolved. PMID:21447756

Distribution and Function of HCN Channels in the Apical Dendritic Tuft of Neocortical Pyramidal Neurons

PubMed Central

Harnett, Mark T.; Magee, Jeffrey C.

2015-01-01

The apical tuft is the most remote area of the dendritic tree of neocortical pyramidal neurons. Despite its distal location, the apical dendritic tuft of layer 5 pyramidal neurons receives substantial excitatory synaptic drive and actively processes corticocortical input during behavior. The properties of the voltage-activated ion channels that regulate synaptic integration in tuft dendrites have, however, not been thoroughly investigated. Here, we use electrophysiological and optical approaches to examine the subcellular distribution and function of hyperpolarization-activated cyclic nucleotide-gated nonselective cation (HCN) channels in rat layer 5B pyramidal neurons. Outside-out patch recordings demonstrated that the amplitude and properties of ensemble HCN channel activity were uniform in patches excised from distal apical dendritic trunk and tuft sites. Simultaneous apical dendritic tuft and trunk whole-cell current-clamp recordings revealed that the pharmacological blockade of HCN channels decreased voltage compartmentalization and enhanced the generation and spread of apical dendritic tuft and trunk regenerative activity. Furthermore, multisite two-photon glutamate uncaging demonstrated that HCN channels control the amplitude and duration of synaptically evoked regenerative activity in the distal apical dendritic tuft. In contrast, at proximal apical dendritic trunk and somatic recording sites, the blockade of HCN channels decreased excitability. Dynamic-clamp experiments revealed that these compartment-specific actions of HCN channels were heavily influenced by the local and distributed impact of the high density of HCN channels in the distal apical dendritic arbor. The properties and subcellular distribution pattern of HCN channels are therefore tuned to regulate the interaction between integration compartments in layer 5B pyramidal neurons. PMID:25609619

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sumrejkanchanakij, Piyamas; Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330; Eto, Kazuhiro

2006-02-03

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, amore » process that was inhibited by p16{sup INK4a}, a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.« less

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells.

PubMed

Sumrejkanchanakij, Piyamas; Eto, Kazuhiro; Ikeda, Masa-Aki

2006-02-03

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.

Tight regulation of a timed nuclear import wave of EKLF by PKCθ and FOE during Pro-E to Baso-E transition.

PubMed

Shyu, Yu-Chiau; Lee, Tung-Liang; Chen, Xin; Hsu, Pang-Hung; Wen, Shau-Ching; Liaw, Yi-Wei; Lu, Chi-Huan; Hsu, Po-Yen; Lu, Mu-Jie; Hwang, JauLang; Tsai, Ming-Daw; Hwang, Ming-Jing; Chen, Jim-Ray; Shen, Che-Kun James

2014-02-24

Erythropoiesis is a highly regulated process during which BFU-E are differentiated into RBCs through CFU-E, Pro-E, PolyCh-E, OrthoCh-E, and reticulocyte stages. Uniquely, most erythroid-specific genes are activated during the Pro-E to Baso-E transition. We show that a wave of nuclear import of the erythroid-specific transcription factor EKLF occurs during the Pro-E to Baso-E transition. We further demonstrate that this wave results from a series of finely tuned events, including timed activation of PKCθ, phosphorylation of EKLF at S68 by P-PKCθ(S676), and sumoylation of EKLF at K74. The latter EKLF modifications modulate its interactions with a cytoplasmic ankyrin-repeat-protein FOE and importinβ1, respectively. The role of FOE in the control of EKLF nuclear import is further supported by analysis of the subcellular distribution patterns of EKLF in FOE-knockout mice. This study reveals the regulatory mechanisms of the nuclear import of EKLF, which may also be utilized in the nuclear import of other factors. Copyright © 2014 Elsevier Inc. All rights reserved.

Cell death monitoring using quantitative optical coherence tomography methods

NASA Astrophysics Data System (ADS)

Farhat, Golnaz; Yang, Victor X. D.; Kolios, Michael C.; Czarnota, Gregory J.

2011-03-01

Cell death is characterized by a series of predictable morphological changes, which modify the light scattering properties of cells. We present a multi-parametric approach to detecting changes in subcellular morphology related to cell death using optical coherence tomography (OCT). Optical coherence tomography data were acquired from acute myeloid leukemia (AML) cells undergoing apoptosis over a period of 48 hours. Integrated backscatter (IB) and spectral slope (SS) were computed from OCT backscatter spectra and statistical parameters were extracted from a generalized gamma (GG) distribution fit to OCT signal intensity histograms. The IB increased by 2-fold over 48 hours with significant increases observed as early as 4 hours. The SS increased in steepness by 2.5-fold with significant changes at 12 hours, while the GG parameters were sensitive to apoptotic changes at 24 to 48 hours. Histology slides indicated nuclear condensation and fragmentation at 24 hours, suggesting the late scattering changes could be related to nuclear structure. A second series of measurements from AML cells treated with cisplatin, colchicine or ionizing radiation suggested that the GG parameters could potentially differentiate between modes of cell death. Distinct cellular morphology was observed in histology slides obtained from cells treated under each condition.

Inter-kingdom prediction certainty evaluation of protein subcellular localization tools: microbial pathogenesis approach for deciphering host microbe interaction.

PubMed

Khan, Abdul Arif; Khan, Zakir; Kalam, Mohd Abul; Khan, Azmat Ali

2018-01-01

Microbial pathogenesis involves several aspects of host-pathogen interactions, including microbial proteins targeting host subcellular compartments and subsequent effects on host physiology. Such studies are supported by experimental data, but recent detection of bacterial proteins localization through computational eukaryotic subcellular protein targeting prediction tools has also come into practice. We evaluated inter-kingdom prediction certainty of these tools. The bacterial proteins experimentally known to target host subcellular compartments were predicted with eukaryotic subcellular targeting prediction tools, and prediction certainty was assessed. The results indicate that these tools alone are not sufficient for inter-kingdom protein targeting prediction. The correct prediction of pathogen's protein subcellular targeting depends on several factors, including presence of localization signal, transmembrane domain and molecular weight, etc., in addition to approach for subcellular targeting prediction. The detection of protein targeting in endomembrane system is comparatively difficult, as the proteins in this location are channelized to different compartments. In addition, the high specificity of training data set also creates low inter-kingdom prediction accuracy. Current data can help to suggest strategy for correct prediction of bacterial protein's subcellular localization in host cell. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Subcellular TSC22D4 localization in cerebellum granule neurons of the mouse depends on development and differentiation.

PubMed

Canterini, Sonia; Bosco, Adriana; Carletti, Valentina; Fuso, Andrea; Curci, Armando; Mangia, Franco; Fiorenza, Maria Teresa

2012-03-01

We previously demonstrated that TSC22D4, a protein encoded by the TGF-β1-activated gene Tsc22d4 (Thg-1pit) and highly expressed in postnatal and adult mouse cerebellum with multiple post-translationally modified protein forms, moves to nucleus when in vitro differentiated cerebellum granule neurons (CGNs) are committed to apoptosis by hyperpolarizing KCl concentrations in the culture medium. We have now studied TSC22D4 cytoplasmic/nuclear localization in CGNs and Purkinje cells: (1) during CGN differentiation/maturation in vivo, (2) during CGN differentiation in vitro, and (3) by in vitro culturing ex vivo cerebellum slices under conditions favoring/inhibiting CGN/Purkinje cell differentiation. We show that TSC22D4 displays both nuclear and cytoplasmic localizations in undifferentiated, early postnatal cerebellum CGNs, irrespectively of CGN proliferation/migration from external to internal granule cell layer, and that it specifically accumulates in the somatodendritic and synaptic compartments when CGNs mature, as indicated by TSC22D4 abundance at the level of adult cerebellum glomeruli and apparent lack in CGN nuclei. These features were also observed in cerebellum slices cultured in vitro under conditions favoring/inhibiting CGN/Purkinje cell differentiation. In vitro TSC22D4 silencing with siRNAs blocked CGN differentiation and inhibited neurite elongation in N1E-115 neuroblastoma cells, pinpointing the relevance of this protein to CGN differentiation.

Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging.

PubMed

Wang, Chuangqi; Choi, Hee June; Kim, Sung-Jin; Desai, Aesha; Lee, Namgyu; Kim, Dohoon; Bae, Yongho; Lee, Kwonmoo

2018-04-27

Cell protrusion is morphodynamically heterogeneous at the subcellular level. However, the mechanism of cell protrusion has been understood based on the ensemble average of actin regulator dynamics. Here, we establish a computational framework called HACKS (deconvolution of heterogeneous activity in coordination of cytoskeleton at the subcellular level) to deconvolve the subcellular heterogeneity of lamellipodial protrusion from live cell imaging. HACKS identifies distinct subcellular protrusion phenotypes based on machine-learning algorithms and reveals their underlying actin regulator dynamics at the leading edge. Using our method, we discover "accelerating protrusion", which is driven by the temporally ordered coordination of Arp2/3 and VASP activities. We validate our finding by pharmacological perturbations and further identify the fine regulation of Arp2/3 and VASP recruitment associated with accelerating protrusion. Our study suggests HACKS can identify specific subcellular protrusion phenotypes susceptible to pharmacological perturbation and reveal how actin regulator dynamics are changed by the perturbation.

Echinococcus granulosus fatty acid binding proteins subcellular localization.

PubMed

Alvite, Gabriela; Esteves, Adriana

2016-05-01

Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Cellular redistribution of Rad51 in response to DNA damage: novel role for Rad51C.

PubMed

Gildemeister, Otto S; Sage, Jay M; Knight, Kendall L

2009-11-13

Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damage-induced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damage-induced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.

A steady-state model of spreading depression predicts the importance of an unknown conductance in specific dendritic domains.

PubMed

Makarova, Julia; Ibarz, José M; Canals, Santiago; Herreras, Oscar

2007-06-15

Spreading depression (SD) is a pathological wave of transient neuronal inactivation. We recently reported that the characteristic sustained complete depolarization is restricted to specific cell domains where the input resistance (R(in)) first becomes negligible before achieving partial recovery, whereas in adjacent, more polarized membranes it drops by much less. The experimental study of the participating membrane channels is hindered by their mixed contribution and heterogeneous distribution. Therefore, we derived a biophysical model to analyze the conductances that replicate the subcellular profile of R(in) during SD. Systematic variation of conductance densities far beyond the ranges reported failed to fit the experimental values. Besides standard potassium, sodium, and Glu-mediated conductances, the initial opening and gradual closing of an as yet undetermined large conductance is required to account for the evolution of R(in). Potassium conductances follow in the relative contribution and their closing during the late phase is also predicted. Large intracellular potential gradients from zero to rest are readily sustained between shunted and adjacent SD-spared membranes, which remain electroregenerative. The gradients are achieved by a combination of high-conductance subcellular domains and transmembrane ion redistribution in extended but discrete dendritic domains. We conclude that the heterogeneous subcellular behavior is due to local membrane properties, some of which may be specifically activated under extreme SD conditions.

PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha.

PubMed

Wahl, Andreas; Schuth, Nora; Pfeiffer, Daniel; Nussberger, Stephan; Jendrossek, Dieter

2012-11-16

Poly(3-hydroxybutyrate) (PHB) granules are important storage compounds of carbon and energy in many prokaryotes which allow survival of the cells in the absence of suitable carbon sources. Formation and subcellular localization of PHB granules was previously assumed to occur randomly in the cytoplasm of PHB accumulating bacteria. However, contradictionary results on subcellular localization of PHB granules in Ralstonia eutropha were published, recently. Here, we provide evidence by transmission electron microscopy that PHB granules are localized in close contact to the nucleoid region in R. eutropha during growth on nutrient broth. Binding of PHB granules to the nucleoid is mediated by PhaM, a PHB granule associated protein with phasin-like properties that is also able to bind to DNA and to phasin PhaP5. Over-expression of PhaM resulted in formation of many small PHB granules that were always attached to the nucleoid region. In contrast, PHB granules of ∆phaM strains became very large and distribution of granules to daughter cells was impaired. Association of PHB granules to the nucleoid region was prevented by over-expression of PhaP5 and clusters of several PHB granules were mainly localized near the cell poles. Subcellular localization of PHB granules is controlled in R. eutropha and depends on the presence and concentrations of at least two PHB granule associated proteins, PhaM and PhaP5.

Internal distribution of Cd in lettuce and resulting effects on Cd trophic transfer to the snail: Achatina fulica.

PubMed

Li, Cheng-Cheng; Dang, Fei; Cang, Long; Zhou, Dong-Mei; Peijnenburg, Willie J G M

2015-09-01

The mechanisms underlying Cd trophic transfer along the soil-lettuce-snail food chain were investigated. The fate of Cd within cells, revealed by assessment of Cd chemical forms and of subcellular partitioning, differed between the two examined lettuce species that we examined (L. longifolia and L. crispa). The species-specific internal Cd fate not only influenced Cd burdens in lettuce, with higher Cd levels in L. crispa, but also affected Cd transfer efficiency to the consumer snail (Achatina fulica). Especially, the incorporation of Cd chemical forms (Cd in the inorganic, water-soluble and pectates and protein-integrated forms) in lettuce could best explain Cd trophic transfer, when compared to dietary Cd levels alone and/or subcellular Cd partitioning. Trophically available metal on the subcellular partitioning base failed to shed light on Cd transfer in this study. After 28-d of exposure, most Cd was trapped in the viscera of Achatina fulica, and cadmium bio-magnification was noted in the snails, as the transfer factor of lettuce-to-snail soft tissue was larger than one. This study provides a first step to apply a chemical speciation approach to dictate the trophic bioavailability of Cd through the soil-plant-snail system, which might be an important pre-requisite for mechanistic understanding of metal trophic transfer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of human cysteine-rich secretory protein 3 (CRISP-3) as a matrix protein in a subset of peroxidase-negative granules of neutrophils and in the granules of eosinophils.

PubMed

Udby, Lene; Calafat, Jero; Sørensen, Ole E; Borregaard, Niels; Kjeldsen, Lars

2002-09-01

Cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) was originally discovered in human neutrophilic granulocytes. We have recently developed a sensitive sandwich enzyme-linked immunosorbent assay for CRISP-3 and demonstrated the presence of CRISP-3 in exocrine secretions. To investigate the subcellular localization and mobilization of CRISP-3 in human neutrophils, we performed subcellular fractionation of resting and activated neutrophils on three-layer Percoll density gradients, release-studies of granule proteins in response to different secretagogues, and double-labeling immunogold electron microscopy. CRISP-3 was found to be localized in a subset of granules with overlapping characteristics of specific and gelatinase granules and mobilized accordingly, thus confirming the hypothesis that peroxidase-negative granules exist as a continuum from specific to gelatinase granules regarding protein content and mobilization. CRISP-3 was found to be a matrix protein, which is stored in granules as glycosylated and as unglycosylated protein. The subcellular distribution of the two forms of CRISP-3 was identical. In addition, CRISP-3 was found as a granule protein in eosinophilic granulocytes. The presence of CRISP-3 in peroxidase-negative granules of neutrophils, in granules of eosinophils, and in exocrine secretions indicates a role in the innate host defense.

Phosphorylation-dependent trafficking of plasma membrane proteins in animal and plant cells.

PubMed

Offringa, Remko; Huang, Fang

2013-09-01

In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples. © 2013 Institute of Botany, Chinese Academy of Sciences.

Subcellular distribution of uranium in the roots of Spirodela punctata and surface interactions

NASA Astrophysics Data System (ADS)

Nie, Xiaoqin; Dong, Faqin; Liu, Ning; Liu, Mingxue; Zhang, Dong; Kang, Wu; Sun, Shiyong; Zhang, Wei; Yang, Jie

2015-08-01

The subcellular distribution of uranium in roots of Spirodela punctata (duckweed) and the process of surface interaction were studied upon exposure to U (0, 5-200 mg/L) at pH 5. The concentration of uranium in each subcelluar fraction increased significantly with increasing solution U level, after 200 mg/L uranium solution treatment 120 h, the proportion of uranium concentration approximate as 8:2:1 in the cell wall organelle and cytosol fractions of roots of S. punctata. OM SEM and EDS showed after 5-200 mg/L U treatment 4-24 h, some intracellular fluid released from the root cells, after 100 mg/L U treatment 48 h, the particles including 35% Fe (wt%) and other organic matters such as EPS released from the cells, most of the uranium bound onto the root surface and contacted with phosphorus ligands and formed as nano-scales U-P lamellar crystal, similar crystal has been found in the cell wall and organelle fractions after 50 mg/L U treatment 120 h. FTIR and XPS analyses result indicates the uranium changed the band position and shapes of phosphate group, and the region of characteristic peak belongs to U(VI) and U(IV) were also observed.

Cloning and expression of hepatic synaptotagmin 1 in mouse.

PubMed

Sancho-Knapik, Sara; Guillén, Natalia; Osada, Jesús

2015-05-15

Mouse hepatic synaptotagmin 1 (SYT1) cDNA was cloned, characterized and compared to the brain one. The hepatic transcript was 1807 bp in length, smaller than the brain, and only encoded by 9 of 11 gene exons. In this regard, 5'-and 3'-untranslated regions were 66 and 476 bp, respectively; the open reading frame of 1266 bp codified for a protein of 421 amino acids, identical to the brain, with a predicted molecular mass of 47.4 kDa and highly conserved across different species. Immunoblotting of protein showed two isoforms of higher molecular masses than the theoretical prediction based on amino acid sequence suggesting posttranslational modifications. Subcellular distribution of protein isoforms corresponded to plasma membrane, lysosomes and microsomes and was identical between the brain and liver. Nonetheless, the highest molecular weight isoform was smaller in the liver, irrespective of subcellular location. Quantitative mRNA tissue distribution showed that it was widely expressed and that the highest values corresponded to the brain, followed by the liver, spleen, abdominal fat, intestine and skeletal muscle. These findings indicate tissue-specific splicing of the gene and posttranslational modification and the variation in expression in the different tissues might suggest a different requirement of SYT1 for the specific function in each organ. Copyright © 2015 Elsevier B.V. All rights reserved.

Dietary toxicity of field-contaminated invertebrates to marine fish: effects of metal doses and subcellular metal distribution.

PubMed

Dang, Fei; Rainbow, Philip S; Wang, Wen-Xiong

2012-09-15

There is growing awareness of the toxicological effects of metal-contaminated invertebrate diets on the health of fish populations in metal-contaminated habitats, yet the mechanisms underlying metal bioaccumulation and toxicity are complex. In the present study, marine fish Terapon jurbua terepon were fed a commercial diet supplemented with specimens of the polychaete Nereis diversicolor or the clam Scrobicularia plana, collected from four metal-impacted estuaries (Tavy, Restronguet Creek, West Looe, Gannel) in southwest England, as environmentally realistic metal sources. A comparative toxicological evaluation of both invertebrates showed that fish fed S. plana for 21 d exhibited evident mortality compared to those fed N. diversicolor. Furthermore, a spatial effect on mortality was observed. Differences in metal doses rather than subcellular metal distributions between N. diversicolor and S. plana appeared to be the cause of such different mortalities. Partial least squares regression was used to evaluate the statistical relationship between multiple-metal doses and fish mortality, revealing that Pb, Fe, Cd and Zn in field-collected invertebrates co-varied most strongly with the observed mortality. This study provides a step toward exploring the underlying mechanism of dietary toxicity and identifying the potential causality in complex metal mixture exposures in the field. Copyright © 2012 Elsevier B.V. All rights reserved.

Accurate prediction of subcellular location of apoptosis proteins combining Chou's PseAAC and PsePSSM based on wavelet denoising.

PubMed

Yu, Bin; Li, Shan; Qiu, Wen-Ying; Chen, Cheng; Chen, Rui-Xin; Wang, Lei; Wang, Ming-Hui; Zhang, Yan

2017-12-08

Apoptosis proteins subcellular localization information are very important for understanding the mechanism of programmed cell death and the development of drugs. The prediction of subcellular localization of an apoptosis protein is still a challenging task because the prediction of apoptosis proteins subcellular localization can help to understand their function and the role of metabolic processes. In this paper, we propose a novel method for protein subcellular localization prediction. Firstly, the features of the protein sequence are extracted by combining Chou's pseudo amino acid composition (PseAAC) and pseudo-position specific scoring matrix (PsePSSM), then the feature information of the extracted is denoised by two-dimensional (2-D) wavelet denoising. Finally, the optimal feature vectors are input to the SVM classifier to predict subcellular location of apoptosis proteins. Quite promising predictions are obtained using the jackknife test on three widely used datasets and compared with other state-of-the-art methods. The results indicate that the method proposed in this paper can remarkably improve the prediction accuracy of apoptosis protein subcellular localization, which will be a supplementary tool for future proteomics research.

Accurate prediction of subcellular location of apoptosis proteins combining Chou’s PseAAC and PsePSSM based on wavelet denoising

PubMed Central

Chen, Cheng; Chen, Rui-Xin; Wang, Lei; Wang, Ming-Hui; Zhang, Yan

2017-01-01

Apoptosis proteins subcellular localization information are very important for understanding the mechanism of programmed cell death and the development of drugs. The prediction of subcellular localization of an apoptosis protein is still a challenging task because the prediction of apoptosis proteins subcellular localization can help to understand their function and the role of metabolic processes. In this paper, we propose a novel method for protein subcellular localization prediction. Firstly, the features of the protein sequence are extracted by combining Chou's pseudo amino acid composition (PseAAC) and pseudo-position specific scoring matrix (PsePSSM), then the feature information of the extracted is denoised by two-dimensional (2-D) wavelet denoising. Finally, the optimal feature vectors are input to the SVM classifier to predict subcellular location of apoptosis proteins. Quite promising predictions are obtained using the jackknife test on three widely used datasets and compared with other state-of-the-art methods. The results indicate that the method proposed in this paper can remarkably improve the prediction accuracy of apoptosis protein subcellular localization, which will be a supplementary tool for future proteomics research. PMID:29296195

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oi, Ami; Katayama, Syouichi; Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Shiga, 525-8577

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed amore » typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. - Highlights: • We investigated the mechanism regulating subcellular localization of CDKL5. • DYRK1A was identified as an enzyme that bound to and phosphorylated CDKL5. • The phosphorylation site of CDKL5 was Ser-308, in the vicinity of the NLS. • When DYRK1A was co-expressed, the cytosolic CDKL5 was significantly increased. • In conclusion, DYRK1A regulates CDKL5 localization via phosphorylation on Ser-308.« less

Integration of Cadmium Accumulation, Subcellular Distribution, and Physiological Responses to Understand Cadmium Tolerance in Apple Rootstocks

PubMed Central

Zhou, Jiangtao; Wan, Huixue; He, Jiali; Lyu, Deguo; Li, Huifeng

2017-01-01

Cadmium (Cd) is a nonessential and highly toxic element causing agricultural problems. However, little information is available about the variation in Cd tolerance among apple rootstocks and its underlying physiological regulation mechanisms. This study investigated Cd accumulation, subcellular distribution, and chemical forms as well as physiological changes among four apple rootstocks exposed to either 0 or 300 μM CdCl2. The results showed that variations in Cd tolerance existed among these rootstocks. Cd exposure caused decline in photosynthesis, chlorophyll and biomass in four apple rootstocks, which was less pronounced in M. baccata, indicating its higher Cd tolerance. This finding was corroborated with higher Cd tolerance indexes (TIs) of the whole plant in M. baccata than those in the other three apple rootstocks. Among the four apple rootstocks, M. baccata displayed the lowest Cd concentrations in roots, wood, and leaves, the smallest total Cd amounts as well as the lowest BCF. In apple rootstocks, it was found that to immobilize Cd in cell wall and soluble fraction (most likely in vacuole) and to convert it into pectate- or protein- integrated forms and undissolved Cd phosphate forms may be the primary strategies to reduce Cd mobility and toxicity. The physiological changes including ROS, carbohydrates and antioxidants were in line with the variations of Cd tolerance among four apple rootstocks. In comparison with the other three apple rootstocks, M. baccata had lower concentrations of ROS in roots and bark, H2O2 in roots and leaves and MDA in roots, wood and bark, but higher concentrations of soluble sugars in bark and starch in roots and leaves, and enhanced antioxidants. These results indicate that M. baccata are more tolerant to Cd stress than the other three apple rootstocks under the current experiment conditions, which is probably related to Cd accumulation, subcellular partitioning and chemical forms of Cd and well-coordinated antioxidant defense mechanisms. PMID:28638400

Designer nanoparticle: nanobiotechnology tool for cell biology

NASA Astrophysics Data System (ADS)

Thimiri Govinda Raj, Deepak B.; Khan, Niamat Ali

2016-09-01

This article discusses the use of nanotechnology for subcellular compartment isolation and its application towards subcellular omics. This technology review significantly contributes to our understanding on use of nanotechnology for subcellular systems biology. Here we elaborate nanobiotechnology approach of using superparamagnetic nanoparticles (SPMNPs) optimized with different surface coatings for subcellular organelle isolation. Using pulse-chase approach, we review that SPMNPs interacted differently with the cell depending on its surface functionalization. The article focuses on the use of functionalized-SPMNPs as a nanobiotechnology tool to isolate high quality (both purity and yield) plasma membranes and endosomes or lysosomes. Such nanobiotechnology tool can be applied in generating subcellular compartment inventories. As a future perspective, this strategy could be applied in areas such as immunology, cancer and stem cell research.

Designer nanoparticle: nanobiotechnology tool for cell biology.

PubMed

Thimiri Govinda Raj, Deepak B; Khan, Niamat Ali

2016-01-01

This article discusses the use of nanotechnology for subcellular compartment isolation and its application towards subcellular omics. This technology review significantly contributes to our understanding on use of nanotechnology for subcellular systems biology. Here we elaborate nanobiotechnology approach of using superparamagnetic nanoparticles (SPMNPs) optimized with different surface coatings for subcellular organelle isolation. Using pulse-chase approach, we review that SPMNPs interacted differently with the cell depending on its surface functionalization. The article focuses on the use of functionalized-SPMNPs as a nanobiotechnology tool to isolate high quality (both purity and yield) plasma membranes and endosomes or lysosomes. Such nanobiotechnology tool can be applied in generating subcellular compartment inventories. As a future perspective, this strategy could be applied in areas such as immunology, cancer and stem cell research.

Expression of c-Fes protein isoforms correlates with differentiation in myeloid leukemias.

PubMed

Carlson, Anne; Berkowitz, Jeanne McAdara; Browning, Damaris; Slamon, Dennis J; Gasson, Judith C; Yates, Karen E

2005-05-01

The cellular fes gene encodes a 93-kilodalton protein-tyrosine kinase (p93) that is expressed in both normal and neoplastic myeloid cells. Increased c-Fes expression is associated with differentiation in normal myeloid cells and cell lines. Our hypothesis was that primary leukemia cells would show a similar pattern of increased expression in more differentiated cells. Therefore, we compared c-Fes expression in cells with an undifferentiated, blast phenotype (acute myelogenous leukemia--AML) to cells with a differentiated phenotype (chronic myelogenous leukemia--CML). Instead of differences in p93 expression levels, we found complex patterns of c-Fes immunoreactive proteins that corresponded with differentiation in normal and leukemic myeloid cells. The "blast" pattern consisted of c-Fes immunoreactive proteins p93, p74, and p70; the "differentiated" pattern showed two additional c-Fes immunoreactive proteins, p67 and p62. Using mRNA from mouse and human cell lines, we found deletion of one or more exons in the c-fes mRNA. Those deletions predicted truncation of conserved domains (CDC15/FCH and SH2) involved in protein-protein interactions. No deletions were found, however, within the kinase domain. We infer that alternative splicing generates a family of c-Fes proteins. This may be a mechanism to direct the c-Fes kinase domain to different subcellular locations and/or substrates at specific stages of myeloid cell differentiation.

Two-photon excited autofluorescence imaging of freshly isolated frog retinas.

PubMed

Lu, Rong-Wen; Li, Yi-Chao; Ye, Tong; Strang, Christianne; Keyser, Kent; Curcio, Christine A; Yao, Xin-Cheng

2011-06-01

The purpose of this study was to investigate cellular sources of autofluorescence signals in freshly isolated frog (Rana pipiens) retinas. Equipped with an ultrafast laser, a laser scanning two-photon excitation fluorescence microscope was employed for sub-cellular resolution examination of both sliced and flat-mounted retinas. Two-photon imaging of retinal slices revealed autofluorescence signals over multiple functional layers, including the photoreceptor layer (PRL), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Using flat-mounted retinas, depth-resolved imaging of individual retinal layers further confirmed multiple sources of autofluorescence signals. Cellular structures were clearly observed at the PRL, ONL, INL, and GCL. At the PRL, the autofluorescence was dominantly recorded from the intracellular compartment of the photoreceptors; while mixed intracellular and extracellular autofluorescence signals were observed at the ONL, INL, and GCL. High resolution autofluorescence imaging clearly revealed mosaic organization of rod and cone photoreceptors; and sub-cellular bright autofluorescence spots, which might relate to connecting cilium, was observed in the cone photoreceptors only. Moreover, single-cone and double-cone outer segments could be directly differentiated.

Intermediate filament proteins of digestive organs: physiology and pathophysiology.

PubMed

Omary, M Bishr

2017-06-01

Intermediate filament proteins (IFs), such as cytoplasmic keratins in epithelial cells and vimentin in mesenchymal cells and the nuclear lamins, make up one of the three major cytoskeletal protein families. Whether in digestive organs or other tissues, IFs share several unique features including stress-inducible overexpression, abundance, cell-selective and differentiation state expression, and association with >80 human diseases when mutated. Whereas most IF mutations cause disease, mutations in simple epithelial keratins 8, 18, or 19 or in lamin A/C predispose to liver disease with or without other tissue manifestations. Keratins serve major functions including protection from apoptosis, providing cellular and subcellular mechanical integrity, protein targeting to subcellular compartments, and scaffolding and regulation of cell-signaling processes. Keratins are essential for Mallory-Denk body aggregate formation that occurs in association with several liver diseases, whereas an alternate type of keratin and lamin aggregation occurs upon liver involvement in porphyria. IF-associated diseases have no known directed therapy, but high-throughput drug screening to identify potential therapies is an appealing ongoing approach. Despite the extensive current knowledge base, much remains to be discovered regarding IF physiology and pathophysiology in digestive and nondigestive organs. Copyright © 2017 the American Physiological Society.

PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes.

PubMed

Yu, Nancy Y; Wagner, James R; Laird, Matthew R; Melli, Gabor; Rey, Sébastien; Lo, Raymond; Dao, Phuong; Sahinalp, S Cenk; Ester, Martin; Foster, Leonard J; Brinkman, Fiona S L

2010-07-01

PSORTb has remained the most precise bacterial protein subcellular localization (SCL) predictor since it was first made available in 2003. However, the recall needs to be improved and no accurate SCL predictors yet make predictions for archaea, nor differentiate important localization subcategories, such as proteins targeted to a host cell or bacterial hyperstructures/organelles. Such improvements should preferably be encompassed in a freely available web-based predictor that can also be used as a standalone program. We developed PSORTb version 3.0 with improved recall, higher proteome-scale prediction coverage, and new refined localization subcategories. It is the first SCL predictor specifically geared for all prokaryotes, including archaea and bacteria with atypical membrane/cell wall topologies. It features an improved standalone program, with a new batch results delivery system complementing its web interface. We evaluated the most accurate SCL predictors using 5-fold cross validation plus we performed an independent proteomics analysis, showing that PSORTb 3.0 is the most accurate but can benefit from being complemented by Proteome Analyst predictions. http://www.psort.org/psortb (download open source software or use the web interface). psort-mail@sfu.ca Supplementary data are available at Bioinformatics online.

Dgat1 and Dgat2 regulate enterocyte triacylglycerol distribution and alter proteins associated with cytoplasmic lipid droplets in response to dietary fat

PubMed Central

Hung, Yu-Han; Carreiro, Alicia L.; Buhman, Kimberly K.

2017-01-01

Enterocytes, the absorptive cells of the small intestine, mediate efficient absorption of dietary fat (triacylglycerol, TAG). The digestive products of dietary fat are taken up by enterocytes, re-esterified into TAG, and packaged on chylomicrons (CMs) for secretion into blood or temporarily stored within cytoplasmic lipid droplets (CLDs). Altered enterocyte TAG distribution impacts susceptibility to high fat diet associated diseases, but molecular mechanisms directing TAG toward these fates are unclear. Two enzymes, acyl CoA: diacylglycerol acyltransferase 1 (Dgat1) and Dgat2, catalyze the final, committed step of TAG synthesis within enterocytes. Mice with intestine-specific overexpression of Dgat1 (Dgat1Int) or Dgat2 (Dgat2Int), or lack of Dgat1 (Dgat1−/−), were previously found to have altered intestinal TAG secretion and storage. We hypothesized that varying intestinal Dgat1 and Dgat2 levels alters TAG distribution in subcellular pools for CM synthesis as well as the morphology and proteome of CLDs. To test this we used ultrastructural and proteomic methods to investigate intracellular TAG distribution and CLD-associated proteins in enterocytes from Dgat1Int, Dgat2Int, and Dgat1−/− mice 2 hours after a 200 μl oral olive oil gavage. We found that varying levels of intestinal Dgat1 and Dgat2 altered TAG pools involved in CM assembly and secretion, the number or size of CLDs present in enterocytes, and the enterocyte CLD proteome. Overall, these results support a model where Dgat1 and Dgat2 function coordinately to regulate the process of dietary fat absorption by preferentially synthesizing TAG for incorporation into distinct subcellular TAG pools in enterocytes. PMID:28249764

Chlordecone Altered Hepatic Disposition of [14C]Cholesterol and Plasma Cholesterol Distribution but not SR-BI or ABCG8 Proteins in Livers of C57BL/6 Mice

PubMed Central

Lee, Junga; Scheri, Richard C.; Curtis, Lawrence R.

2011-01-01

Organochlorine (OC) insecticides continue to occur in tissues of humans and wildlife throughout the world although they were banned in the United States a few decades ago. Low doses of the OC insecticide chlordecone (CD) alter hepatic disposition of lipophilic xenobiotics and perturb lipid homeostasis in rainbow trout, mice and rats. CD pretreatment altered tissue and hepatic subcellular distribution of exogenous [14C]cholesterol (CH) equivalents 4 and 16 h after a bolus intraperitoneal (ip) injection of 5 ml corn oil/kg that contained 10 mg CH/kg. CD pretreatment altered tissue distribution of exogenously administered [14C]CH by decreased hepatic and renal accumulation, and increased biliary excretion up to 300%. Biliary excretion of polar [14C]CH metabolites was not altered by CD. CD pretreatment decreased subcellular distribution of [14C]CH equivalents in hepatic cytosol and microsomes and lipoprotein-rich fraction-to-homogenate ratio. CD pretreatment increased the ratio of [14C]CH equivalents in high density lipoprotein (HDL) to that in plasma and reduced [14C]CH equivalents in the non-HDL fraction 4 h after a bolus lipid dose. CD pretreatment increased plasma non-HDL total CH by 80% 4 h after a bolus lipid dose. Scavenger receptor class B type I (SR-BI) and ATPbinding cassette transporter G8 (ABCG8) proteins were quantified by western blotting in hepatic membranes from control and CD treated mice. Liver membrane contents of SR-BI and ABCG8 proteins were unchanged by CD pretreatment. The data demonstrated that a single dose of CD altered CH homeostasis and lipoprotein metabolism. PMID:18387646

Chlordecone altered hepatic disposition of [14C]cholesterol and plasma cholesterol distribution but not SR-BI or ABCG8 proteins in livers of C57BL/6 mice.

PubMed

Lee, Junga; Scheri, Richard C; Curtis, Lawrence R

2008-06-15

Organochlorine (OC) insecticides continue to occur in tissues of humans and wildlife throughout the world although they were banned in the United States a few decades ago. Low doses of the OC insecticide chlordecone (CD) alter hepatic disposition of lipophilic xenobiotics and perturb lipid homeostasis in rainbow trout, mice and rats. CD pretreatment altered tissue and hepatic subcellular distribution of exogenous [(14)C]cholesterol (CH) equivalents 4 and 16 h after a bolus intraperitoneal (ip) injection of 5 ml corn oil/kg that contained 10 mg CH/kg. CD pretreatment altered tissue distribution of exogenously administered [(14)C]CH by decreased hepatic and renal accumulation, and increased biliary excretion up to 300%. Biliary excretion of polar [(14)C]CH metabolites was not altered by CD. CD pretreatment decreased subcellular distribution of [(14)C]CH equivalents in hepatic cytosol and microsomes and lipoprotein-rich fraction-to-homogenate ratio. CD pretreatment increased the ratio of [(14)C]CH equivalents in high density lipoprotein (HDL) to that in plasma and reduced [(14)C]CH equivalents in the non-HDL fraction 4 h after a bolus lipid dose. CD pretreatment increased plasma non-HDL total CH by 80% 4 h after a bolus lipid dose. Scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter G8 (ABCG8) proteins were quantified by western blotting in hepatic membranes from control and CD treated mice. Liver membrane contents of SR-BI and ABCG8 proteins were unchanged by CD pretreatment. The data demonstrated that a single dose of CD altered CH homeostasis and lipoprotein metabolism.

Distribution and function of HCN channels in the apical dendritic tuft of neocortical pyramidal neurons.

PubMed

Harnett, Mark T; Magee, Jeffrey C; Williams, Stephen R

2015-01-21

The apical tuft is the most remote area of the dendritic tree of neocortical pyramidal neurons. Despite its distal location, the apical dendritic tuft of layer 5 pyramidal neurons receives substantial excitatory synaptic drive and actively processes corticocortical input during behavior. The properties of the voltage-activated ion channels that regulate synaptic integration in tuft dendrites have, however, not been thoroughly investigated. Here, we use electrophysiological and optical approaches to examine the subcellular distribution and function of hyperpolarization-activated cyclic nucleotide-gated nonselective cation (HCN) channels in rat layer 5B pyramidal neurons. Outside-out patch recordings demonstrated that the amplitude and properties of ensemble HCN channel activity were uniform in patches excised from distal apical dendritic trunk and tuft sites. Simultaneous apical dendritic tuft and trunk whole-cell current-clamp recordings revealed that the pharmacological blockade of HCN channels decreased voltage compartmentalization and enhanced the generation and spread of apical dendritic tuft and trunk regenerative activity. Furthermore, multisite two-photon glutamate uncaging demonstrated that HCN channels control the amplitude and duration of synaptically evoked regenerative activity in the distal apical dendritic tuft. In contrast, at proximal apical dendritic trunk and somatic recording sites, the blockade of HCN channels decreased excitability. Dynamic-clamp experiments revealed that these compartment-specific actions of HCN channels were heavily influenced by the local and distributed impact of the high density of HCN channels in the distal apical dendritic arbor. The properties and subcellular distribution pattern of HCN channels are therefore tuned to regulate the interaction between integration compartments in layer 5B pyramidal neurons. Copyright © 2015 the authors 0270-6474/15/351024-14$15.00/0.

Dgat1 and Dgat2 regulate enterocyte triacylglycerol distribution and alter proteins associated with cytoplasmic lipid droplets in response to dietary fat.

PubMed

Hung, Yu-Han; Carreiro, Alicia L; Buhman, Kimberly K

2017-06-01

Enterocytes, the absorptive cells of the small intestine, mediate efficient absorption of dietary fat (triacylglycerol, TAG). The digestive products of dietary fat are taken up by enterocytes, re-esterified into TAG, and packaged on chylomicrons (CMs) for secretion into blood or temporarily stored within cytoplasmic lipid droplets (CLDs). Altered enterocyte TAG distribution impacts susceptibility to high fat diet associated diseases, but molecular mechanisms directing TAG toward these fates are unclear. Two enzymes, acyl CoA: diacylglycerol acyltransferase 1 (Dgat1) and Dgat2, catalyze the final, committed step of TAG synthesis within enterocytes. Mice with intestine-specific overexpression of Dgat1 (Dgat1 Int ) or Dgat2 (Dgat2 Int ), or lack of Dgat1 (Dgat1 -/- ), were previously found to have altered intestinal TAG secretion and storage. We hypothesized that varying intestinal Dgat1 and Dgat2 levels alters TAG distribution in subcellular pools for CM synthesis as well as the morphology and proteome of CLDs. To test this we used ultrastructural and proteomic methods to investigate intracellular TAG distribution and CLD-associated proteins in enterocytes from Dgat1 Int , Dgat2 Int , and Dgat1 -/- mice 2h after a 200μl oral olive oil gavage. We found that varying levels of intestinal Dgat1 and Dgat2 altered TAG pools involved in CM assembly and secretion, the number or size of CLDs present in enterocytes, and the enterocyte CLD proteome. Overall, these results support a model where Dgat1 and Dgat2 function coordinately to regulate the process of dietary fat absorption by preferentially synthesizing TAG for incorporation into distinct subcellular TAG pools in enterocytes. Copyright © 2017 Elsevier B.V. All rights reserved.

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes

PubMed Central

Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

2016-01-01

ABSTRACT Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes. PMID:27433972

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes.

PubMed

Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

2016-10-01

Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.

Tomato Ribonuclease LX with the Functional Endoplasmic Reticulum Retention Motif HDEF Is Expressed during Programmed Cell Death Processes, Including Xylem Differentiation, Germination, and Senescence1

PubMed Central

Lehmann, Karin; Hause, Bettina; Altmann, Dorit; Köck, Margret

2001-01-01

We have studied the subcellular localization of the acid S-like ribonuclease (RNase) LX in tomato (Lycopersicon esculentum Mill.) cells using a combination of biochemical and immunological methods. It was found that the enzyme, unexpectedly excluded from highly purified vacuoles, accumulates in the endoplasmic reticulum. The evidence that RNase LX is a resident of the endoplasmic reticulum (ER) is supported by an independent approach showing that the C-terminal peptide HDEF of RNase LX acts as an alternative ER retention signal in plants. For functional testing, the cellular distribution of chimeric protein constructs based on a marker protein, Brazil nut (Bertholletia excelsa) 2S albumin, was analyzed immunochemically in transgenic tobacco (Nicotiana tabacum) plants. Here, we report that the peptide motif is necessary and sufficient to accumulate 2S albumin constructs of both vacuolar and extracellular final destinations in the ER. We have shown immunochemically that RNase LX is specifically expressed during endosperm mobilization and leaf and flower senescence. Using immunofluorescence, RNase LX protein was detected in immature tracheary elements, suggesting a function in xylem differentiation. These results support a physiological function of RNase LX in selective cell death processes that are also thought to involve programmed cell death. It is assumed that RNase LX accumulates in an ER-derived compartment and is released by membrane disruption into the cytoplasma of those cells that are intended to undergo autolysis. These processes are accompanied by degradation of cellular components supporting a metabolic recycling function of the intracellular RNase LX. PMID:11598219

Androgen Receptor Functional Analyses by High Throughput Imaging: Determination of Ligand, Cell Cycle, and Mutation-Specific Effects

PubMed Central

Szafran, Adam T.; Szwarc, Maria; Marcelli, Marco; Mancini, Michael A.

2008-01-01

Background Understanding how androgen receptor (AR) function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate cancer, androgen insensitivity syndrome, AIS), and in the analysis of environmental endocrine disruptors. Methodology/Principal Findings We report the development of a high throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variation were 5–24%, with Z' values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, demonstrated that nuclear translocation and nuclear “speckling” were linked with transcriptional output, and specific ligands were noted to differentially affect measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations demonstrated a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions. Conclusions/Significance HT imaging-based multiplex screening will provide a rapid, systems-level analysis of compounds/RNAi that may differentially affect wild type AR or clinically relevant AR mutations. PMID:18978937

7 CFR 340.8 - Container requirements for the movement of regulated articles.

Code of Federal Regulations, 2011 CFR

2011-01-01

... requirements—(1) Plants and plant parts. All plants or plant parts, except seeds, cells, and subcellular... strength. (3) Live microorganisms and/or etiologic agents, cells, or subcellular elements. All regulated articles which are live (non-inactivated) microorganisms, or etiologic agents, cells, or subcellular...

7 CFR 340.8 - Container requirements for the movement of regulated articles.

Code of Federal Regulations, 2014 CFR

2014-01-01

... requirements—(1) Plants and plant parts. All plants or plant parts, except seeds, cells, and subcellular... strength. (3) Live microorganisms and/or etiologic agents, cells, or subcellular elements. All regulated articles which are live (non-inactivated) microorganisms, or etiologic agents, cells, or subcellular...

7 CFR 340.8 - Container requirements for the movement of regulated articles.

Code of Federal Regulations, 2013 CFR

2013-01-01

... requirements—(1) Plants and plant parts. All plants or plant parts, except seeds, cells, and subcellular... strength. (3) Live microorganisms and/or etiologic agents, cells, or subcellular elements. All regulated articles which are live (non-inactivated) microorganisms, or etiologic agents, cells, or subcellular...

7 CFR 340.8 - Container requirements for the movement of regulated articles.

Code of Federal Regulations, 2012 CFR

2012-01-01

... requirements—(1) Plants and plant parts. All plants or plant parts, except seeds, cells, and subcellular... strength. (3) Live microorganisms and/or etiologic agents, cells, or subcellular elements. All regulated articles which are live (non-inactivated) microorganisms, or etiologic agents, cells, or subcellular...

Cellular and subcellular localization of uncoupling protein 2 in the human kidney.

PubMed

Nigro, Michelangelo; De Sanctis, Claudia; Formisano, Pietro; Stanzione, Rosita; Forte, Maurizio; Capasso, Giovambattista; Gigliotti, Giuseppe; Rubattu, Speranza; Viggiano, Davide

2018-06-23

The uncoupling protein-2 (UCP2) is an anion transporter that plays a key role in the control of intracellular oxidative stress. In animal models UCP2 downregulation has several pathological sequelae, particularly affecting the vasculature and the kidney. Specifically, in these models kidney damage is highly favored in the absence of UCP2 in the context of experimental hypertension. Confirmations of these data in humans awaits further information, as no data are yet available concerning the cell-type and subcellular expression in the human kidney. In the present study, we aimed to characterize the UCP2 protein distribution in human kidney biopsies. In humans UCP2 is mainly localized in proximal convoluted tubule cells, with an intracytoplasmic punctate staining. UCP2 positive puncta are often localized at the interface between the endoplasmic reticulum and the mitochondria. Glomerular structures do not express UCP2 at detectable levels. The expression of UCP2 in proximal tubular cells may explain their relative propensity to damage in pathological conditions including the hypertensive disease.

Trehalose Alters Subcellular Trafficking and the Metabolism of the Alzheimer-associated Amyloid Precursor Protein.

PubMed

Tien, Nguyen T; Karaca, Ilker; Tamboli, Irfan Y; Walter, Jochen

2016-05-13

The disaccharide trehalose is commonly considered to stimulate autophagy. Cell treatment with trehalose could decrease cytosolic aggregates of potentially pathogenic proteins, including mutant huntingtin, α-synuclein, and phosphorylated tau that are associated with neurodegenerative diseases. Here, we demonstrate that trehalose also alters the metabolism of the Alzheimer disease-related amyloid precursor protein (APP). Cell treatment with trehalose decreased the degradation of full-length APP and its C-terminal fragments. Trehalose also reduced the secretion of the amyloid-β peptide. Biochemical and cell biological experiments revealed that trehalose alters the subcellular distribution and decreases the degradation of APP C-terminal fragments in endolysosomal compartments. Trehalose also led to strong accumulation of the autophagic marker proteins LC3-II and p62, and decreased the proteolytic activation of the lysosomal hydrolase cathepsin D. The combined data indicate that trehalose decreases the lysosomal metabolism of APP by altering its endocytic vesicular transport. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Nanodosimetric track structure in homogeneous extended beams.

PubMed

Conte, V; Moro, D; Colautti, P; Grosswendt, B

2015-09-01

Physical aspects of particle track structure are important in determining the induction of clustered damage in relevant subcellular structures like the DNA and higher-order genomic structures. The direct measurement of track-structure properties of ionising radiation is feasible today by counting the number of ionisations produced inside a small gas volume. In particular, the so-called track-nanodosimeter, installed at the TANDEM-ALPI accelerator complex of LNL, measures ionisation cluster-size distributions in a simulated subcellular structure of dimensions 20 nm, corresponding approximately to the diameter of the chromatin fibre. The target volume is irradiated by pencil beams of primary particles passing at specified impact parameter. To directly relate these measured track-structure data to radiobiological measurements performed in broad homogeneous particle beams, these data can be integrated over the impact parameter. This procedure was successfully applied to 240 MeV carbon ions and compared with Monte Carlo simulations for extended fields. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Subcellular localization of proflavine derivative and induction of oxidative stress--in vitro studies.

PubMed

Ipóthová, Z; Paulíková, H; Cižeková, L; Hunáková, L; Labudová, M; Grolmusová, A; Janovec, L; Imrich, J

2013-11-01

Acridines have been studied for several decades because of their numerous biological effects, especially anticancer activity. Recently, cytotoxicity of novel acridine derivatives, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was confirmed for leukemic cell lines [Bioorg. Med. Chem.2011, 19, 1790]. The mechanism of action of the most cytotoxic hexyl-AcrDIM was studied in this paper focusing attention on a subcellular distribution of the drug. Accumulation of hexyl-AcrDIM in mitochondria was confirmed after labeling mitochondria with MitoRED using ImageStream Imaging Flow Cytometer. The derivative significantly decreased intracellular ATP level (reduction of ATP level was decreased by vitamin E), and induced oxidative stress (ROS production detected by DHE assay) as well as cell cycle arrest in the S-phase (flow cytometry analysis) already after short-time incubation and induction of apoptosis. Cytotoxicity of hexyl-AcrDIM is closely connected with induction of oxidative stress in cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Adipocyte aminopeptidases in obesity and fasting.

PubMed

Alponti, Rafaela Fadoni; Silveira, Paulo Flavio

2015-11-05

This study checked the existence of a diverse array of aminopeptidase (AP) enzymes in high (HDM) and low (LDM) density microsomal and plasma membrane (MF) fractions from adipocytes of control, monosodium glutamate obese and food deprived rats. Gene expression was detected for ArgAP, AspAP, MetAP, and two AlaAP (APM and PSA). APM and PSA had the highest catalytic efficiency, whereas AspAP the highest affinity. Subcellular distribution of AP activities depended on metabolic status. Comparing catalytic levels, AspAP in HDM, LDM and MF was absent in obese and control under food deprivation; PSA in LDM was 3.5-times higher in obese than in normally fed control and control and obese under food deprivation; MetAP in MF was 4.5-times higher in obese than in food deprived obese. Data show new AP enzymes genetically expressed in subcellular compartments of adipocytes, three of them with altered catalytic levels that respond to whole-body energetic demands. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Subcellular analysis of interaction between breast cancer cells and drug by digital holography

NASA Astrophysics Data System (ADS)

Zhao, Jie; Lin, Qiaowen; Wang, Dayong; Wang, Yunxin; Ouyang, Liting; Guo, Sha; Yao, Qian

2017-10-01

Digital holographic microscopy is a promising quantitative phase-contrast imaging technique, which exhibits the advantages of non-destruction, full field of view, quasi-real time, and don't need dye and external marker to the living biological sample. In this paper, the inverted off-axis image-plane digital holography with pre-magnification is built up to study the living MDA-MB-231 breast cancer cells. The lateral resolution of the proposed experimental setup is 0.87μm, which is verified by the standard USAF test target. Then the system is used to visualize the interaction between living breast cancer cells and drug. The blebbing is observed after the cells are treated by paclitaxel drug, and the distribution of the paclitaxel inside the cells is detected, which is near the cytomembrane, or in other words the end of the microtubules. It will stop the mitosis and cause the death of the cells. It is helpful to reveal the anticancer mechanism of paclitaxel in the subcellular scale.

Multicoloured fluorescent indicators for live-cell and in vivo imaging of inorganic mercury dynamics.

PubMed

Tao, Rongkun; Shi, Mei; Zou, Yejun; Cheng, Di; Wang, Qiaohui; Liu, Renmei; Wang, Aoxue; Zhu, Jiahuan; Deng, Lei; Hu, Hanyang; Chen, Xianjun; Du, Jiulin; Zhu, Weiping; Zhao, Yuzheng; Yang, Yi

2018-06-01

Engineered fluorescent indicators for visualizing mercury ion (Hg 2+ ) are powerful tools to illustrate the intracellular distribution and serious toxicity of the ion. However, the sensitive and specific detection of Hg 2+ in living cells and in vivo is challenging. This paper reported the development of fluorescent indicators for Hg 2+ in green or red color by inserting a circularly permuted fluorescent protein into a highly mercury-specific repressor. These sensors provided a rapid, sensitive, specific, and real-time read-out of Hg 2+ dynamics in solutions, bacteria, subcellular organelles of mammalian cells, and zebrafish, thereby providing a useful new method for Hg 2+ detection and bioimaging. In conjunction with the hydrogen peroxide sensor HyPer, we found mercury uptake would trigger subcellular oxidative events at the single-cell level, and provided visual evidence of the causality of mercury and oxidative damage. These sensors would paint the landscape of mercury toxicity to cell functions. Copyright © 2018 Elsevier Inc. All rights reserved.

Distinct subcellular patterns of neprilysin protein and activity in the brains of Alzheimer’s disease patients, transgenic mice and cultured human neuronal cells

PubMed Central

Zhou, Li; Wei, Chunsheng; Huang, Wei; Bennett, David A; Dickson, Dennis W; Wang, Rui; Wang, Dengshun

2013-01-01

We investigated the subcellular distribution of NEP protein and activity in brains of human individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD dementia, as well as double transgenic mice and human neuronal cell line treated with Aβ and 4-hydroxy-2-nonenal (HNE). Total cortical neuronal-related NEP was significantly increased in MCI compared to NCI brains. NeuN was decreased in both MCI and AD, consistent with neuronal loss occurring in MCI and AD. Negative relationship between NEP protein and NeuN in MCI brains, and positive correlation between NEP and pan-cadherin in NCI and MCI brains, suggesting the increased NEP expression in NCI and MCI might be due to membrane associated NEP in non-neuronal cells. In subcellular extracts, NEP protein decreased in cytoplasmic fractions in MCI and AD, but increased in membrane fractions, with a significant increase in the membrane/cytoplasmic ratio of NEP protein in AD brains. By contrast, NEP activity was decreased in AD. Similar results were observed in AD-mimic transgenic mice. Studies of SH-SY5Y neuroblastoma showed an up-regulation of NEP protein in the cytoplasmic compartment induced by HNE and Aβ; however, NEP activity decreased in cytoplasmic fractions. Activity of NEP in membrane fractions increased at 48 hours and then significantly decreased after treatment with HNE and Aβ. The cytoplasmic/membrane ratio of NEP protein increased at 24 hours and then decreased in both HNE and Aβ treated cells. Both HNE and Aβ up-regulate NEP expression, but NEP enzyme activity did not show the same increase, possibly indicating immature cytoplasmic NEP is less active than membrane associated NEP. These observations indicate that modulation of NEP protein levels and its subcellular location influence the net proteolytic activity and this complex association might participate in deficiency of Aβ degradation that is associated with amyloid deposition in AD. PMID:24093058

Automated processing of label-free Raman microscope images of macrophage cells with standardized regression for high-throughput analysis.

PubMed

Milewski, Robert J; Kumagai, Yutaro; Fujita, Katsumasa; Standley, Daron M; Smith, Nicholas I

2010-11-19

Macrophages represent the front lines of our immune system; they recognize and engulf pathogens or foreign particles thus initiating the immune response. Imaging macrophages presents unique challenges, as most optical techniques require labeling or staining of the cellular compartments in order to resolve organelles, and such stains or labels have the potential to perturb the cell, particularly in cases where incomplete information exists regarding the precise cellular reaction under observation. Label-free imaging techniques such as Raman microscopy are thus valuable tools for studying the transformations that occur in immune cells upon activation, both on the molecular and organelle levels. Due to extremely low signal levels, however, Raman microscopy requires sophisticated image processing techniques for noise reduction and signal extraction. To date, efficient, automated algorithms for resolving sub-cellular features in noisy, multi-dimensional image sets have not been explored extensively. We show that hybrid z-score normalization and standard regression (Z-LSR) can highlight the spectral differences within the cell and provide image contrast dependent on spectral content. In contrast to typical Raman imaging processing methods using multivariate analysis, such as single value decomposition (SVD), our implementation of the Z-LSR method can operate nearly in real-time. In spite of its computational simplicity, Z-LSR can automatically remove background and bias in the signal, improve the resolution of spatially distributed spectral differences and enable sub-cellular features to be resolved in Raman microscopy images of mouse macrophage cells. Significantly, the Z-LSR processed images automatically exhibited subcellular architectures whereas SVD, in general, requires human assistance in selecting the components of interest. The computational efficiency of Z-LSR enables automated resolution of sub-cellular features in large Raman microscopy data sets without compromise in image quality or information loss in associated spectra. These results motivate further use of label free microscopy techniques in real-time imaging of live immune cells.

Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions

PubMed Central

Nielsen, Joachim; Farup, Jean; Rahbek, Stine Klejs; de Paoli, Frank Vincenzo; Vissing, Kristian

2015-01-01

Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthesis, as described by the product of glycogen particle size and number, is dependent on the time course of recovery after exercise and carbohydrate availability. In the present study, we investigated the subcellular distribution of glycogen in fibers with high (type I) and low (type II) mitochondrial content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h) enhanced storage of intramyofibrillar glycogen (defined as glycogen particles located within the myofibrils) of type I fibers, which was associated with an increase in the number of particles. In contrast, late in recovery (post 48 h), intermyofibrillar, intramyofibrillar and subsarcolemmal glycogen in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular localization, muscle fiber’s oxidative capacity, and the time course of recovery. The early enhanced storage of intramyofibrillar glycogen after the eccentric contractions may entail important implications for muscle function and fatigue resistance. PMID:25996774

A Quantitative Spatial Proteomics Analysis of Proteome Turnover in Human Cells*

PubMed Central

Boisvert, François-Michel; Ahmad, Yasmeen; Gierliński, Marek; Charrière, Fabien; Lamont, Douglas; Scott, Michelle; Barton, Geoff; Lamond, Angus I.

2012-01-01

Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ∼20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function. PMID:21937730

Distribution and Characterization of Antigens Found in Subcellular Fractions of African Trypanosomes

DTIC Science & Technology

1983-08-01

glycoproteln of Trypanosoma brucel synthesized with a c-termlnal hydrophob Ic tall absent from purified glycoproteln. Nature Zfifi: 624-626. 3...Cardosa de Almeida, M.L. and Turner, M.J. 1983. The membrane form of variant surface glycoprotelns of Trypanosoma brucel . Nature 202: 349- 352. 4...Blochem. 131; 1-15. 7. Godfrey, D.G. 1967. Phosphol Iplds of Trypanosoma lewlsl,. I,. vlvaxP L. congolense and L brucel . Expt. Parasit. 20

PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha

PubMed Central

2012-01-01

Background Poly(3-hydroxybutyrate) (PHB) granules are important storage compounds of carbon and energy in many prokaryotes which allow survival of the cells in the absence of suitable carbon sources. Formation and subcellular localization of PHB granules was previously assumed to occur randomly in the cytoplasm of PHB accumulating bacteria. However, contradictionary results on subcellular localization of PHB granules in Ralstonia eutropha were published, recently. Results Here, we provide evidence by transmission electron microscopy that PHB granules are localized in close contact to the nucleoid region in R. eutropha during growth on nutrient broth. Binding of PHB granules to the nucleoid is mediated by PhaM, a PHB granule associated protein with phasin-like properties that is also able to bind to DNA and to phasin PhaP5. Over-expression of PhaM resulted in formation of many small PHB granules that were always attached to the nucleoid region. In contrast, PHB granules of ∆phaM strains became very large and distribution of granules to daughter cells was impaired. Association of PHB granules to the nucleoid region was prevented by over-expression of PhaP5 and clusters of several PHB granules were mainly localized near the cell poles. Conclusion Subcellular localization of PHB granules is controlled in R. eutropha and depends on the presence and concentrations of at least two PHB granule associated proteins, PhaM and PhaP5. PMID:23157596

Predicting protein submitochondrial locations using a K-Nearest neighbor method based on the Bit-Score weighted euclidean distance

USDA-ARS?s Scientific Manuscript database

Mitochondria are essential subcellular organelles found in eukaryotic cells. Knowing information on a protein’s subcellular or sub subcellular location provides in-depth insights about the microenvironment where it interacts with other molecules and is crucial for inferring the protein’s function. T...

Predicting plant protein subcellular multi-localization by Chou's PseAAC formulation based multi-label homolog knowledge transfer learning.

PubMed

Mei, Suyu

2012-10-07

Recent years have witnessed much progress in computational modeling for protein subcellular localization. However, there are far few computational models for predicting plant protein subcellular multi-localization. In this paper, we propose a multi-label multi-kernel transfer learning model for predicting multiple subcellular locations of plant proteins (MLMK-TLM). The method proposes a multi-label confusion matrix and adapts one-against-all multi-class probabilistic outputs to multi-label learning scenario, based on which we further extend our published work MK-TLM (multi-kernel transfer learning based on Chou's PseAAC formulation for protein submitochondria localization) for plant protein subcellular multi-localization. By proper homolog knowledge transfer, MLMK-TLM is applicable to novel plant protein subcellular localization in multi-label learning scenario. The experiments on plant protein benchmark dataset show that MLMK-TLM outperforms the baseline model. Unlike the existing models, MLMK-TLM also reports its misleading tendency, which is important for comprehensive survey of model's multi-labeling performance. Copyright © 2012 Elsevier Ltd. All rights reserved.

Sub-cellular force microscopy in single normal and cancer cells.

PubMed

Babahosseini, H; Carmichael, B; Strobl, J S; Mahmoodi, S N; Agah, M

2015-08-07

This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. Copyright © 2015 Elsevier Inc. All rights reserved.

Nuclear overexpression of the overexpressed in lung cancer 1 predicts worse prognosis in gastric adenocarcinoma.

PubMed

Wang, Jue; Shen, Hongchang; Fu, Guobin; Zhao, Dandan; Wang, Weibo

2017-02-07

We have performed this retrospective study to elucidate whether elevated expression of the overexpressed in lung cancer 1 (OLC1) was related to the clinicopathological parameters and prognosis of patients with gastric adenocarcinoma. Additionally, different effects of various subcellular OLC1 expression on gastric adeno-carcinogenesis were focused on in our study. Both overall and subcellular expression of OLC1 was evaluated by immunohistochemistry(IHC) via tissue microarrays from total 393 samples. The Kaplan-Meier method and Cox's proportional hazard model were exerted to further explore the correlation between OLC1 and prognosis. Total overexpression of OLC1 was significantly associated with stage (P = 0.004) and differentiation (P = 0.009), and only the strong total expression could predict a poor prognosis (HR = 1.31, P = 0.04). There were significant associations found between nuclear overexpression and tumor invasion depth(P = 0.002), lymph node (P < 0.001), stage (P = 0.004), differentiation (P < 0.001) and smoking history (P = 0.045). Furthermore, over-expressed nuclear OLC1 protein could be an independent risk factor for gastric adenocarcinoma (univariate: HR = 1.43, P = 0.003; multivariate: HR = 1.39, P = 0.011). In general, both total and nuclear overexpression of OLC1 could be the signs of gastric adeno-carcinogenesis, which might be served as the biomarkers for diagnosis at an early stage, even at the onset of tumorigenesis. Rather than the total expression, nuclear overexpression of OLC1 was correlated with most clinicopathological parameters and could predict a poor overall survival as an independent factor for prognosis, which made it a more effective and sensitive biomarker for gastric adenocarcinoma.

Morphine Regulated Synaptic Networks Revealed by Integrated Proteomics and Network Analysis*

PubMed Central

Stockton, Steven D.; Gomes, Ivone; Liu, Tong; Moraje, Chandrakala; Hipólito, Lucia; Jones, Matthew R.; Ma'ayan, Avi; Morón, Jose A.; Li, Hong; Devi, Lakshmi A.

2015-01-01

Despite its efficacy, the use of morphine for the treatment of chronic pain remains limited because of the rapid development of tolerance, dependence and ultimately addiction. These undesired effects are thought to be because of alterations in synaptic transmission and neuroplasticity within the reward circuitry including the striatum. In this study we used subcellular fractionation and quantitative proteomics combined with computational approaches to investigate the morphine-induced protein profile changes at the striatal postsynaptic density. Over 2,600 proteins were identified by mass spectrometry analysis of subcellular fractions enriched in postsynaptic density associated proteins from saline or morphine-treated striata. Among these, the levels of 34 proteins were differentially altered in response to morphine. These include proteins involved in G-protein coupled receptor signaling, regulation of transcription and translation, chaperones, and protein degradation pathways. The altered expression levels of several of these proteins was validated by Western blotting analysis. Using Genes2Fans software suite we connected the differentially expressed proteins with proteins identified within the known background protein-protein interaction network. This led to the generation of a network consisting of 116 proteins with 40 significant intermediates. To validate this, we confirmed the presence of three proteins predicted to be significant intermediates: caspase-3, receptor-interacting serine/threonine protein kinase 3 and NEDD4 (an E3-ubiquitin ligase identified as a neural precursor cell expressed developmentally down-regulated protein 4). Because this morphine-regulated network predicted alterations in proteasomal degradation, we examined the global ubiquitination state of postsynaptic density proteins and found it to be substantially altered. Together, these findings suggest a role for protein degradation and for the ubiquitin/proteasomal system in the etiology of opiate dependence and addiction. PMID:26149443

Sensitivity of tumor cells towards CIGB-300 anticancer peptide relies on its nucleolar localization.

PubMed

Perera, Yasser; Costales, Heydi C; Diaz, Yakelin; Reyes, Osvaldo; Farina, Hernan G; Mendez, Lissandra; Gómez, Roberto E; Acevedo, Boris E; Gomez, Daniel E; Alonso, Daniel F; Perea, Silvio E

2012-04-01

CIGB-300 is a novel anticancer peptide that impairs the casein kinase 2-mediated phosphorylation by direct binding to the conserved phosphoacceptor site on their substrates. Previous findings indicated that CIGB-300 inhibits tumor cell proliferation in vitro and induces tumor growth delay in vivo in cancer animal models. Interestingly, we had previously demonstrated that the putative oncogene B23/nucleophosmin (NPM) is the major intracellular target for CIGB-300 in a sensitive human lung cancer cell line. However, the ability of this peptide to target B23/NPM in cancer cells with differential CIGB-300 response phenotype remained to be determined. Interestingly, in this work, we evidenced that CIGB-300's antiproliferative activity on tumor cells strongly correlates with its nucleolar localization, the main subcellular localization of the previously identified B23/NPM target. Likewise, using CIGB-300 equipotent doses (concentration that inhibits 50% of proliferation), we demonstrated that this peptide interacts and inhibits B23/NPM phosphorylation in different cancer cell lines as evidenced by in vivo pull-down and metabolic labeling experiments. Moreover, such inhibition was followed by a fast apoptosis on CIGB-300-treated cells and also an impairment of cell cycle progression mainly after 5 h of treatment. Altogether, our data not only validates B23/NPM as a main target for CIGB-300 in cancer cells but also provides the first experimental clues to explain their differential antiproliferative response. Importantly, our findings suggest that further improvements to this cell penetrating peptide-based drug should entail its more efficient intracellular delivery at such subcellular localization. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

Differential subcellular membrane recruitment of Src may specify its downstream signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diesbach, Philippe de; Medts, Thierry; Carpentier, Sarah

2008-04-15

Most Src family members are diacylated and constitutively associate with membrane 'lipid rafts' that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at 'rafts' remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to 'lipid rafts'; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 deg. C and floated into sucrose density gradients like caveolin-1 andmore » flotillin-2, i.e. 'lipid rafts'. By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ({approx} 70%) cholesterol extraction with methyl-{beta}-cyclodextrin (M{beta}CD) did not abolish 'rafts' floatation, but strongly decreased Src association with floating 'rafts' and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to M{beta}CD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at 'non-raft' domains on endosomes, then via PI3-kinase-Akt on a distinct set of 'rafts' at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.« less

Cutting edge: rescue of pre-TCR but not mature TCR signaling in mice expressing membrane-targeted SLP-76.

PubMed

Bezman, Natalie A; Baker, Rebecca G; Lenox, Laurie E; Jordan, Martha S; Koretzky, Gary A

2009-05-01

SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) organizes signaling from immunoreceptors, including the platelet collagen receptor, the pre-TCR, and the TCR, and is required for T cell development. In this study we examine a mouse in which wild-type SLP-76 is replaced with a mutant constitutively targeted to the cell membrane. Membrane-targeted SLP-76 (MTS) supports ITAM signaling in platelets and from the pre-TCR. Signaling from the mature TCR, however, is defective in MTS thymocytes, resulting in failed T cell differentiation. Defective thymic selection by MTS is not rescued by a SLP-76 mutant whose localization is restricted to the cytosol. Thus, fixed localization of SLP-76 reveals differential requirements for the subcellular localization of signaling complexes downstream of the pre-TCR vs mature TCR.

Simplified Enrichment of Plasma Membrane Proteins from Arabidopsis thaliana Seedlings Using Differential Centrifugation and Brij-58 Treatment.

PubMed

Collins, Carina A; Leslie, Michelle E; Peck, Scott C; Heese, Antje

2017-01-01

The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.

Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16.

PubMed

Pfeiffer, Daniel; Wahl, Andreas; Jendrossek, Dieter

2011-11-01

A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1. © 2011 Blackwell Publishing Ltd.

Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments

PubMed Central

1985-01-01

The endocytic compartments of the asialoglycoprotein (ASGP) pathway in rat hepatocytes were studied using a combined morphological and biochemical approach in the isolated perfused liver. Use of electron microscopic tracers and a temperature-shift protocol to synchronize ligand entry confirmed the route of ASGP internalization observed in our previous in vivo studies (1) and established conditions under which we could label the contents of successive compartments in the pathway for subcellular fractionation studies. Three endosomal compartments were demonstrated in which ASGPs appear after they enter the cell via coated pits and vesicles but before they reach their site of degradation in lysosomes. These three compartments could be distinguished by their location within the hepatocyte, by their morphological appearance in situ, and by their density in sucrose gradients. The distributions of ASGP receptors, both accessible and latent (revealed by detergent permeabilization), were also examined and compared with that of ligand during subcellular fractionation. Most accessible ASGP receptors co-distributed with conventional plasma membrane markers. However, hepatocytes contain a substantial intracellular pool of latent ASGP binding sites that exceeds the number of cell surface receptors and whose presence is not dependent on ASGP exposure. The distribution of these latent ASGP receptors on sucrose gradients (detected either immunologically or by binding assays) was coincident with that of ligand sequestered within the early endosome compartments. In addition, both early endosomes and the membrane vesicles containing latent ASGP receptors had high cholesterol content, because both shifted markedly in density upon exposure to digitonin. PMID:2866191

Jmy regulates oligodendrocyte differentiation via modulation of actin cytoskeleton dynamics.

PubMed

Azevedo, Maria M; Domingues, Helena S; Cordelières, Fabrice P; Sampaio, Paula; Seixas, Ana I; Relvas, João B

2018-05-06

During central nervous system development, oligodendrocytes form structurally and functionally distinct actin-rich protrusions that contact and wrap around axons to assemble myelin sheaths. Establishment of axonal contact is a limiting step in myelination that relies on the oligodendrocyte's ability to locally coordinate cytoskeletal rearrangements with myelin production, under the control of a transcriptional differentiation program. The molecules that provide fine-tuning of actin dynamics during oligodendrocyte differentiation and axon ensheathment remain largely unidentified. We performed transcriptomics analysis of soma and protrusion fractions from rat brain oligodendrocyte progenitors and found a subcellular enrichment of mRNAs in newly-formed protrusions. Approximately 30% of protrusion-enriched transcripts encode proteins related to cytoskeleton dynamics, including the junction mediating and regulatory protein Jmy, a multifunctional regulator of actin polymerization. Here, we show that expression of Jmy is upregulated during myelination and is required for the assembly of actin filaments and protrusion formation during oligodendrocyte differentiation. Quantitative morphodynamics analysis of live oligodendrocytes showed that differentiation is driven by a stereotypical actin network-dependent "cellular shaping" program. Disruption of actin dynamics via knockdown of Jmy leads to a program fail resulting in oligodendrocytes that do not acquire an arborized morphology and are less efficient in contacting neurites and forming myelin wraps in co-cultures with neurons. Our findings provide new mechanistic insight into the relationship between cell shape dynamics and differentiation in development. © 2018 Wiley Periodicals, Inc.

Multi-Label Learning via Random Label Selection for Protein Subcellular Multi-Locations Prediction.

PubMed

Wang, Xiao; Li, Guo-Zheng

2013-03-12

Prediction of protein subcellular localization is an important but challenging problem, particularly when proteins may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular localization methods are only used to deal with the single-location proteins. In the past few years, only a few methods have been proposed to tackle proteins with multiple locations. However, they only adopt a simple strategy, that is, transforming the multi-location proteins to multiple proteins with single location, which doesn't take correlations among different subcellular locations into account. In this paper, a novel method named RALS (multi-label learning via RAndom Label Selection), is proposed to learn from multi-location proteins in an effective and efficient way. Through five-fold cross validation test on a benchmark dataset, we demonstrate our proposed method with consideration of label correlations obviously outperforms the baseline BR method without consideration of label correlations, indicating correlations among different subcellular locations really exist and contribute to improvement of prediction performance. Experimental results on two benchmark datasets also show that our proposed methods achieve significantly higher performance than some other state-of-the-art methods in predicting subcellular multi-locations of proteins. The prediction web server is available at http://levis.tongji.edu.cn:8080/bioinfo/MLPred-Euk/ for the public usage.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhaskar,; Kumari, Neeti; Goyal, Neena, E-mail: neenacdri@yahoo.com

Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complexmore » (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests that, this gene may have a role in maintaining the structural dynamics of cytoskeleton of parasite.« less

Ion pump sorting in polarized renal epithelial cells.

PubMed

Caplan, M J

2001-08-01

The plasma membranes of renal epithelial cells are divided into distinct apical and basolateral domains, which contain different inventories of ion transport proteins. Without this polarity vectorial ion and fluid transport would not be possible. Little is known of the signals and mechanisms that renal epithelial cells use to establish and maintain polarized distributions of their ion transport proteins. Analysis of ion pump sorting reveals that multiple complex signals participate in determining and regulating these proteins' subcellular localizations.

Intracrine action of angiotensin II in mesangial cells: subcellular distribution of angiotensin II receptor subtypes AT1 and AT2.

PubMed

da Silva Novaes, Antônio; Ribeiro, Rosemara Silva; Pereira, Luciana Guilhermino; Borges, Fernanda Teixeira; Boim, Mirian Aparecida

2018-02-17

Biological effects of angiotensin II (AngII) such as regulation of AngII target genes may be triggered by interaction of AngII with intracellular AngII receptor types 1 and 2 (AT 1 and AT 2 ), defined as intracrine response. The aim of this study was to examine the presence of AT 1 and AT 2 receptors in nuclear membrane of human mesangial cells (HMCs) and evaluate the possible biological effects mediated by intracellular AT 1 through an intracrine mechanism. Subcellular distribution of AT 1 and AT 2 was evaluated by immunofluorescence and by western blot in isolated nuclear extract. Endogenous intracellular synthesis of AngII was stimulated by high glucose (HG). Effects of HG were analyzed in the presence of candesartan, which prevents AngII internalization. Both receptors were found in nuclear membrane. Fluorescein isothiocyanate (FITC)-labeled AngII added to isolated nuclei produced a fluorescence that was reduced in the presence of losartan or PD-123319 and quenched in the presence of both inhibitors simultaneously. HG induced overexpression of fibronectin and increased cell proliferation in the presence of candesartan, indicating an intracrine action of AngII induced by HG. Results showed the presence of nuclear receptors in HMCs that can be activated by AngII through an intracrine response independent of cytoplasmic membrane AngII receptors.

A Novel Mutation of Human Liver Alanine:Glyoxylate Aminotransferase Causes Primary Hyperoxaluria Type 1: Immunohistochemical Quantification and Subcellular Distribution

PubMed Central

Kawai, Chikage; Minatogawa, Yohsuke; Akiyoshi, Hidetaka; Hirose, Shinichi; Suehiro, Tsunatoshi; Tone, Shigenobu

2012-01-01

A novel alanine:glyoxylate aminotransferase (AGT) mutation involved in primary hyperoxaluria type 1 (PH1) was studied in Japanese patients. Two mutations in exon 7, c.751T>A and c.752G>A, lead to a W251K amino acid substitution. Proband 1 (patient 1) was homozygous for the W251K mutation allele (DDBJ Accession No. AB292648), and AGT-specific activity in the patient’s liver was very low. To reveal the cause of the low enzymatic activity, the intracellular localization of AGT (W251K) was studied using immunohistochemistry and immunoelectron microscopy. The latter analysis showed that patient 2 had only one-fifth of the normal AGT expression per catalase, suggesting impairment of AGT (W251K) dependent transport into peroxisomes. Peroxisomal transport of human AGT is believed to be dependent on the presence of the type 1 peroxisomal targeting sequence. The C-terminal tripeptide of AGT, KKL is necessary for peroxisomal targeting. In cultured cells, EGFP-AGT (W251K) localized both in the peroxisome and cytosol. These results were consistent with the data obtained from liver analysis of patient 2. The subcellular distribution of AGT (W251K) and the results from a random mutagenesis study suggest that KKL is necessary for peroxisomal targeting of human AGT, but additional signal other than KKL may be necessary. PMID:22685354

Multiple-labelling immunoEM using different sizes of colloidal gold: alternative approaches to test for differential distribution and colocalization in subcellular structures.

PubMed

Mayhew, Terry M; Lucocq, John M

2011-03-01

Various methods for quantifying cellular immunogold labelling on transmission electron microscope thin sections are currently available. All rely on sound random sampling principles and are applicable to single immunolabelling across compartments within a given cell type or between different experimental groups of cells. Although methods are also available to test for colocalization in double/triple immunogold labelling studies, so far, these have relied on making multiple measurements of gold particle densities in defined areas or of inter-particle nearest neighbour distances. Here, we present alternative two-step approaches to codistribution and colocalization assessment that merely require raw counts of gold particles in distinct cellular compartments. For assessing codistribution over aggregate compartments, initial statistical evaluation involves combining contingency table and chi-squared analyses to provide predicted gold particle distributions. The observed and predicted distributions allow testing of the appropriate null hypothesis, namely, that there is no difference in the distribution patterns of proteins labelled by different sizes of gold particle. In short, the null hypothesis is that of colocalization. The approach for assessing colabelling recognises that, on thin sections, a compartment is made up of a set of sectional images (profiles) of cognate structures. The approach involves identifying two groups of compartmental profiles that are unlabelled and labelled for one gold marker size. The proportions in each group that are also labelled for the second gold marker size are then compared. Statistical analysis now uses a 2 × 2 contingency table combined with the Fisher exact probability test. Having identified double labelling, the profiles can be analysed further in order to identify characteristic features that might account for the double labelling. In each case, the approach is illustrated using synthetic and/or experimental datasets and can be refined to correct observed labelling patterns to specific labelling patterns. These simple and efficient approaches should be of more immediate utility to those interested in codistribution and colocalization in multiple immunogold labelling investigations.

Intracellular Protein Shuttling: A Mechanism Relevant for Myelin Repair in Multiple Sclerosis?

PubMed Central

Göttle, Peter; Küry, Patrick

2015-01-01

A prominent feature of demyelinating diseases such as multiple sclerosis (MS) is the degeneration and loss of previously established functional myelin sheaths, which results in impaired signal propagation and axonal damage. However, at least in early disease stages, partial replacement of lost oligodendrocytes and thus remyelination occur as a result of resident oligodendroglial precursor cell (OPC) activation. These cells represent a widespread cell population within the adult central nervous system (CNS) that can differentiate into functional myelinating glial cells to restore axonal functions. Nevertheless, the spontaneous remyelination capacity in the adult CNS is inefficient because OPCs often fail to generate new oligodendrocytes due to the lack of stimulatory cues and the presence of inhibitory factors. Recent studies have provided evidence that regulated intracellular protein shuttling is functionally involved in oligodendroglial differentiation and remyelination activities. In this review we shed light on the role of the subcellular localization of differentiation-associated factors within oligodendroglial cells and show that regulation of intracellular localization of regulatory factors represents a crucial process to modulate oligodendroglial maturation and myelin repair in the CNS. PMID:26151843

Optogenetic Tools for Subcellular Applications in Neuroscience.

PubMed

Rost, Benjamin R; Schneider-Warme, Franziska; Schmitz, Dietmar; Hegemann, Peter

2017-11-01

The ability to study cellular physiology using photosensitive, genetically encoded molecules has profoundly transformed neuroscience. The modern optogenetic toolbox includes fluorescent sensors to visualize signaling events in living cells and optogenetic actuators enabling manipulation of numerous cellular activities. Most optogenetic tools are not targeted to specific subcellular compartments but are localized with limited discrimination throughout the cell. Therefore, optogenetic activation often does not reflect context-dependent effects of highly localized intracellular signaling events. Subcellular targeting is required to achieve more specific optogenetic readouts and photomanipulation. Here we first provide a detailed overview of the available optogenetic tools with a focus on optogenetic actuators. Second, we review established strategies for targeting these tools to specific subcellular compartments. Finally, we discuss useful tools and targeting strategies that are currently missing from the optogenetics repertoire and provide suggestions for novel subcellular optogenetic applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device.

PubMed

Schlimpert, Susan; Flärdh, Klas; Buttner, Mark

2016-02-28

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

Ultrastructural characterization of atrial natriuretic peptide receptors (ANP-R) mRNA expression in rat kidney cortex.

PubMed

Grandclément, B; Morel, G

1998-06-01

Atrial natriuretic peptide (ANP) and two complementary peptides named brain natriuretic peptide and C-type natriuretic peptide are involved in diuresis, natriuresis, hypotension and vasorelaxation. Their actions are mediated by highly selective and specific ANP receptors. Three subtypes have been characterized and cloned: ANP receptor A, -B and -C. In the present study, the mRNA for each subtype was detected by ultrastructural in situ hybridization on ultrathin sections of Lowicryl-embedded tissue and frozen tissue. The distribution of mRNA (visualized by gold particles) for each subtype was found to differ in different cells of the nephron. The three subtypes of this receptor family were expressed in all the parts of the nephron, but their expression levels were different. The ANPR-A mRNA was the most abundant in cells of glomerulus, proximal and distal tubules. The subtype C was the least expressed mRNA in glomerulus. In contrast, the subcellular localization of the three mRNAs was similar; they were found in the cytoplasmic matrix and the euchromatin of the nucleus. In conclusion, the differential expression of these mRNAs in kidney cortex indicates that these three peptides act directly in differing parts of nephron regions which are the glomerulus, the proximal and distal tubules.

Commensal-Associated Molecular Patterns Induce Selective Toll-Like Receptor-Trafficking from Apical Membrane to Cytoplasmic Compartments in Polarized Intestinal Epithelium

PubMed Central

Cario, Elke; Brown, Dennis; McKee, Mary; Lynch-Devaney, Kathryn; Gerken, Guido; Podolsky, Daniel K.

2002-01-01

Commensal-associated molecular patterns, the major products of nonpathogenic bacteria, are present at high concentrations at the apical surface of the intestinal epithelium. However, the nature of the interaction of commensal-associated molecular patterns with the lumenal surface of the epithelium has not been defined. We have recently demonstrated that intestinal epithelial cells constitutively express several Toll-like receptors (TLRs) in vitro and in vivo that seem to be the key receptors responsible for immune cell activation in response to various bacterial products. In this study we characterize the subcellular distribution of two major TLRs, TLR2 and TLR4, and their ligand-specific dynamic regulation in the model human intestinal epithelial cell line T84. Immunocytochemical studies indicate that TLR2 and TLR4 are constitutively expressed at the apical pole of differentiated T84 cells. After stimulation with lipopolysaccharide or peptidoglycan, TLRs selectively traffic to cytoplasmic compartments near the basolateral membrane. Thus, we demonstrate that TLRs are positioned at the apical pole where they are poised to monitor the sensitive balance of the lumenal microbial array. The results of this dynamic epithelial surveillance can then be conveyed to the underlying cell populations of the lamina propria via these innate immune pattern recognition receptors. PMID:11786410

Nucleocytoplasmic transfer of cyclin dependent kinase 5 and its binding to puromycin-sensitive aminopeptidase in Dictyostelium discoideum.

PubMed

Huber, Robert J; O'Day, Danton H

2011-08-01

The Dictyostelium discoideum homolog of mammalian cyclin dependent kinase 5 (Cdk5) has previously been shown to be required for optimal growth and differentiation in this model organism, however, the subcellular localization of the protein has not previously been studied. In this study, immunolocalizations and a GFP fusion construct localized Cdk5 predominantly to the nucleus of vegetative cells. Western blots showed that Cdk5 was present in both nuclear and non-nuclear fractions, suggesting a functional role in both cellular locales. During the early stages of mitosis, Cdk5 gradually moved from a punctate nucleoplasmic distribution to localize adjacent to the inner nuclear envelope. During anaphase and telophase, Cdk5 localized to the cytoplasm and was not detected in the nucleoplasm. Cdk5 returned to the nucleus during cytokinesis. Proteolytic activity has been shown to be a critical regulator of the cell cycle. Immunoprecipitations coupled with immunolocalizations identified puromycin-sensitive aminopeptidase A (PsaA) as a potential Cdk5 binding partner in Dictyostelium. Immunoprecipitations also identified two phosphotyrosine proteins (35 and 18 kDa) that may interact with Cdk5 in vivo. Together, this work provides new insight into the localization of Cdk5, its function during cell division, and its binding to a proteolytic enzyme in Dictyostelium.

How do bacteria localize proteins to the cell pole?

PubMed Central

Laloux, Géraldine; Jacobs-Wagner, Christine

2014-01-01

ABSTRACT It is now well appreciated that bacterial cells are highly organized, which is far from the initial concept that they are merely bags of randomly distributed macromolecules and chemicals. Central to their spatial organization is the precise positioning of certain proteins in subcellular domains of the cell. In particular, the cell poles – the ends of rod-shaped cells – constitute important platforms for cellular regulation that underlie processes as essential as cell cycle progression, cellular differentiation, virulence, chemotaxis and growth of appendages. Thus, understanding how the polar localization of specific proteins is achieved and regulated is a crucial question in bacterial cell biology. Often, polarly localized proteins are recruited to the poles through their interaction with other proteins or protein complexes that were already located there, in a so-called diffusion-and-capture mechanism. Bacteria are also starting to reveal their secrets on how the initial pole ‘recognition’ can occur and how this event can be regulated to generate dynamic, reproducible patterns in time (for example, during the cell cycle) and space (for example, at a specific cell pole). Here, we review the major mechanisms that have been described in the literature, with an emphasis on the self-organizing principles. We also present regulation strategies adopted by bacterial cells to obtain complex spatiotemporal patterns of protein localization. PMID:24345373

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

PubMed Central

Schlimpert, Susan; Flärdh, Klas; Buttner, Mark

2016-01-01

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series. PMID:26967231

alpha2-chimaerin, a Cdc42/Rac1 regulator, is selectively expressed in the rat embryonic nervous system and is involved in neuritogenesis in N1E-115 neuroblastoma cells.

PubMed

Hall, C; Michael, G J; Cann, N; Ferrari, G; Teo, M; Jacobs, T; Monfries, C; Lim, L

2001-07-15

Neuronal differentiation involves Rac and Cdc42 GTPases. alpha-Chimaerin, a Rac/Cdc42 regulator, occurs as alpha1- and alternatively spliced Src homology 2 (SH2) domain-containing alpha2-isoforms. alpha2-chimaerin mRNA was highly expressed in the rat embryonic nervous system, especially in early postmitotic neurons. alpha1-chimaerin mRNA was undetectable before embryonic day 16.5. Adult alpha2-chimaerin mRNA was restricted to neurons within specific brain regions, with highest expression in the entorhinal cortex. alpha2-chimaerin protein localized to neuronal perikarya, dendrites, and axons. The overall pattern of alpha2-chimaerin mRNA expression resembles that of cyclin-dependent kinase regulator p35 (CDK5/p35) which participates in neuronal differentiation and with which chimaerin interacts. To determine whether alpha2-chimaerin may have a role in neuronal differentiation and the relevance of the SH2 domain, the morphological effects of both chimaerin isoforms were investigated in N1E-115 neuroblastoma cells. When plated on poly-lysine, transient alpha2-chimaerin but not alpha1-chimaerin transfectants formed neurites. Permanent alpha2-chimaerin transfectants generated neurites whether or not they were stimulated by serum starvation, and many cells were enlarged. Permanent alpha1-chimaerin transfectants displayed numerous microspikes and contained F-actin clusters, a Cdc42-phenotype, but generated few neurites. In neuroblastoma cells, alpha2-chimaerin was predominantly soluble with some being membrane-associated, whereas alpha1-chimaerin was absent from the cytosol, being membrane- and cytoskeleton-associated, paralleling their subcellular distribution in brain. Transient transfection with alpha2-chimaerin mutated in the SH2 domain (N94H) generated an alpha1-chimaerin-like phenotype, protein partitioned in the particulate fraction, and in NGF-stimulated pheochromocytoma cell line 12 (PC12) cells, neurite formation was inhibited. These results indicate a role for alpha2-chimaerin in morphological differentiation for which its SH2 domain is vital.

Modeling of Protein Subcellular Localization in Bacteria

NASA Astrophysics Data System (ADS)

Xu, Xiaohua; Kulkarni, Rahul

2006-03-01

Specific subcellular localization of proteins is a vital component of important bacterial processes: e.g. the Min proteins which regulate cell division in E. coli and Spo0J-Soj system which is critical for sporulation in B. subtilis. We examine how the processes of diffusion and membrane attachment contribute to protein subcellular localization for the above systems. We use previous experimental results to suggest minimal models for these processes. For the minimal models, we derive analytic expressions which provide insight into the processes that determine protein subcellular localization. Finally, we present the results of numerical simulations for the systems studied and make connections to the observed experiemental phenomenology.

LOCATE: a mouse protein subcellular localization database

PubMed Central

Fink, J. Lynn; Aturaliya, Rajith N.; Davis, Melissa J.; Zhang, Fasheng; Hanson, Kelly; Teasdale, Melvena S.; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Teasdale, Rohan D.

2006-01-01

We present here LOCATE, a curated, web-accessible database that houses data describing the membrane organization and subcellular localization of proteins from the FANTOM3 Isoform Protein Sequence set. Membrane organization is predicted by the high-throughput, computational pipeline MemO. The subcellular locations of selected proteins from this set were determined by a high-throughput, immunofluorescence-based assay and by manually reviewing >1700 peer-reviewed publications. LOCATE represents the first effort to catalogue the experimentally verified subcellular location and membrane organization of mammalian proteins using a high-throughput approach and provides localization data for ∼40% of the mouse proteome. It is available at . PMID:16381849

Molecular mechanism of serine/threonine protein phosphatase 1 (PP1cα-PP1r7) in spermatogenesis of Toxocara canis.

PubMed

Ma, Guang Xu; Zhou, Rong Qiong; Song, Zhen Hui; Zhu, Hong Hong; Zhou, Zuo Yong; Zeng, Yuan Qin

2015-09-01

Toxocariasis is one of the most important, but neglected, zoonoses, which is mainly caused by Toxocara canis. To better understand the role of serine/threonine protein phosphatase 1 (PP1) in reproductive processes of male adult T. canis, differential expression analysis was used to reveal the profiles of PP1 catalytic subunit α (PP1cα) gene Tc-stp-1 and PP1 regulatory subunit 7 (PP1r7) gene TcM-1309. Indirect fluorescence immunocytochemistry was carried out to determine the subcellular distribution of PP1cα. Double-stranded RNA interference (RNAi) assays were employed to illustrate the function and mechanism of PP1cα in male adult reproduction. Real-time quantitative PCR (qPCR) showed transcriptional consistency of Tc-stp-1 and TcM-1309 in sperm-producing germline tissues and localization research showed cytoplasmic distribution of PP1cα in sf9 cells, which indicated relevant involvements of PP1cα and PP1r7 in spermatogenesis. Moreover, spatiotemporal transcriptional differences of Tc-stp-1 were determined by gene knockdown analysis, which revealed abnormal morphologies and blocked meiotic divisions of spermatocytes by phenotypic aberration scanning, thereby highlighting the crucial involvement of PP1cα in spermatogenesis. These results revealed a PP1cα-PP1r7 mechanism by which PP1 regulates kinetochore-microtubule interactions in spermatogenesis and provided important clues to identify novel drug or vaccine targets for toxocariasis control. Copyright © 2015 Elsevier B.V. All rights reserved.

Ecto-nucleoside triphosphate diphosphohydrolase 3 in the ventral and lateral hypothalamic area of female rats: morphological characterization and functional implications

PubMed Central

Kiss, David S; Zsarnovszky, Attila; Horvath, Krisztina; Gyorffy, Andrea; Bartha, Tibor; Hazai, Diana; Sotonyi, Peter; Somogyi, Virag; Frenyo, Laszlo V; Diano, Sabrina

2009-01-01

Background Based on its distribution in the brain, ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) may play a role in the hypothalamic regulation of homeostatic systems, including feeding, sleep-wake behavior and reproduction. To further characterize the morphological attributes of NTPDase3-immunoreactive (IR) hypothalamic structures in the rat brain, here we investigated: 1.) The cellular and subcellular localization of NTPDase3; 2.) The effects of 17β-estradiol on the expression level of hypothalamic NTPDase3; and 3.) The effects of NTPDase inhibition in hypothalamic synaptosomal preparations. Methods Combined light- and electron microscopic analyses were carried out to characterize the cellular and subcellular localization of NTPDase3-immunoreactivity. The effects of estrogen on hypothalamic NTPDase3 expression was studied by western blot technique. Finally, the effects of NTPDase inhibition on mitochondrial respiration were investigated using a Clark-type oxygen electrode. Results Combined light- and electron microscopic analysis of immunostained hypothalamic slices revealed that NTPDase3-IR is linked to ribosomes and mitochondria, is predominantly present in excitatory axon terminals and in distinct segments of the perikaryal plasma membrane. Immunohistochemical labeling of NTPDase3 and glutamic acid decarboxylase (GAD) indicated that γ-amino-butyric-acid- (GABA) ergic hypothalamic neurons do not express NTPDase3, further suggesting that in the hypothalamus, NTPDase3 is predominantly present in excitatory neurons. We also investigated whether estrogen influences the expression level of NTPDase3 in the ventrobasal and lateral hypothalamus. A single subcutaneous injection of estrogen differentially increased NTPDase3 expression in the medial and lateral parts of the hypothalamus, indicating that this enzyme likely plays region-specific roles in estrogen-dependent hypothalamic regulatory mechanisms. Determination of mitochondrial respiration rates with and without the inhibition of NTPDases confirmed the presence of NTPDases, including NTPDase3 in neuronal mitochondria and showed that blockade of mitochondrial NTPDase functions decreases state 3 mitochondrial respiration rate and total mitochondrial respiratory capacity. Conclusion Altogether, these results suggest the possibility that NTPDases, among them NTPDase3, may play an estrogen-dependent modulatory role in the regulation of intracellular availability of ATP needed for excitatory neuronal functions including neurotransmission. PMID:19383175

Effect of recombinant human gamma interferon on intracellular activities of antibiotics against Listeria monocytogenes in the human macrophage cell line THP-1.

PubMed Central

Scorneaux, B; Ouadrhiri, Y; Anzalone, G; Tulkens, P M

1996-01-01

Listeria monocytogenes is a facultative intracellular pathogen which enters cells by endocytosis and reaches phagolysosomes from where it escapes and multiplies in the cytosol of untreated cells. Exposure of macrophages to gamma interferon (IFN-gamma) restricts L. monocytogenes to phagosomes and prevents its intracellular multiplication. We have tested whether IFN-gamma also modulates the susceptibility of L. monocytogenes to antibiotics. We selected drugs from three different classes displaying marked properties concerning their cellular accumulation and subcellular distribution, namely, ampicillin (not accumulated by cells but present in cytosol), azithromycin (largely accumulated by cells but mostly restricted to lysosomes), and sparfloxacin (accumulated to a fair extent but detected only in cytosol). We used a continuous line of myelomonocytic cells (THP-1 macrophages), which display specific surface receptors for IFN-gamma, and examined the activity of these antibiotics against L. monocytogenes Hly+ (virulent variant) and L. monocytogenes Hly- (a nonvirulent variant defective in hemolysin production). Untreated THP-1 and phorbol myristate acetate-differentiated THP-1 were permissive for infection and multiplication of intracellular L. monocytogenes Hly+ (virulent variant). All three antibiotics tested were bactericidal against this Listeria strain when added to an extracellular concentration of 10x their MIC. After preexposure of THP-1 to IFN-gamma, L. monocytogenes Hly+ was still phagocytosed but no longer grew intracellularly. The activity of ampicillin became almost undetectable (antagonistic effect), and that of azithromycin was unchanged (additive effect with that of IFN-gamma), whereas that of sparfloxacin was markedly enhanced (synergy). A similar behavior (lack of bacterial growth, associated with a loss of activity of ampicillin, an enhanced activity of sparfloxacin, and unchanged activity of azithromycin) was observed in cells infected with L. monocytogenes Hly-. This modulation of antibiotic activity, which we ascribe to the change of subcellular localization of L. monocytogenes caused by IFN-gamma or by the lack of virulence factor, could result from a change in bacterial responsiveness to antibiotics, a modification of the drug activity, or differences in drug bioavailabilities between cytosol and phagosomes. PMID:8723471

Finding the Subcellular Location of Barley, Wheat, Rice and Maize Proteins: The Compendium of Crop Proteins with Annotated Locations (cropPAL).

PubMed

Hooper, Cornelia M; Castleden, Ian R; Aryamanesh, Nader; Jacoby, Richard P; Millar, A Harvey

2016-01-01

Barley, wheat, rice and maize provide the bulk of human nutrition and have extensive industrial use as agricultural products. The genomes of these crops each contains >40,000 genes encoding proteins; however, the major genome databases for these species lack annotation information of protein subcellular location for >80% of these gene products. We address this gap, by constructing the compendium of crop protein subcellular locations called crop Proteins with Annotated Locations (cropPAL). Subcellular location is most commonly determined by fluorescent protein tagging of live cells or mass spectrometry detection in subcellular purifications, but can also be predicted from amino acid sequence or protein expression patterns. The cropPAL database collates 556 published studies, from >300 research institutes in >30 countries that have been previously published, as well as compiling eight pre-computed subcellular predictions for all Hordeum vulgare, Triticum aestivum, Oryza sativa and Zea mays protein sequences. The data collection including metadata for proteins and published studies can be accessed through a search portal http://crop-PAL.org. The subcellular localization information housed in cropPAL helps to depict plant cells as compartmentalized protein networks that can be investigated for improving crop yield and quality, and developing new biotechnological solutions to agricultural challenges. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Surface-charge-dependent cell localization and cytotoxicity of cerium oxide nanoparticles.

PubMed

Asati, Atul; Santra, Santimukul; Kaittanis, Charalambos; Perez, J Manuel

2010-09-28

Cerium oxide nanoparticles (nanoceria) have shown great potential as antioxidant and radioprotective agents for applications in cancer therapy. Recently, various polymer-coated nanoceria preparations have been developed to improve their aqueous solubility and allow for surface functionalization of these nanoparticles. However, the interaction of polymer-coated nanoceria with cells, their uptake mechanism, and subcellular localization are poorly understood. Herein, we engineered polymer-coated cerium oxide nanoparticles with different surface charges (positive, negative, and neutral) and studied their internalization and toxicity in normal and cancer cell lines. The results showed that nanoceria with a positive or neutral charge enters most of the cell lines studied, while nanoceria with a negative charge internalizes mostly in the cancer cell lines. Moreover, upon entry into the cells, nanoceria is localized to different cell compartments (e.g., cytoplasm and lysosomes) depending on the nanoparticle's surface charge. The internalization and subcellular localization of nanoceria plays a key role in the nanoparticles' cytotoxicity profile, exhibiting significant toxicity when they localize in the lysosomes of the cancer cells. In contrast, minimal toxicity is observed when they localize into the cytoplasm or do not enter the cells. Taken together, these results indicate that the differential surface-charge-dependent localization of nanoceria in normal and cancer cells plays a critical role in the nanoparticles' toxicity profile.

Laser Surgery: Organelles to Organs

NASA Astrophysics Data System (ADS)

Berns, Michael W. D.

1998-03-01

Understanding the physical mechanisms of light interaction with biological molecules and structure has resulted in the application of photons to a wide variety of biological and medical problems ranging from subcellular manipulation/surgery to the successful diagnosis and treatment of human disease. Mechanisms such as the generation and transfer of heat, light-driven chemistry (photochemistry), high peak power acoustic-mechanical effects, high photon-energy induced bond breaking, and optical induced forces through momentum transfer, are being utilized in single cells at the microscopic (submicron and micron) level as well as the macroscopic level in tissue and organs. At the subcellular level, focused laser microbeams (laser scissors and tweezers) are being used to cut and move chromosomes to study genetic function as well as to clone and sequence genes. The same laser technology is being used to manipulate a variety of cell organelles such as mitochondria, cell membranes, nucleoli, and mitochondria in order to study their functions in cell physiology. At the tissue level, lasers are being used to diagnose and treat malignancy in combination with light-activated drugs, to ablate cornea and other hard and soft tissue through ultraviolet photoablation, to selectively ablate structures within the skin under controlled heating/cooling conditions, and to differentiate normal from abnormal tissue using a variety of fluorescence detection and light scattering techniques.

Nonreceptor Protein-Tyrosine Kinases in Neutrophil Activation

PubMed

Welch; Mauran; Maridonneau-Parini

1996-06-01

Nonreceptor protein-tyrosine kinases are involved in the regulation of almost all neutrophil responses such as adhesion, chemotaxis, priming, oxidative burst, and degranulation. Here, we show that phagocytosis is also regulated by protein-tyrosine kinase activity. Using various protein-tyrosine kinase inhibitors, we further demonstrate that opsonized zymosan-induced degranulation of specific and azurophil granules is regulated by protein-tyrosine kinase activity, whereas phorbol ester-induced degranulation is not. Several of the nonreceptor protein-tyrosine kinases involving in neutrophil signal transduction are known, including Fgr, Hck, Lyn, Yes, and Syk. Among these, Hck and Fgr are localized on the azurophil and specific granules, suggesting the involvement of these two protein-tyrosine kinases in the regulation of degranulation. In this report, we characterize some of the molecular properties of Hck and Fgr. We discuss the methods generally used for the measurement of protein-tyrosine kinase activities in neutrophils highlighting precautions against proteolysis. In addition, we show that in subcellular fractions of retinoic acid-differentiated neutrophil-like NB4 cells, the 59- and 61-kDa forms of Hck are attached to the membranes of their respective compartments by different mechanisms. Finally, we discuss the functional roles of protein-tyrosine kinases in the regulation of neutrophil activation and speculate on the importance of their subcellular localization.

Commitment of Scaffold Proteins in the Onco-Biology of Human Colorectal Cancer and Liver Metastases after Oxaliplatin-Based Chemotherapy.

PubMed

Rotoli, Deborah; Morales, Manuel; Ávila, Julio; Maeso, María Del Carmen; García, María Del Pino; Mobasheri, Ali; Martín-Vasallo, Pablo

2017-04-22

Scaffold proteins play pivotal roles in the regulation of signaling pathways, integrating external and internal stimuli to various cellular outputs. We report the pattern of cellular and subcellular expression of scaffoldins angiomotin-like 2 (AmotL2), FK506 binding protein 5 (FKBP51) and IQ motif containing GTPase-activating protein 1 (IQGAP1) in colorectal cancer (CRC) and metastases in liver resected after oxaliplatin-based chemotherapy (CT). Positive immunostaining for the three scaffoldins was found in most cells in healthy colon, tumor, healthy liver and metastasized liver. The patterns of expression of AmotL2, FKBP51 and IQGAP1 show the greatest variability in immune system cells and neurons and glia cells and the least in blood vessel cells. The simultaneous subcellular localization in tumor cells and other cell types within the tumor suggest an involvement of these three scaffoldins in cancer biology, including a role in Epithelial Mesenchymal Transition. The display in differential localization and quantitative expression of AmotL2, FKBP51, and IQGAP1 could be used as biomarkers for more accurate tumor staging and as potential targets for anti-cancer therapeutics by blocking or slowing down their interconnecting functions. Tough further research needs to be done in order to improve these assessments.

Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

NASA Astrophysics Data System (ADS)

Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

2014-07-01

Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

RABA Members Act in Distinct Steps of Subcellular Trafficking of the FLAGELLIN SENSING2 Receptor[W

PubMed Central

Choi, Seung-won; Tamaki, Takayuki; Ebine, Kazuo; Uemura, Tomohiro; Ueda, Takashi; Nakano, Akihiko

2013-01-01

Cell surface proteins play critical roles in the perception of environmental stimuli at the plasma membrane (PM) and ensuing signal transduction. Intracellular localization of such proteins must be strictly regulated, which requires elaborate integration of exocytic and endocytic trafficking pathways. Subcellular localization of Arabidopsis thaliana FLAGELLIN SENSING2 (FLS2), a receptor that recognizes bacterial flagellin, also depends on membrane trafficking. However, our understanding about the mechanisms involved is still limited. In this study, we visualized ligand-induced endocytosis of FLS2 using green fluorescent protein (GFP)-tagged FLS2 expressed in Nicotiana benthamiana. Upon treatment with the flg22 peptide, internalized FLS2-GFP from the PM was transported to a compartment with properties intermediate between the trans-Golgi network (TGN) and the multivesicular endosome. This compartment gradually discarded the TGN characteristics as it continued along the trafficking pathway. We further found that FLS2 endocytosis involves distinct RABA/RAB11 subgroups at different steps. Moreover, we demonstrated that transport of de novo–synthesized FLS2 to the PM also involves a distinct RABA/RAB11 subgroup. Our results demonstrate the complex regulatory system for properly localizing FLS2 and functional differentiation in RABA members in endo- and exocytosis. PMID:23532067

Isolation of the Lateral Border Recycling Compartment using a diaminobenzidine-induced density shift

PubMed Central

Sullivan, David P.; Rüffer, Claas; Muller, William A.

2014-01-01

The migration of leukocytes across the endothelium and into tissue is critical to mounting an inflammatory response. The Lateral Border Recycling Compartment (LBRC), a complex vesicular-tubule invagination of the plasma membrane found at endothelial cell borders, plays an important role in the this process. Although a few proteins have been shown to be present in the LBRC, no unique marker is known. Here we detail methods that can be used to characterize a subcellular compartment that lacks an identifying marker. Initial characterization of the LBRC was performed using standard subcellular fractionation with sucrose gradients and took advantage of the observation that the compartment migrated at a lower density than other membrane compartments. To isolate larger quantities of the compartment, we modified a classic technique known as a diaminobenzidine (DAB)-induced density shift. The DAB-induced density shift allowed for specific isolation of membranes labeled with HRP conjugated antibody. Because the LBRC could be differentially labeled at 4°C and 37°C, we were able to identify proteins that are enriched in the compartment, despite lacking a unique marker. These methods serve as a model to others studying poorly characterized compartments and organelles and are applicable to a wide variety of biological systems. PMID:24915828

Divergent lactate dehydrogenase isoenzyme profile in cellular compartments of primate forebrain structures.

PubMed

Duka, Tetyana; Collins, Zachary; Anderson, Sarah M; Raghanti, Mary Ann; Ely, John J; Hof, Patrick R; Wildman, Derek E; Goodman, Morris; Grossman, Lawrence I; Sherwood, Chet C

2017-07-01

The compartmentalization and association of lactate dehydrogenase (LDH) with specific cellular structures (e.g., synaptosomal, sarcoplasmic or mitochondrial) may play an important role in brain energy metabolism. Our previous research revealed that LDH in the synaptosomal fraction shifts toward the aerobic isoforms (LDH-B) among the large-brained haplorhine primates compared to strepsirrhines. Here, we further analyzed the subcellular localization of LDH in primate forebrain structures using quantitative Western blotting and ELISA. We show that, in cytosolic and mitochondrial subfractions, LDH-B expression level was relatively elevated and LDH-A declined in haplorhines compared to strepsirrhines. LDH-B expression in mitochondrial fractions of the neocortex was preferentially increased, showing a particularly significant rise in the ratio of LDH-B to LDH-A in chimpanzees and humans. We also found a significant correlation between the protein levels of LDH-B in mitochondrial fractions from haplorhine neocortex and the synaptosomal LDH-B that suggests LDH isoforms shift from a predominance of A-subunits toward B-subunits as part of a system that spatially buffers dynamic energy requirements of brain cells. Our results indicate that there is differential subcellular compartmentalization of LDH isoenzymes that evolved among different primate lineages to meet the energy requirements in neocortical and striatal cells. Copyright © 2017 Elsevier Inc. All rights reserved.

A Molecular Smart Surface for Spatio-Temporal Studies of Cell Mobility

PubMed Central

Lee, Eun-ju; Luo, Wei; Chan, Eugene W. L.; Yousaf, Muhammad N.

2015-01-01

Active migration in both healthy and malignant cells requires the integration of information derived from soluble signaling molecules with positional information gained from interactions with the extracellular matrix and with other cells. How a cell responds and moves involves complex signaling cascades that guide the directional functions of the cytoskeleton as well as the synthesis and release of proteases that facilitate movement through tissues. The biochemical events of the signaling cascades occur in a spatially and temporally coordinated manner then dynamically shape the cytoskeleton in specific subcellular regions. Therefore, cell migration and invasion involve a precise but constantly changing subcellular nano-architecture. A multidisciplinary effort that combines new surface chemistry and cell biological tools is required to understand the reorganization of cytoskeleton triggered by complex signaling during migration. Here we generate a class of model substrates that modulate the dynamic environment for a variety of cell adhesion and migration experiments. In particular, we use these dynamic substrates to probe in real-time how the interplay between the population of cells, the initial pattern geometry, ligand density, ligand affinity and integrin composition affects cell migration and growth. Whole genome microarray analysis indicates that several classes of genes ranging from signal transduction to cytoskeletal reorganization are differentially regulated depending on the nature of the surface conditions. PMID:26030281

Structural analysis of alterations in zebrafish muscle differentiation induced by simvastatin and their recovery with cholesterol.

PubMed

Campos, Laise M; Rios, Eduardo A; Midlej, Victor; Atella, Georgia C; Herculano-Houzel, Suzana; Benchimol, Marlene; Mermelstein, Claudia; Costa, Manoel Luís

2015-06-01

In vitro studies show that cholesterol is essential to myogenesis. We have been using zebrafish to overcome the limitations of the in vitro approach and to study the sub-cellular structures and processes involved during myogenesis. We use simvastatin--a drug widely used to prevent high levels of cholesterol and cardiovascular disease--during zebrafish skeletal muscle formation. Simvastatin is an efficient inhibitor of cholesterol synthesis that has various myotoxic consequences. Here, we employed simvastatin concentrations that cause either mild or severe morphological disturbances to observe changes in the cytoskeleton (intermediate filaments and microfilaments), extracellular matrix and adhesion markers by confocal microscopy. With low-dose simvastatin treatment, laminin was almost normal, and alpha-actinin was reduced in the myofibrils. With high simvastatin doses, laminin and vinculin were reduced and appeared discontinuous along the septa, with almost no myofibrils, and small amounts of desmin accumulating close to the septa. We also analyzed sub-cellular alterations in the embryos by electron microscopy, and demonstrate changes in embryo and somite size, septa shape, and in myofibril structure. These effects could be reversed by the addition of exogenous cholesterol. These results contribute to the understanding of the mechanisms of action of simvastatin in muscle cells in particular, and in the study of myogenesis in general. © The Author(s) 2015.

Structural Analysis of Alterations in Zebrafish Muscle Differentiation Induced by Simvastatin and Their Recovery with Cholesterol

PubMed Central

Campos, Laise M.; Rios, Eduardo A.; Midlej, Victor; Atella, Georgia C.; Herculano-Houzel, Suzana; Benchimol, Marlene; Mermelstein, Claudia; Costa, Manoel Luís

2015-01-01

In vitro studies show that cholesterol is essential to myogenesis. We have been using zebrafish to overcome the limitations of the in vitro approach and to study the sub-cellular structures and processes involved during myogenesis. We use simvastatin—a drug widely used to prevent high levels of cholesterol and cardiovascular disease—during zebrafish skeletal muscle formation. Simvastatin is an efficient inhibitor of cholesterol synthesis that has various myotoxic consequences. Here, we employed simvastatin concentrations that cause either mild or severe morphological disturbances to observe changes in the cytoskeleton (intermediate filaments and microfilaments), extracellular matrix and adhesion markers by confocal microscopy. With low-dose simvastatin treatment, laminin was almost normal, and alpha-actinin was reduced in the myofibrils. With high simvastatin doses, laminin and vinculin were reduced and appeared discontinuous along the septa, with almost no myofibrils, and small amounts of desmin accumulating close to the septa. We also analyzed sub-cellular alterations in the embryos by electron microscopy, and demonstrate changes in embryo and somite size, septa shape, and in myofibril structure. These effects could be reversed by the addition of exogenous cholesterol. These results contribute to the understanding of the mechanisms of action of simvastatin in muscle cells in particular, and in the study of myogenesis in general. PMID:25786435

Protein subcellular localization prediction using multiple kernel learning based support vector machine.

PubMed

Hasan, Md Al Mehedi; Ahmad, Shamim; Molla, Md Khademul Islam

2017-03-28

Predicting the subcellular locations of proteins can provide useful hints that reveal their functions, increase our understanding of the mechanisms of some diseases, and finally aid in the development of novel drugs. As the number of newly discovered proteins has been growing exponentially, which in turns, makes the subcellular localization prediction by purely laboratory tests prohibitively laborious and expensive. In this context, to tackle the challenges, computational methods are being developed as an alternative choice to aid biologists in selecting target proteins and designing related experiments. However, the success of protein subcellular localization prediction is still a complicated and challenging issue, particularly, when query proteins have multi-label characteristics, i.e., if they exist simultaneously in more than one subcellular location or if they move between two or more different subcellular locations. To date, to address this problem, several types of subcellular localization prediction methods with different levels of accuracy have been proposed. The support vector machine (SVM) has been employed to provide potential solutions to the protein subcellular localization prediction problem. However, the practicability of an SVM is affected by the challenges of selecting an appropriate kernel and selecting the parameters of the selected kernel. To address this difficulty, in this study, we aimed to develop an efficient multi-label protein subcellular localization prediction system, named as MKLoc, by introducing multiple kernel learning (MKL) based SVM. We evaluated MKLoc using a combined dataset containing 5447 single-localized proteins (originally published as part of the Höglund dataset) and 3056 multi-localized proteins (originally published as part of the DBMLoc set). Note that this dataset was used by Briesemeister et al. in their extensive comparison of multi-localization prediction systems. Finally, our experimental results indicate that MKLoc not only achieves higher accuracy than a single kernel based SVM system but also shows significantly better results than those obtained from other top systems (MDLoc, BNCs, YLoc+). Moreover, MKLoc requires less computation time to tune and train the system than that required for BNCs and single kernel based SVM.

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization.

PubMed

Roberts, Brock; Haupt, Amanda; Tucker, Andrew; Grancharova, Tanya; Arakaki, Joy; Fuqua, Margaret A; Nelson, Angelique; Hookway, Caroline; Ludmann, Susan A; Mueller, Irina A; Yang, Ruian; Horwitz, Rick; Rafelski, Susanne M; Gunawardane, Ruwanthi N

2017-10-15

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1-4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line-generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. © 2017 Roberts, Haupt, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Three-dimensional imaging of cholesterol and sphingolipids within a Madin-Darby canine kidney cell

DOE PAGES

Yeager, Ashley N.; Weber, Peter K.; Kraft, Mary L.

2016-01-08

Metabolic stable isotope incorporation and secondary ion mass spectrometry(SIMS) depth profiling performed on a Cameca NanoSIMS 50 were used to image the 18O-cholesterol and 15N-sphingolipid distributions within a portion of a Madin-Darby canine kidney (MDCK) cell. Three-dimensional representations of the component-specific isotope distributions show clearly defined regions of 18O-cholesterol and 15N-sphingolipid enrichment that seem to be separate subcellular compartments. Furthermore, the low levels of nitrogen-containing secondary ions detected at the 18O-enriched regions suggest that these 18O-cholesterol-rich structures may be lipiddroplets, which have a core consisting of cholesterol esters and triacylglycerides.

Three-dimensional imaging of cholesterol and sphingolipids within a Madin-Darby canine kidney cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeager, Ashley N.; Weber, Peter K.; Kraft, Mary L.

Metabolic stable isotope incorporation and secondary ion mass spectrometry(SIMS) depth profiling performed on a Cameca NanoSIMS 50 were used to image the 18O-cholesterol and 15N-sphingolipid distributions within a portion of a Madin-Darby canine kidney (MDCK) cell. Three-dimensional representations of the component-specific isotope distributions show clearly defined regions of 18O-cholesterol and 15N-sphingolipid enrichment that seem to be separate subcellular compartments. Furthermore, the low levels of nitrogen-containing secondary ions detected at the 18O-enriched regions suggest that these 18O-cholesterol-rich structures may be lipiddroplets, which have a core consisting of cholesterol esters and triacylglycerides.

Quantitative Analysis of Cell Nucleus Organisation

PubMed Central

Shiels, Carol; Adams, Niall M; Islam, Suhail A; Stephens, David A; Freemont, Paul S

2007-01-01

There are almost 1,300 entries for higher eukaryotes in the Nuclear Protein Database. The proteins' subcellular distribution patterns within interphase nuclei can be complex, ranging from diffuse to punctate or microspeckled, yet they all work together in a coordinated and controlled manner within the three-dimensional confines of the nuclear volume. In this review we describe recent advances in the use of quantitative methods to understand nuclear spatial organisation and discuss some of the practical applications resulting from this work. PMID:17676980

Focal calcium monitoring with targeted nanosensors at the cytosolic side of endoplasmic reticulum

NASA Astrophysics Data System (ADS)

Hou, Yanyan; Arai, Satoshi; Takei, Yoshiaki; Murata, Atsushi; Takeoka, Shinji; Suzuki, Madoka

2016-01-01

Ca2+ distribution is spatially and temporally non-uniform inside cells due to cellular compartmentalization. However, Ca2+ sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N3-fura-2) to a specific site of subcellular compartments to realize focal Ca2+ sensing. Using scAVD (single-chain avidin)-biotin interaction and a copper-free click reaction system, we linked N3-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca2+ sensors conjugated with four N3-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N3-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N3-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N3-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca2+ concentration, ER-localized N3-fura-2 successfully sensed the Ca2+ level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N3-fura-2 to subcellular compartments and the ability of sensing focal Ca2+ level changes with the specifically targeted Ca2+ sensors.

Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells

PubMed Central

Riskin, Arieh; Mond, Yehudit

2015-01-01

Background Studying the biological pathways involved in mammalian milk production during lactation could have many clinical implications. The mammary gland is unique in its requirement for transport of free glucose into the cell for the synthesis of lactose, the primary carbohydrate in milk. Objective To study GLUT1 trafficking and subcellular targeting in living mammary epithelial cells (MEC) in culture. Methods Immunocytochemistry was used to study GLUT1 hormonally regulated subcellular targeting in human MEC (HMEC). To study GLUT1 targeting and recycling in living mouse MEC (MMEC) in culture, we constructed fusion proteins of GLUT1 and green fluorescent protein (GFP) and expressed them in CIT3 MMEC. Cells were maintained in growth medium (GM), or exposed to secretion medium (SM), containing prolactin. Results GLUT1 in HMEC localized primarily to the plasma membrane in GM. After exposure to prolactin for 4 days, GLUT1 was targeted intracellularly and demonstrated a perinuclear distribution, co-localizing with lactose synthetase. The dynamic trafficking of GFP-GLUT1 fusion proteins in CIT3 MMEC suggested a basal constitutive GLUT1 recycling pathway between an intracellular pool and the cell surface that targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly within 90–110 minutes. Conclusions Our studies suggest intracellular targeting of GLUT1 to the central vesicular transport system upon exposure to prolactin. The existence of a dynamic prolactin-induced sorting machinery for GLUT1 could be important for transport of free glucose into the Golgi for lactose synthesis during lactation. PMID:26886772

Spreading the news: subcellular and organellar reactive oxygen species production and signalling.

PubMed

Mignolet-Spruyt, Lorin; Xu, Enjun; Idänheimo, Niina; Hoeberichts, Frank A; Mühlenbock, Per; Brosché, Mikael; Van Breusegem, Frank; Kangasjärvi, Jaakko

2016-06-01

As plants are sessile organisms that have to attune their physiology and morphology continuously to varying environmental challenges in order to survive and reproduce, they have evolved complex and integrated environment-cell, cell-cell, and cell-organelle signalling circuits that regulate and trigger the required adjustments (such as alteration of gene expression). Although reactive oxygen species (ROS) are essential components of this network, their pathways are not yet completely unravelled. In addition to the intrinsic chemical properties that define the array of interaction partners, mobility, and stability, ROS signalling specificity is obtained via the spatiotemporal control of production and scavenging at different organellar and subcellular locations (e.g. chloroplasts, mitochondria, peroxisomes, and apoplast). Furthermore, these cellular compartments may crosstalk to relay and further fine-tune the ROS message. Hence, plant cells might locally and systemically react upon environmental or developmental challenges by generating spatiotemporally controlled dosages of certain ROS types, each with specific chemical properties and interaction targets, that are influenced by interorganellar communication and by the subcellular location and distribution of the involved organelles, to trigger the suitable acclimation responses in association with other well-established cellular signalling components (e.g. reactive nitrogen species, phytohormones, and calcium ions). Further characterization of this comprehensive ROS signalling matrix may result in the identification of new targets and key regulators of ROS signalling, which might be excellent candidates for engineering or breeding stress-tolerant plants. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Human skeletal muscle glycogen utilization in exhaustive exercise: role of subcellular localization and fibre type

PubMed Central

Nielsen, Joachim; Holmberg, Hans-Christer; Schrøder, Henrik D; Saltin, Bengt; Ørtenblad, Niels

2011-01-01

Abstract Although glycogen is known to be heterogeneously distributed within skeletal muscle cells, there is presently little information available about the role of fibre types, utilization and resynthesis during and after exercise with respect to glycogen localization. Here, we tested the hypothesis that utilization of glycogen with different subcellular localizations during exhaustive arm and leg exercise differs and examined the influence of fibre type and carbohydrate availability on its subsequent resynthesis. When 10 elite endurance athletes (22 ± 1 years, = 68 ± 5 ml kg−1 min−1, mean ± SD) performed one hour of exhaustive arm and leg exercise, transmission electron microscopy revealed more pronounced depletion of intramyofibrillar than of intermyofibrillar and subsarcolemmal glycogen. This phenomenon was the same for type I and II fibres, although at rest prior to exercise, the former contained more intramyofibrillar and subsarcolemmal glycogen than the latter. In highly glycogen-depleted fibres, the remaining small intermyofibrillar and subsarcolemmal glycogen particles were often found to cluster in groupings. In the recovery period, when the athletes received either a carbohydrate-rich meal or only water the impaired resynthesis of glycogen with water alone was associated primarily with intramyofibrillar glycogen. In conclusion, after prolonged high-intensity exercise the depletion of glycogen is dependent on subcellular localization. In addition, the localization of glycogen appears to be influenced by fibre type prior to exercise, as well as carbohydrate availability during the subsequent period of recovery. These findings provide insight into the significance of fibre type-specific compartmentalization of glycogen metabolism in skeletal muscle during exercise and subsequent recovery. PMID:21486810

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bamunusinghe, Devinka, E-mail: dbamu001@ucr.ed; Hemenway, Cynthia L., E-mail: cindy_hemenway@ncsu.ed; Nelson, Richard S., E-mail: rsnelson@noble.or

Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A subset of TGBp3 resides in the ER atmore » the same location as the replicase. Sucrose gradient fractionation showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization represents a region where both proteins may be synthesized and/or function. There is no evidence to indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies on ER-derived membrane recruitment and membrane proliferation.« less

Simulation of Arrhythmogenic Effect of Rogue RyRs in Failing Heart by Using a Coupled Model

PubMed Central

Lu, Luyao; Xia, Ling; Zhu, Xiuwei

2012-01-01

Cardiac cells with heart failure are usually characterized by impairment of Ca2+ handling with smaller SR Ca2+ store and high risk of triggered activities. In this study, we developed a coupled model by integrating the spatiotemporal Ca2+ reaction-diffusion system into the cellular electrophysiological model. With the coupled model, the subcellular Ca2+ dynamics and global cellular electrophysiology could be simultaneously traced. The proposed coupled model was then applied to study the effects of rogue RyRs on Ca2+ cycling and membrane potential in failing heart. The simulation results suggested that, in the presence of rogue RyRs, Ca2+ dynamics is unstable and Ca2+ waves are prone to be initiated spontaneously. These release events would elevate the membrane potential substantially which might induce delayed afterdepolarizations or triggered action potentials. Moreover, the variation of membrane potential depolarization is indicated to be dependent on the distribution density of rogue RyR channels. This study provides a new possible arrhythmogenic mechanism for heart failure from subcellular to cellular level. PMID:23056145

The Fibroblast Growth Factor 14·Voltage-gated Sodium Channel Complex Is a New Target of Glycogen Synthase Kinase 3 (GSK3)*

PubMed Central

Shavkunov, Alexander S.; Wildburger, Norelle C.; Nenov, Miroslav N.; James, Thomas F.; Buzhdygan, Tetyana P.; Panova-Elektronova, Neli I.; Green, Thomas A.; Veselenak, Ronald L.; Bourne, Nigel; Laezza, Fernanda

2013-01-01

The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na+ (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14-dependent regulation of Na+ currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novel GSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions. PMID:23640885

Signaling induced by hop/STI-1 depends on endocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Americo, Tatiana A.; Chiarini, Luciana B.; Linden, Rafael

The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrP{sup C}), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrP{sup C} and activation of the Erk pathway. We tested whether clathrin-mediated endocytosis affects signaling induced by hop/STI-1. Both hyperosmolarity induced by sucrose and monodansyl-cadaverine blocked Erk activity induced by hop/STI-1, without affecting the high basal Akt activity typical of A172. The endocytosis inhibitors also affected the sub-cellular distribution of phosphorylated Erk, consistent with blockade of the latter's activity. The data indicate that signaling inducedmore » by hop/STI-1 depends on endocytosis. These findings are consistent with a role of sub-cellular trafficking in signal transduction following engagement by PrP{sup C} by ligands such as hop/STI-1, and may help help unravel both the functions of the prion protein, as well as possible loss-of-function components of prion diseases.« less

Fluorescent probe based subcellular distribution of Cu(II) ions in living electrotrophs isolated from Cu(II)-reduced biocathodes of microbial fuel cells.

PubMed

Tao, Ye; Xue, Hua; Huang, Liping; Zhou, Peng; Yang, Wei; Quan, Xie; Yuan, Jinxiu

2017-02-01

Based on the four indigenous electrotrophs (Stenotrophomonas maltophilia JY1, Citrobacter sp. JY3, Pseudomonas aeruginosa JY5 and Stenotrophomonas sp. JY6) isolated from well adapted Cu(II)-reduced biocathodes of microbial fuel cells (MFCs), a rhodamine based Cu(II) fluorescent probe was used to imaginably and quantitatively track subcellular Cu(II) ions in these electrotrophs. Cathodic electrons led to more Cu(II) ions (14.3-30.1%) in the intracellular sites at operation time of 2-3h with Cu(II) removal rates of 2.90-3.64mg/Lh whereas the absence of cathodic electrons prolonged the appearance of more Cu(II) ions (16.6-22.5%) to 5h with Cu(II) removal rates of 1.96-2.28mg/Lh. This study illustrates that cathodic electrons directed more Cu(II) ions for quicker entrance into the electrotrophic cytoplasm, and gives an alternative approach for developing imaging and functionally tracking Cu(II) ions in the electrotrophs of MFCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Localization of arginine decarboxylase in tobacco plants.

PubMed

Bortolotti, Cristina; Cordeiro, Alexandra; Alcázar, Rubén; Borrell, Antoni; Culiañez-Macià, Francisco A.; Tiburcio, Antonio F.; Altabella, Teresa

2004-01-01

The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli, and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types.

Subcellular localization for Gram positive and Gram negative bacterial proteins using linear interpolation smoothing model.

PubMed

Saini, Harsh; Raicar, Gaurav; Dehzangi, Abdollah; Lal, Sunil; Sharma, Alok

2015-12-07

Protein subcellular localization is an important topic in proteomics since it is related to a protein׳s overall function, helps in the understanding of metabolic pathways, and in drug design and discovery. In this paper, a basic approximation technique from natural language processing called the linear interpolation smoothing model is applied for predicting protein subcellular localizations. The proposed approach extracts features from syntactical information in protein sequences to build probabilistic profiles using dependency models, which are used in linear interpolation to determine how likely is a sequence to belong to a particular subcellular location. This technique builds a statistical model based on maximum likelihood. It is able to deal effectively with high dimensionality that hinders other traditional classifiers such as Support Vector Machines or k-Nearest Neighbours without sacrificing performance. This approach has been evaluated by predicting subcellular localizations of Gram positive and Gram negative bacterial proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

BUSCA: an integrative web server to predict subcellular localization of proteins.

PubMed

Savojardo, Castrense; Martelli, Pier Luigi; Fariselli, Piero; Profiti, Giuseppe; Casadio, Rita

2018-04-30

Here, we present BUSCA (http://busca.biocomp.unibo.it), a novel web server that integrates different computational tools for predicting protein subcellular localization. BUSCA combines methods for identifying signal and transit peptides (DeepSig and TPpred3), GPI-anchors (PredGPI) and transmembrane domains (ENSEMBLE3.0 and BetAware) with tools for discriminating subcellular localization of both globular and membrane proteins (BaCelLo, MemLoci and SChloro). Outcomes from the different tools are processed and integrated for annotating subcellular localization of both eukaryotic and bacterial protein sequences. We benchmark BUSCA against protein targets derived from recent CAFA experiments and other specific data sets, reporting performance at the state-of-the-art. BUSCA scores better than all other evaluated methods on 2732 targets from CAFA2, with a F1 value equal to 0.49 and among the best methods when predicting targets from CAFA3. We propose BUSCA as an integrated and accurate resource for the annotation of protein subcellular localization.

MultiP-Apo: A Multilabel Predictor for Identifying Subcellular Locations of Apoptosis Proteins

PubMed Central

Li, Hui; Wang, Rong; Gan, Yong

2017-01-01

Apoptosis proteins play an important role in the mechanism of programmed cell death. Predicting subcellular localization of apoptosis proteins is an essential step to understand their functions and identify drugs target. Many computational prediction methods have been developed for apoptosis protein subcellular localization. However, these existing works only focus on the proteins that have one location; proteins with multiple locations are either not considered or assumed as not existing when constructing prediction models, so that they cannot completely predict all the locations of the apoptosis proteins with multiple locations. To address this problem, this paper proposes a novel multilabel predictor named MultiP-Apo, which can predict not only apoptosis proteins with single subcellular location but also those with multiple subcellular locations. Specifically, given a query protein, GO-based feature extraction method is used to extract its feature vector. Subsequently, the GO feature vector is classified by a new multilabel classifier based on the label-specific features. It is the first multilabel predictor ever established for identifying subcellular locations of multilocation apoptosis proteins. As an initial study, MultiP-Apo achieves an overall accuracy of 58.49% by jackknife test, which indicates that our proposed predictor may become a very useful high-throughput tool in this area. PMID:28744305

MultiP-Apo: A Multilabel Predictor for Identifying Subcellular Locations of Apoptosis Proteins.

PubMed

Wang, Xiao; Li, Hui; Wang, Rong; Zhang, Qiuwen; Zhang, Weiwei; Gan, Yong

2017-01-01

Apoptosis proteins play an important role in the mechanism of programmed cell death. Predicting subcellular localization of apoptosis proteins is an essential step to understand their functions and identify drugs target. Many computational prediction methods have been developed for apoptosis protein subcellular localization. However, these existing works only focus on the proteins that have one location; proteins with multiple locations are either not considered or assumed as not existing when constructing prediction models, so that they cannot completely predict all the locations of the apoptosis proteins with multiple locations. To address this problem, this paper proposes a novel multilabel predictor named MultiP-Apo, which can predict not only apoptosis proteins with single subcellular location but also those with multiple subcellular locations. Specifically, given a query protein, GO-based feature extraction method is used to extract its feature vector. Subsequently, the GO feature vector is classified by a new multilabel classifier based on the label-specific features. It is the first multilabel predictor ever established for identifying subcellular locations of multilocation apoptosis proteins. As an initial study, MultiP-Apo achieves an overall accuracy of 58.49% by jackknife test, which indicates that our proposed predictor may become a very useful high-throughput tool in this area.

Sensitivity of Neural Stem Cell Survival, Differentiation and Neurite Outgrowth within 3D Hydrogels to Environmental Heavy Metals

PubMed Central

Tasneem, Sameera; Farrell, Kurt; Lee, Moo-Yeal; Kothapalli, Chandrasekhar R.

2015-01-01

We investigated the sensitivity of embryonic murine neural stem cells exposed to 10 pM – 10 μM concentrations of three heavy metals (Cd, Hg, Pb), continuously for 14 days within 3D collagen hydrogels. Critical endpoints for neurogenesis such as survival, differentiation and neurite outgrowth were assessed. Results suggest significant compromise in cell viability within the first four days at concentrations ≥ 10 nM, while lower concentrations induced a more delayed effect. Mercury and lead suppressed neural differentiation at as low as 10 pM concentration within 7 days, while all three metals inhibited neural and glial differentiation by day 14. Neurite outgrowth remained unaffected at lower cadmium or mercury concentrations (≤ 100 pM), but was completely repressed beyond day 1 at higher concentrations. Higher metal concentrations (≥ 100 pM) suppressed NSC differentiation to motor or dopaminergic neurons. Cytokines and chemokines released by NSCs, and the sub-cellular mechanisms by which metals induce damage to NSCs have been quantified and correlated to phenotypic data. The observed degree of toxicity in NSC cultures is in the order: lead > mercury > cadmium. Results point to the use of biomimetic 3D culture models to screen the toxic effects of heavy metals during developmental stages, and investigate their underlying mechanistic pathways. PMID:26621541

Seawater acidification aggravated cadmium toxicity in the oyster Crassostrea gigas: Metal bioaccumulation, subcellular distribution and multiple physiological responses.

PubMed

Cao, Ruiwen; Liu, Yongliang; Wang, Qing; Dong, Zhijun; Yang, Dinglong; Liu, Hui; Ran, Wen; Qu, Yi; Zhao, Jianmin

2018-06-17

Mounting evidence has demonstrated the combined effects of ocean acidification (OA) and other environmental stressors on marine organisms. Although metal pollution is widely distributed in coasts and estuaries, the combined effects of OA and metal pollution have received little attention until recent years. In this study, the accumulation and subcellular distribution of cadmium (Cd) and the physiological responses of the oyster Crassostrea gigas were investigated after 31 days of exposure to OA and Cd, either alone or in combination. Increased Cd accumulation was found both in gills (about 57% increase at pH 7.8, 22% increase at pH 7.6) and digestive glands (about 38% increase at pH 7.8, 22% increase at pH 7.6) of C. gigas under elevated pCO 2 exposure. Although a similar total Cd accumulation pattern was seen in oyster gills and digestive glands, a higher partition of Cd in the BIM (biologically inactive metal) fractions of gills (about 60%) was found in Cd-exposed treatments compared to the digestive glands (about 45%), which might correspond to the generally lower toxicity in gills. Moreover, synergetic effects of Cd and OA on the oxidative stresses, histopathological damage, and apoptosis of exposed oysters were observed in this study, which might be explained by significant interactions of these two factors on increased generation of ROS. These findings demonstrated that OA could aggravate the toxicity of metals in marine organisms, with significant implications for coastal benthic ecosystems regarding the widespread metal contamination and the concurrent increase of acidified seawater. Copyright © 2018 Elsevier B.V. All rights reserved.

An approach to estimate spatial distribution of analyte within cells using spectrally-resolved fluorescence microscopy.

PubMed

Sharma, Dharmendar Kumar; Irfanullah, Mir; Basu, Santanu Kumar; Madhu, Sheri; De, Suman; Jadhav, Sameer; Ravikanth, Mangalampalli; Chowdhury, Arindam

2017-01-18

While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in cellular media due to strong cross-talk between energetically separated detection channels.

Sex Differences in the Subcellular Distribution of Corticotropin-Releasing Factor Receptor 1 in the Rat Hippocampus following Chronic Immobilization Stress.

PubMed

McAlinn, Helena R; Reich, Batsheva; Contoreggi, Natalina H; Kamakura, Renata Poulton; Dyer, Andreina G; McEwen, Bruce S; Waters, Elizabeth M; Milner, Teresa A

2018-07-15

Corticotropin-releasing factor receptors (CRFR1) contribute to stress-induced adaptations in hippocampal structure and function that can affect learning and memory processes. Our prior studies showed that female rats with elevated estrogens compared to males have more plasmalemmal CRFR1 in CA1 pyramidal cells, suggesting a greater sensitivity to stress. Here, we examined the distribution of hippocampal CRFR1 following chronic immobilization stress (CIS) in female and male rats using immuno-electron microscopy. Without stress, total CRFR1 dendritic levels were higher in females in CA1 and in males in the hilus; moreover, plasmalemmal CRFR1 was elevated in pyramidal cell dendrites in CA1 in females and in CA3 in males. Following CIS, near-plasmalemmal CRFR1 increased in CA1 pyramidal cell dendrites in males but not to levels of control or CIS females. In CA3 and the hilus, CIS decreased cytoplasmic and total CRFR1 in dendrites in males only. These results suggest that in naive rats, CRF could induce a greater activation of CA1 pyramidal cells in females than males. Moreover, after CIS, which leads to even greater sex differences in CRFR1 by trafficking it to different subcellular compartments, CRF could enhance activation of CA1 pyramidal cells in males but to a lesser extent than either unstressed or CIS females. Additionally, CA3 pyramidal cells and inhibitory interneurons in males have heightened sensitivity to CRF, regardless of stress state. These sex differences in CRFR1 distribution and trafficking in the hippocampus may contribute to reported sex differences in hippocampus-dependent learning processes in baseline conditions and following chronic stress. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

An approach to estimate spatial distribution of analyte within cells using spectrally-resolved fluorescence microscopy

NASA Astrophysics Data System (ADS)

Sharma, Dharmendar Kumar; Irfanullah, Mir; Basu, Santanu Kumar; Madhu, Sheri; De, Suman; Jadhav, Sameer; Ravikanth, Mangalampalli; Chowdhury, Arindam

2017-03-01

While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in cellular media due to strong cross-talk between energetically separated detection channels. Dedicated to Professor Kankan Bhattacharyya.

Multilabel learning via random label selection for protein subcellular multilocations prediction.

PubMed

Wang, Xiao; Li, Guo-Zheng

2013-01-01

Prediction of protein subcellular localization is an important but challenging problem, particularly when proteins may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular localization methods are only used to deal with the single-location proteins. In the past few years, only a few methods have been proposed to tackle proteins with multiple locations. However, they only adopt a simple strategy, that is, transforming the multilocation proteins to multiple proteins with single location, which does not take correlations among different subcellular locations into account. In this paper, a novel method named random label selection (RALS) (multilabel learning via RALS), which extends the simple binary relevance (BR) method, is proposed to learn from multilocation proteins in an effective and efficient way. RALS does not explicitly find the correlations among labels, but rather implicitly attempts to learn the label correlations from data by augmenting original feature space with randomly selected labels as its additional input features. Through the fivefold cross-validation test on a benchmark data set, we demonstrate our proposed method with consideration of label correlations obviously outperforms the baseline BR method without consideration of label correlations, indicating correlations among different subcellular locations really exist and contribute to improvement of prediction performance. Experimental results on two benchmark data sets also show that our proposed methods achieve significantly higher performance than some other state-of-the-art methods in predicting subcellular multilocations of proteins. The prediction web server is available at >http://levis.tongji.edu.cn:8080/bioinfo/MLPred-Euk/ for the public usage.

Idas, a Novel Phylogenetically Conserved Geminin-related Protein, Binds to Geminin and Is Required for Cell Cycle Progression*

PubMed Central

Pefani, Dafni-Eleutheria; Dimaki, Maria; Spella, Magda; Karantzelis, Nickolas; Mitsiki, Eirini; Kyrousi, Christina; Symeonidou, Ioanna-Eleni; Perrakis, Anastassis; Taraviras, Stavros; Lygerou, Zoi

2011-01-01

Development and homeostasis of multicellular organisms relies on an intricate balance between cell proliferation and differentiation. Geminin regulates the cell cycle by directly binding and inhibiting the DNA replication licensing factor Cdt1. Geminin also interacts with transcriptional regulators of differentiation and chromatin remodelling factors, and its balanced interactions are implicated in proliferation-differentiation decisions during development. Here, we describe Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology), a previously uncharacterised coiled-coil protein related to Geminin. We show that human Idas localizes to the nucleus, forms a complex with Geminin both in cells and in vitro through coiled-coil mediated interactions, and can change Geminin subcellular localization. Idas does not associate with Cdt1 and prevents Geminin from binding to Cdt1 in vitro. Idas depletion from cells affects cell cycle progression; cells accumulate in S phase and are unable to efficiently progress to mitosis. Idas protein levels decrease in anaphase, whereas its overexpression causes mitotic defects. During development, we show that Idas exhibits high level expression in the choroid plexus and the cortical hem of the mouse telencephalon. Our data highlight Idas as a novel Geminin binding partner, implicated in cell cycle progression, and a putative regulator of proliferation-differentiation decisions during development. PMID:21543332

Idas, a novel phylogenetically conserved geminin-related protein, binds to geminin and is required for cell cycle progression.

PubMed

Pefani, Dafni-Eleutheria; Dimaki, Maria; Spella, Magda; Karantzelis, Nickolas; Mitsiki, Eirini; Kyrousi, Christina; Symeonidou, Ioanna-Eleni; Perrakis, Anastassis; Taraviras, Stavros; Lygerou, Zoi

2011-07-01

Development and homeostasis of multicellular organisms relies on an intricate balance between cell proliferation and differentiation. Geminin regulates the cell cycle by directly binding and inhibiting the DNA replication licensing factor Cdt1. Geminin also interacts with transcriptional regulators of differentiation and chromatin remodelling factors, and its balanced interactions are implicated in proliferation-differentiation decisions during development. Here, we describe Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology), a previously uncharacterised coiled-coil protein related to Geminin. We show that human Idas localizes to the nucleus, forms a complex with Geminin both in cells and in vitro through coiled-coil mediated interactions, and can change Geminin subcellular localization. Idas does not associate with Cdt1 and prevents Geminin from binding to Cdt1 in vitro. Idas depletion from cells affects cell cycle progression; cells accumulate in S phase and are unable to efficiently progress to mitosis. Idas protein levels decrease in anaphase, whereas its overexpression causes mitotic defects. During development, we show that Idas exhibits high level expression in the choroid plexus and the cortical hem of the mouse telencephalon. Our data highlight Idas as a novel Geminin binding partner, implicated in cell cycle progression, and a putative regulator of proliferation-differentiation decisions during development.

Human Embryonic Stem Cell-Derived Cardiomyocytes Self-Arrange with Areas of Different Subtypes During Differentiation.

PubMed

Vestergaard, Maj Linea; Grubb, Søren; Koefoed, Karen; Anderson-Jenkins, Zoe; Grunnet-Lauridsen, Kristina; Calloe, Kirstine; Clausen, Christian; Christensen, Søren Tvorup; Møllgård, Kjeld; Andersen, Claus Yding

2017-11-01

The derivation of functional cardiomyocytes (CMs) from human embryonic stem cells (hESCs) represents a unique way of studying human cardiogenesis, including the development of CM subtypes. In this study, we investigated the development and organization of hESC-derived cardiomyocytes (hESC-CMs) and examined how the expression levels of CM subtypes correspond to human in vivo cardiogenesis. Beating clusters were used to determine cardiac differentiation, which was evaluated by the expression of cardiac genes GATA4 and TNNT2 and subcellular localization of GATA4 and NKX2.5. Sharp electrode recordings to determine action potentials (APs) further revealed spatial organization of intracluster CM subtypes (ie, complex clusters). Nodal-, atrial-, and ventricular-like AP morphologies were detected within distinct regions of complex clusters. The ability of different CM subtypes to self-organize was documented by immunohistochemical analyses and a differential spatial expression of β-III tubulin, myosin light chain 2v (MLC-2V), and α-smooth muscle actin (α-SMA). Furthermore, all hESC-CM subtypes formed expressed primary cilia, which are known to coordinate cellular signaling pathways during cardiomyogenesis and heart development. This study expands the foundation for studying regulatory pathways for spatial and temporal CM differentiation during human cardiogenesis.

Tracking the Spatial and Functional Gradient of Monocyte-To-Macrophage Differentiation in Inflamed Lung.

PubMed

Sen, Debasish; Jones, Stephen M; Oswald, Erin M; Pinkard, Henry; Corbin, Kaitlin; Krummel, Matthew F

2016-01-01

Myeloid-derived cells such as monocytes, dendritic cells (DCs), and macrophages are at the heart of the immune effector function in an inflammatory response. But because of the lack of an efficient imaging system to trace these cells live during their migration and maturation in their native environment at sub-cellular resolution, our knowledge is limited to data available from specific time-points analyzed by flow cytometry, histology, genomics and other immunological methods. Here, we have developed a ratiometric imaging method for measuring monocyte maturation in inflamed mouse lungs in situ using real-time using 2-photon imaging and complementary methods. We visualized that while undifferentiated monocytes were predominantly found only in the vasculature, a semi-differentiated monocyte/macrophage population could enter the tissue and resembled more mature and differentiated populations by morphology and surface phenotype. As these cells entered and differentiated, they were already selectively localized near inflamed airways and their entry was associated with changes in motility and morphology. We were able to visualize these during the act of differentiation, a process that can be demonstrated in this way to be faster on a per-cell basis under inflammatory conditions. Finally, our in situ analyses demonstrated increases, in the differentiating cells, for both antigen uptake and the ability to mediate interactions with T cells. This work, while largely confirming proposed models for in situ differentiation, provides important in situ data on the coordinated site-specific recruitment and differentiation of these cells and helps elaborate the predominance of immune pathology at the airways. Our novel imaging technology to trace immunogenic cell maturation in situ will complement existing information available on in situ differentiation deduced from other immunological methods, and assist better understanding of the spatio-temporal cellular behavior during an inflammatory response.

The Impact of Phosphorus Supply on Selenium Uptake During Hydroponics Experiment of Winter Wheat (Triticum aestivum) in China.

PubMed

Liu, Hongen; Shi, Zhiwei; Li, Jinfeng; Zhao, Peng; Qin, Shiyu; Nie, Zhaojun

2018-01-01

Selenium (Se) is a necessary trace element for humans and animals, and Se fertilization is an efficient way to increase Se concentration in the edible parts of crops, thus enhance the beneficiary effects of Se in human and animal health. Due to the similarity of physical and chemical properties between phosphate () and selenite (), phosphorus (P) supply often significantly impacts the absorption of Se in plants, but little is known about how P supply influences the subcellular distribution and chemical forms of Se. In this study, the effects of P supply on subcellular distribution and chemical forms of Se in winter wheat were investigated in a hydroponic trial with medium Se level (0.1 mg Se L -1 ). P was applied with three concentrations (0.31, 3.1, and 31 mg P L -1 ) in the experiment. The results showed that increasing P supply significantly decreased the concentration and accumulation of Se in the roots, stems, and leaves of winter wheat. An increase in P supply significantly inhibited Se accumulation in the root cell wall, but enhanced Se distribution in the organelles and soluble fraction of root cells. These findings suggest that increased P supply inhibited the root-to-shoot transport of Se. An increase in P supply enhanced Se accumulation in the cell wall of plant stems (both apical and axillary stem) and cell organelles of plants leaves, but inhibited Se distribution in the soluble fraction of stems and leaves. This suggests that P supply enhances Se transportation across the cell membrane in shoots of winter wheat. In addition, increased P supply also altered the chemical forms of Se in tissues of winter wheat. These findings will help in understanding of the regulation grain Se accumulation and provide a practical way to enhance Se intake for humans inform Se-enriched grains.

Expression and sub-cellular localization of an epigenetic regulator, co-activator arginine methyltransferase 1 (CARM1), is associated with specific breast cancer subtypes and ethnicity

PubMed Central

2013-01-01

Background Co-Activator Arginine Methyltransferase 1(CARM1) is an Estrogen Receptor (ER) cofactor that remodels chromatin for gene regulation via methylation of Histone3. We investigated CARM1 levels and localization across breast cancer tumors in a cohort of patients of either European or African ancestry. Methods We analyzed CARM1 levels using tissue microarrays with over 800 histological samples from 549 female cancer patients from the US and Nigeria, Africa. We assessed associations between CARM1 expression localized to the nucleus and cytoplasm for 11 distinct variables, including; ER status, Progesterone Receptor status, molecular subtypes, ethnicity, HER2+ status, other clinical variables and survival. Results We found that levels of cytoplasmic CARM1 are distinct among tumor sub-types and increased levels are associated with ER-negative (ER-) status. Higher nuclear CARM1 levels are associated with HER2 receptor status. EGFR expression also correlates with localization of CARM1 into the cytoplasm. This suggests there are distinct functions of CARM1 among molecular tumor types. Our data reveals a basal-like subtype association with CARM1, possibly due to expression of Epidermal Growth Factor Receptor (EGFR). Lastly, increased cytoplasmic CARM1, relative to nuclear levels, appear to be associated with self-identified African ethnicity and this result is being further investigated using quantified genetic ancestry measures. Conclusions Although it is known to be an ER cofactor in breast cancer, CARM1 expression levels are independent of ER. CARM1 has distinct functions among molecular subtypes, as is indicative of its sub-cellular localization and it may function in subtype etiology. These sub-cellular localization patterns, indicate a novel role beyond its ER cofactor function in breast cancer. Differential localization among ethnic groups may be due to ancestry-specific polymorphisms which alter the gene product. PMID:23663560

Lipase genes in Mucor circinelloides: identification, sub-cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation.

PubMed

Zan, Xinyi; Tang, Xin; Chu, Linfang; Zhao, Lina; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

2016-10-01

Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/β-hydrolase_3 family were the conserved lipase family in M. circinelloides. Additionally, some lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospholipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform for the selection of candidate lipase genes for further detailed functional study.

Variation of Herbivore-Induced Volatile Terpenes among Arabidopsis Ecotypes Depends on Allelic Differences and Subcellular Targeting of Two Terpene Synthases, TPS02 and TPS031[W][OA

PubMed Central

Huang, Mengsu; Abel, Christian; Sohrabi, Reza; Petri, Jana; Haupt, Ina; Cosimano, John; Gershenzon, Jonathan; Tholl, Dorothea

2010-01-01

When attacked by insects, plants release mixtures of volatile compounds that are beneficial for direct or indirect defense. Natural variation of volatile emissions frequently occurs between and within plant species, but knowledge of the underlying molecular mechanisms is limited. We investigated intraspecific differences of volatile emissions induced from rosette leaves of 27 accessions of Arabidopsis (Arabidopsis thaliana) upon treatment with coronalon, a jasmonate mimic eliciting responses similar to those caused by insect feeding. Quantitative variation was found for the emission of the monoterpene (E)-β-ocimene, the sesquiterpene (E,E)-α-farnesene, the irregular homoterpene 4,8,12-trimethyltridecatetra-1,3,7,11-ene, and the benzenoid compound methyl salicylate. Differences in the relative emissions of (E)-β-ocimene and (E,E)-α-farnesene from accession Wassilewskija (Ws), a high-(E)-β-ocimene emitter, and accession Columbia (Col-0), a trace-(E)-β-ocimene emitter, were attributed to allelic variation of two closely related, tandem-duplicated terpene synthase genes, TPS02 and TPS03. The Ws genome contains a functional allele of TPS02 but not of TPS03, while the opposite is the case for Col-0. Recombinant proteins of the functional Ws TPS02 and Col-0 TPS03 genes both showed (E)-β-ocimene and (E,E)-α-farnesene synthase activities. However, differential subcellular compartmentalization of the two enzymes in plastids and the cytosol was found to be responsible for the ecotype-specific differences in (E)-β-ocimene/(E,E)-α-farnesene emission. Expression of the functional TPS02 and TPS03 alleles is induced in leaves by elicitor and insect treatment and occurs constitutively in floral tissues. Our studies show that both pseudogenization in the TPS family and subcellular segregation of functional TPS enzymes control the variation and plasticity of induced volatile emissions in wild plant species. PMID:20463089

Activity-Dependent Subcellular Cotrafficking of the Small GTPase Rem2 and Ca2+/CaM-Dependent Protein Kinase IIα

PubMed Central

Flynn, Robyn; Labrie-Dion, Etienne; Bernier, Nikolas; Colicos, Michael A.; De Koninck, Paul; Zamponi, Gerald W.

2012-01-01

Background Rem2 is a small monomeric GTP-binding protein of the RGK family, whose known functions are modulation of calcium channel currents and alterations of cytoskeletal architecture. Rem2 is the only RGK protein found predominantly in the brain, where it has been linked to synaptic development. We wished to determine the effect of neuronal activity on the subcellular distribution of Rem2 and its interacting partners. Results We show that Rem2 undergoes activity-and N-Methyl-D-Aspartate Receptor (NMDAR)-dependent translocation in rat hippocampal neurons. This redistribution of Rem2, from a diffuse pattern to one that is highly punctate, is dependent on Ca2+ influx, on binding to calmodulin (CaM), and also involves an auto-inhibitory domain within the Rem2 distal C-terminus region. We found that Rem2 can bind to Ca2+/CaM-dependent protein kinase IIα (CaMKII) a in Ca2+/CaM-dependent manner. Furthermore, our data reveal a spatial and temporal correlation between NMDAR-dependent clustering of Rem2 and CaMKII in neurons, indicating co-assembly and co-trafficking in neurons. Finally, we show that inhibiting CaMKII aggregation in neurons and HEK cells reduces Rem2 clustering, and that Rem2 affects the baseline distribution of CaMKII in HEK cells. Conclusions Our data suggest a novel function for Rem2 in co-trafficking with CaMKII, and thus potentially expose a role in neuronal plasticity. PMID:22815963

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A).

PubMed

Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

2017-01-08

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. Copyright © 2016 Elsevier Inc. All rights reserved.

Influence of metal exposure history on the bioaccumulation and subcellular distribution of aqueous cadmium in the insect Hydropsyche californica

USGS Publications Warehouse

Cain, D.J.; Buchwalter, D.B.; Luoma, S.N.

2006-01-01

The influence of metal exposure history on rates of aqueous Cd accumulation, elimination, and subcellular distribution was examined in the aquatic insect Hydropsyche californica. Specimens were obtained from a reference site and a metal-contaminated site and returned to the laboratory where they were continuously exposed to aqueous Cd (518 ng/L, nominal) for 6 d, followed by 9 d of depuration. Rates of Cd accumulation and elimination were similar in insects from the two sites. Efflux rate constants, ke, ranged from 0.20 to 0.24/d (t1/2 ??? 3 d). Immediately following exposure, the cytosol accounted for 40% of the body burden in insects from both sites; however, 89 ?? 2% of the cytosolic Cd was associated with metallothionein-like proteins (MTLP) in insects from the contaminated site, compared to 60 ?? 0% in insects from the reference site. The concentration of Cd bound to non-MTLPs (representing potentially Cd-sensitive proteins) was significantly greater in the insects from the reference site (134 ?? 7 ng/g) than in those from the contaminated site (42 ?? 2 ng/g). At the end of the depuration period, 90% of the accumulated Cd body burden had been eliminated, and Cd concentrations in MTLPs and non-MTLPs were similar between the sites. Results suggested that differences in exposure history had no influence on the bioaccumulation of Cd, but did affect the concentrations of Cd bound to MTLP during Cd exposure in these insects. ?? 2006 SETAC.

Binding domain-driven intracellular trafficking of sterols for synthesis of steroid hormones, bile acids and oxysterols.

PubMed

Midzak, Andrew; Papadopoulos, Vassilios

2014-09-01

Steroid hormones, bioactive oxysterols and bile acids are all derived from the biological metabolism of lipid cholesterol. The enzymatic pathways generating these compounds have been an area of intense research for almost a century, as cholesterol and its metabolites have substantial impacts on human health. Owing to its high degree of hydrophobicity and the chemical properties that it confers to biological membranes, the distribution of cholesterol in cells is tightly controlled, with subcellular organelles exhibiting highly divergent levels of cholesterol. The manners in which cells maintain such sterol distributions are of great interest in the study of steroid and bile acid synthesis, as limiting cholesterol substrate to the enzymatic pathways is the principal mechanism by which production of steroids and bile acids is regulated. The mechanisms by which cholesterol moves within cells, however, remain poorly understood. In this review, we examine the subcellular machinery involved in cholesterol metabolism to steroid hormones and bile acid, relating it to both lipid- and protein-based mechanisms facilitating intracellular and intraorganellar cholesterol movement and delivery to these pathways. In particular, we examine evidence for the involvement of specific protein domains involved in cholesterol binding, which impact cholesterol movement and metabolism in steroidogenesis and bile acid synthesis. A better understanding of the physical mechanisms by which these protein- and lipid-based systems function is of fundamental importance to understanding physiological homeostasis and its perturbation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Estrogen levels regulate the subcellular distribution of phosphorylated Akt in hippocampal CA1 dendrites.

PubMed

Znamensky, Vladimir; Akama, Keith T; McEwen, Bruce S; Milner, Teresa A

2003-03-15

In addition to genomic pathways, estrogens may regulate gene expression by activating specific signal transduction pathways, such as that involving phosphatidylinositol 3-kinase (PI3-K) and the subsequent phosphorylation of Akt (protein kinase B). The Akt pathway regulates various cellular events, including the initiation of protein synthesis. Our previous studies showed that synaptogenesis in hippocampal CA1 pyramidal cell dendritic spines is highest when brain estrogen levels are highest. To address the role of Akt in this process, the subcellular distribution of phosphorylated Akt immunoreactivity (pAkt-I) in the hippocampus of female rats across the estrous cycle and male rats was analyzed by light microscopy (LM) and electron microscopy (EM). By LM, the density of pAkt-I in stratum radiatum of CA1 was significantly higher in proestrus rats (or in estrogen-supplemented ovariectomized females) compared with diestrus, estrus, or male rats. By EM, pAkt-I was found throughout the shafts and in select spines of stratum radiatum dendrites. Quantitative ultrastructural analysis identifying pAkt-I with immunogold particles revealed that proestrus rats compared with diestrus, estrus, and male rats contained significantly higher pAkt-I associated with (1) dendritic spines (both cytoplasm and plasmalemma), (2) spine apparati located within 0.1 microm of dendritic spine bases, (3) endoplasmic reticula and polyribosomes in the cytoplasm of dendritic shafts, and (4) the plasmalemma of dendritic shafts. These findings suggest that estrogens may regulate spine formation in CA1 pyramidal neurons via Akt-mediated signaling events.

Cellular Distribution and Subcellular Localization of Molecular Components of Vesicular Transmitter Release in Horizontal Cells of Rabbit Retina

PubMed Central

HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN H.; BRECHA, NICHOLAS C.

2010-01-01

The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of γ-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca2+-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells. PMID:15912504

Immunogold labeling reveals subcellular localisation of silica nanoparticles in a human blood-brain barrier model

NASA Astrophysics Data System (ADS)

Ye, Dong; Anguissola, Sergio; O'Neill, Tiina; Dawson, Kenneth A.

2015-05-01

Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles. Electronic supplementary information (ESI) available: Nanoparticle characterisation data, preservation of cellular structures, staining controls, optimisation of size amplification via the silver enhancement, and more imaging results from anti-clathrin and anti-caveolin 1 immunolabeling. See DOI: 10.1039/c5nr01539a

Mammalian follicular development and atresia: role of apoptosis.

PubMed

Asselin, E; Xiao, C W; Wang, Y F; Tsang, B K

2000-01-01

The regulation of follicular development and atresia is a complex process and involves interactions between endocrine factors (gonadotropins) and intraovarian regulators (sex steroids, growth factors and cytokines) in the control of follicular cell fate (i.e. proliferation, differentiation and programmed cell death). Granulosa and theca cells are key players in this fascinating process. As atresia is the fate of most follicles, understanding of how these physiological regulators participate in determining the destiny of the follicle (to degenerate or to ovulate) at cellular and subcellular levels is fundamental. This short review summarizes the role of intraovarian modulators of programmed cell death in the induction of atresia during follicular development. Copyright 2000 S. Karger AG, Basel

Subcellular analysis by laser ablation electrospray ionization mass spectrometry

DOEpatents

Vertes, Akos; Stolee, Jessica A; Shrestha, Bindesh

2014-12-02

In various embodiments, a method of laser ablation electrospray ionization mass spectrometry (LAESI-MS) may generally comprise micro-dissecting a cell comprising at least one of a cell wall and a cell membrane to expose at least one subcellular component therein, ablating the at least one subcellular component by an infrared laser pulse to form an ablation plume, intercepting the ablation plume by an electrospray plume to form ions, and detecting the ions by mass spectrometry.

Evaluation on subcellular partitioning and biodynamics of pulse copper toxicity in tilapia reveals impacts of a major environmental disturbance.

PubMed

Ju, Yun-Ru; Yang, Ying-Fei; Tsai, Jeng-Wei; Cheng, Yi-Hsien; Chen, Wei-Yu; Liao, Chung-Min

2017-07-01

Fluctuation exposure of trace metal copper (Cu) is ubiquitous in aquatic environments. The purpose of this study was to investigate the impacts of chronically pulsed exposure on biodynamics and subcellular partitioning of Cu in freshwater tilapia (Oreochromis mossambicus). Long-term 28-day pulsed Cu exposure experiments were performed to explore subcellular partitioning and toxicokinetics/toxicodynamics of Cu in tilapia. Subcellular partitioning linking with a metal influx scheme was used to estimate detoxification and elimination rates. A biotic ligand model-based damage assessment model was used to take into account environmental effects and biological mechanisms of Cu toxicity. We demonstrated that the probability causing 50% of susceptibility risk in response to pulse Cu exposure in generic Taiwan aquaculture ponds was ~33% of Cu in adverse physiologically associated, metabolically active pool, implicating no significant susceptibility risk for tilapia. We suggest that our integrated ecotoxicological models linking chronic exposure measurements with subcellular partitioning can facilitate a risk assessment framework that provides a predictive tool for preventive susceptibility reduction strategies for freshwater fish exposed to pulse metal stressors.

Abiotic Stress Tolerance in Plants: Myriad Roles of Ascorbate Peroxidase

PubMed Central

Pandey, Saurabh; Fartyal, Dhirendra; Agarwal, Aakrati; Shukla, Tushita; James, Donald; Kaul, Tanushri; Negi, Yogesh K.; Arora, Sandeep; Reddy, Malireddy K.

2017-01-01

One of the most significant manifestations of environmental stress in plants is the increased production of Reactive Oxygen Species (ROS). These ROS, if allowed to accumulate unchecked, can lead to cellular toxicity. A battery of antioxidant molecules is present in plants for keeping ROS levels under check and to maintain the cellular homeostasis under stress. Ascorbate peroxidase (APX) is a key antioxidant enzyme of such scavenging systems. It catalyses the conversion of H2O2 into H2O, employing ascorbate as an electron donor. The expression of APX is differentially regulated in response to environmental stresses and during normal plant growth and development as well. Different isoforms of APX show differential response to environmental stresses, depending upon their sub-cellular localization, and the presence of specific regulatory elements in the upstream regions of the respective genes. The present review delineates role of APX isoforms with respect to different types of abiotic stresses and its importance as a key antioxidant enzyme in maintaining cellular homeostasis. PMID:28473838

Behavior-dependent specialization of identified hippocampal interneurons

PubMed Central

Lapray, Damien; Lasztoczi, Balint; Lagler, Michael; Viney, Tim James; Katona, Linda; Valenti, Ornella; Hartwich, Katja; Borhegyi, Zsolt; Somogyi, Peter; Klausberger, Thomas

2012-01-01

A large variety of GABAergic interneurons control information processing in hippocampal circuits governing the formation of neuronal representations. Whether distinct hippocampal interneuron types contribute differentially to information-processing during behavior is not known. We employed a novel technique for recording and labeling interneurons and pyramidal cells in drug-free, freely-moving rats. Recorded parvalbumin-expressing basket interneurons innervate somata and proximal pyramidal cell dendrites, whereas nitric-oxide-synthase- and neuropeptide-Y-expressing ivy cells provide synaptic and extrasynaptic dendritic modulation. Basket and ivy cells showed distinct spike timing dynamics, firing at different rates and times during theta and ripple oscillations. Basket but not ivy cells changed their firing rates during movement, sleep and quiet wakefulness, suggesting that basket cells coordinate cell assemblies in a behavioral state-contingent manner, whereas persistently-firing ivy cells might control network excitability and homeostasis. Different interneuron types provide GABA to specific subcellular domains at defined times and rates, thus differentially controlling network activity during behavior. PMID:22864613

Measurement of particle size distribution in mammalian cells in vitro by use of polarized light spectroscopy

NASA Astrophysics Data System (ADS)

Bartlett, Matthew; Huang, George; Larcom, Lyndon; Jiang, Huabei

2004-02-01

We demonstrate the feasibility of measuring the particle size distribution (PSD) of internal cell structures in vitro. We use polarized light spectroscopy to probe the internal morphology of mammalian breast cancer (MCF7) and cervical cancer (Siha) cells. We find that graphing the least-squared error versus the scatterer size provides insight into cell scattering. A nonlinear optimization scheme is used to determine the PSD iteratively. The results suggest that 2-μm particles (possibly the mitochondria) contribute most to the scattering. Other subcellular structures, such as the nucleoli and the nucleus, may also contribute significantly. We reconstruct the PSD of the mitochondria, as verified by optical microscopy. We also demonstrate the angle dependence of the PSD.

DNA Damage Dependence on the Subcellular Distribution of Low-Energy Beta Emitters

NASA Astrophysics Data System (ADS)

Cutaia, Claudia; Alloni, Daniele; Mariotti, Luca; Friedland, Werner; Ottolenghi, Andrea

One of the main issues of low-energy internal emitters is related to the short ranges of beta particles, compared to the dimensions of the biological targets (e.g. the cell nucleus). Also depending on the chemical form, the radionuclide may be more concentrated in the cytoplasm of the target cell (in our calculations a human fibroblast in interphase) and consequently the conventional dosimetry may overestimate the dose to the nucleus; whereas if the radionuclide is more concentrated in the nuclei of the cells there is a risk of underestimating the nucleus dose. The computer code PARTRAC was modified to calculate the energy depositions in the nucleus and the DNA damage for different relative concentrations of the radionuclide in the nucleus and in the cytoplasm. The nuclides considered in the simulations were Tritium (the electrons emitted due to the β - decay have an average energy of 5.7 keV, corresponding to an average range of 0.42 µm) and Nickel-63 (the electrons emitted have an average energy of 17 keV corresponding to an average range of 5 µm). In the case of Tritium, the dose in the nucleus due the tracks generated outside this region is 15% of the average dose in the cell, whereas in the case of Nickel-63 the dose in the nucleus resulted to be 64% of the average dose in the cell. The distributions of DNA fragments as a function of the relative concentration of the nuclides in the nucleus and in the cytoplasm, were also calculated. In the same conditions, the number of complex lesions (which have a high probability of inducing lethal damage to the cells) per Gy (circa 0.5-1) and the total number of double strand breaks (DSBs) per Gy (circa 40) were also calculated. To complete the characterization of the effects of internal emitters inside the cell the distributions of DSBs per chromosome were studied for different radionuclide distributions in the cell. The results obtained from these simulations show the possible overestimation or underestimation of the risk, (particularly for Tritium intake), due to the distribution of the low energy emitters at subcellular levels.

Structure and function of yeast glutaredoxin 2 depend on postranslational processing and are related to subcellular distribution.

PubMed

Porras, Pablo; McDonagh, Brian; Pedrajas, Jose Rafael; Bárcena, J Antonio; Padilla, C Alicia

2010-04-01

We have previously shown that glutaredoxin 2 (Grx2) from Saccharomyces cerevisiae localizes at 3 different subcellular compartments, cytosol, mitochondrial matrix and outer membrane, as the result of different postranslational processing of one single gene. Having set the mechanism responsible for this remarkable phenomenon, we have now aimed at defining whether this diversity of subcellular localizations correlates with differences in structure and function of the Grx2 isoforms. We have determined the N-terminal sequence of the soluble mitochondrial matrix Grx2 by mass spectrometry and have determined the exact cleavage site by Mitochondrial Processing Peptidase (MPP). As a consequence of this cleavage, the mitochondrial matrix Grx2 isoform possesses a basic tetrapeptide extension at the N-terminus compared to the cytosolic form. A functional relationship to this structural difference is that mitochondrial Grx2 displays a markedly higher activity in the catalysis of GSSG reduction by the mitochondrial dithiol dihydrolipoamide. We have prepared Grx2 mutants affected on key residues inside the presequence to direct the protein to one single cellular compartment; either the cytosol, the mitochondrial membrane or the matrix and have analyzed their functional phenotypes. Strains expressing Grx2 only in the cytosol are equally sensitive to H(2)O(2) as strains lacking the gene, whereas those expressing Grx2 exclusively in the mitochondrial matrix are more resistant. Mutations on key basic residues drastically affect the cellular fate of the protein, showing that evolutionary diversification of Grx2 structural and functional properties are strictly dependent on the sequence of the targeting signal peptide. Copyright 2009 Elsevier B.V. All rights reserved.

Estimating the magnitude of near-membrane PDE4 activity in living cells.

PubMed

Xin, Wenkuan; Feinstein, Wei P; Britain, Andrea L; Ochoa, Cristhiaan D; Zhu, Bing; Richter, Wito; Leavesley, Silas J; Rich, Thomas C

2015-09-15

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments. Copyright © 2015 the American Physiological Society.

Live-cell visualization of intracellular interaction between a nuclear migration protein (hNUDC) and the thrombopoietin receptor (Mpl).

PubMed

Zheng, Yuan-Bin; Xiao, Ying-Ying; Tan, Peng; Zhang, Qing; Xu, Peilin

2012-01-01

We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.

Live-Cell Visualization of Intracellular Interaction between a Nuclear Migration Protein (hNUDC) and the Thrombopoietin Receptor (Mpl)

PubMed Central

Zheng, Yuan-Bin; Xiao, Ying-Ying; Tan, Peng; Zhang, Qing; Xu, Peilin

2012-01-01

We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway. PMID:23284788

Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells.

PubMed

Beckmann, Anja; Grissmer, Alexander; Krause, Elmar; Tschernig, Thomas; Meier, Carola

2016-03-01

Pannexins (Panx) are proteins with a similar membrane topology to connexins, the integral membrane protein of gap junctions. Panx1 channels are generally of major importance in a large number of system and cellular processes and their function has been thoroughly characterized. In contrast, little is known about channel structure and subcellular distribution. We therefore determine the subcellular localization of Panx1 channels in cultured cells and aim at the identification of channel morphology in vitro. Using freeze-fracture replica immunolabeling on EYFP-Panx1-overexpressing HEK 293 cells, large particles were identified in plasma membranes, which were immunogold-labeled using either GFP or Panx1 antibodies. There was no labeling or particles in the nuclear membranes of these cells, pointing to plasma membrane localization of Panx1-EYFP channels. The assembly of particles was irregular, this being in contrast to the regular pattern of gap junctions. The fact that no counterparts were identified on apposing cells, which would have been indicative of intercellular signaling, supported the idea of Panx1 channels within one membrane. Control cells (transfected with EYFP only, non-transfected) were devoid of both particles and immunogold labeling. Altogether, this study provides the first demonstration of Panx1 channel morphology and assembly in intact cells. The identification of Panx1 channels as large particles within the plasma membrane provides the knowledge required to enable recognition of Panx1 channels in tissues in future studies. Thus, these results open up new avenues for the detailed analysis of the subcellular localization of Panx1 and of its nearest neighbors such as purinergic receptors in vivo.

The DUF1669 domain of FAM83 family proteins anchor casein kinase 1 isoforms.

PubMed

Fulcher, Luke J; Bozatzi, Polyxeni; Tachie-Menson, Theresa; Wu, Kevin Z L; Cummins, Timothy D; Bufton, Joshua C; Pinkas, Daniel M; Dunbar, Karen; Shrestha, Sabin; Wood, Nicola T; Weidlich, Simone; Macartney, Thomas J; Varghese, Joby; Gourlay, Robert; Campbell, David G; Dingwell, Kevin S; Smith, James C; Bullock, Alex N; Sapkota, Gopal P

2018-05-22

Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Dynamic Fluctuations in Subcellular Localization of the Hippo Pathway Effector Yorkie In Vivo.

PubMed

Manning, Samuel A; Dent, Lucas G; Kondo, Shu; Zhao, Ziqing W; Plachta, Nicolas; Harvey, Kieran F

2018-05-21

The Hippo pathway is an evolutionarily conserved signaling network that integrates diverse cues to control organ size and cell fate. The central downstream pathway protein in Drosophila is the transcriptional co-activator Yorkie (YAP and TAZ in humans), which regulates gene expression with the Scalloped/TEA domain family member (TEAD) transcription factors [1-8]. A central regulatory step in the Hippo pathway is phosphorylation of Yorkie by the NDR family kinase Warts, which promotes Yorkie cytoplasmic localization by stimulating association with 14-3-3 proteins [9-12]. Numerous reports have purported a static model of Hippo signaling whereby, upon Hippo activation, Yorkie/YAP/TAZ become cytoplasmic and therefore inactive, and upon Hippo repression, Yorkie/YAP/TAZ transit to the nucleus and are active. However, we have little appreciation for the dynamics of Yorkie/YAP/TAZ subcellular localization because most studies have been performed in fixed cells and tissues. To address this, we used live multiphoton microscopy to investigate the dynamics of an endogenously tagged Yorkie-Venus protein in growing epithelial organs. We found that the majority of Yorkie rapidly traffics between the cytoplasm and nucleus, rather than being statically localized in either compartment. In addition, discrete cell populations within the same organ display different rates of Yorkie nucleo-cytoplasmic shuttling. By assessing Yorkie dynamics in warts mutant tissue, we found that the Hippo pathway regulates Yorkie subcellular distribution by regulating its rate of nuclear import. Furthermore, Yorkie's localization fluctuates dramatically throughout the cell cycle, being predominantly cytoplasmic during interphase and, unexpectedly, chromatin enriched during mitosis. Yorkie's association with mitotic chromatin is Scalloped dependent, suggesting a potential role in mitotic bookmarking. Copyright © 2018 Elsevier Ltd. All rights reserved.

Sci-Thur AM: YIS – 06: A Monte Carlo study of macro- and microscopic dose descriptors and the microdosimetric spread using detailed cellular models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oliver, Patricia; Thomson, Rowan

2016-08-15

Purpose: To develop Monte Carlo models of cell clusters to investigate the relationships between macro- and microscopic dose descriptors, quantify the microdosimetric spread in energy deposition for subcellular targets, and determine how these results depend on the computational model. Methods: Microscopic tissue structure is modelled as clusters of 13 to 150 cells, with cell (nuclear) radii between 5 and 10 microns (2 and 9 microns). Energy imparted per unit mass (specific energy or dose) is scored in the nucleus (D{sub nuc}) and cytoplasm (D{sub cyt}) for incident photon energies from 20 to 370 keV. Dose-to-water (D{sub w,m}) and dose-to-medium (D{submore » m,m}) are compared to D{sub nuc} and D{sub cyt}. Single cells and single nuclear cavities are also simulated. Results: D{sub nuc} and D{sub cyt} are sensitive to the surrounding environment with deviations of up to 13% for a single nucleus/cell compared with a multicellular cluster. These dose descriptors vary with cell and nucleus size by up to 10%. D{sub nuc} and D{sub cyt} differ from D{sub w,m} and D{sub m,m} by up to 32%. The microdosimetric spread is sensitive to whether cells are arranged randomly or in a hexagonal lattice, and whether subcellular compartment sizes are sampled from a normal distribution or are constant throughout the cluster. Conclusions: D{sub nuc} and D{sub cyt} are sensitive to cell morphology, elemental composition and the presence of surrounding cells. The microdosimetric spread was investigated using realistic elemental compositions for the nucleus and cytoplasm, and depends strongly on subcellular compartment size, source energy and dose.« less

Estimating the magnitude of near-membrane PDE4 activity in living cells

PubMed Central

Xin, Wenkuan; Feinstein, Wei P.; Britain, Andrea L.; Ochoa, Cristhiaan D.; Zhu, Bing; Richter, Wito; Leavesley, Silas J.

2015-01-01

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments. PMID:26201952

5D imaging approaches reveal the formation of distinct intracellular cAMP spatial gradients

NASA Astrophysics Data System (ADS)

Rich, Thomas C.; Annamdevula, Naga; Trinh, Kenny; Britain, Andrea L.; Mayes, Samuel A.; Griswold, John R.; Deal, Joshua; Hoffman, Chase; West, Savannah; Leavesley, Silas J.

2017-02-01

Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions. Several lines of evidence suggest that the distribution of cAMP within cells is not uniform. However, to date, no studies have measured the kinetics of 3D cAMP distributions within cells. This is largely due to the low signal-tonoise ratio of FRET-based probes. We previously reported that hyperspectral imaging improves the signal-to-noise ratio of FRET measurements. Here we utilized hyperspectral imaging approaches to measure FRET signals in five dimensions (5D) - three spatial (x, y, z), wavelength (λ), and time (t) - allowing us to visualize cAMP gradients in pulmonary endothelial cells. cAMP levels were measured using a FRET-based sensor (H188) comprised of a cAMP binding domain sandwiched between FRET donor and acceptor - Turquoise and Venus fluorescent proteins. We observed cAMP gradients in response to 0.1 or 1 μM isoproterenol, 0.1 or 1 μM PGE1, or 50 μM forskolin. Forskolin- and isoproterenol-induced cAMP gradients formed from the apical (high cAMP) to basolateral (low cAMP) face of cells. In contrast, PGE1-induced cAMP gradients originated from both the basolateral and apical faces of cells. Data suggest that 2D (x,y) studies of cAMP compartmentalization may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D (x,y,z) studies are required to assess mechanisms of signaling specificity. Results demonstrate that 5D imaging technologies are powerful tools for measuring biochemical processes in discrete subcellular domains.

In situ enzymatic activity of transglutaminase isoforms on brain tissue sections of rodents: A new approach to monitor differences in post-translational protein modifications during neurodegeneration.

PubMed

Schulze-Krebs, Anja; Canneva, Fabio; Schnepf, Rebecca; Dobner, Julia; Dieterich, Walburga; von Hörsten, Stephan

2016-01-15

Mammalian transglutaminases (TGs) catalyze the irreversible post-translational modifications of proteins, the most prominent of which is the calcium-dependent formation of covalent acyl transfers between the γ-carboxamide group of glutamine and the ε-amino-group of lysine (GGEL-linkage). In the central nervous system, at least four TG isoforms are present and some of them are differentially expressed under pathological conditions in human patients. However, the precise TG-isoform-dependent enzymatic activities in the brain as well as their anatomical distribution are unknown. Specificity of the used biotinylated peptides was analyzed using an in vitro assay. Isoform-specific TG activity was evaluated in in vitro and in situ studies, using brain extracts and native brain tissue obtained from rodents. Our method allowed us to reveal in vitro and in situ TG-isoform-dependent enzymatic activity in brain extracts and tissue of rats and mice, with a specific focus on TG6. In situ activity of this isoform varied between BACHD mice in comparison to their wt controls. TG isozyme-specific activity can be detected by isoform-specific biotinylated peptides in brain tissue sections of rodents to reveal differences in the anatomical and/or subcellular distribution of TG activity. Our findings yield the basis for a broader application of this method for the screening of pathological expression and activity of TGs in a variety of animal models of human diseases, as in the case of neurodegenerative conditions such as Huntington׳s, Parkinson׳s and Alzheimer׳s, where protein modification is involved as a key mechanism of disease progression. Copyright © 2015 Elsevier B.V. All rights reserved.

Axodendritic sorting and pathological missorting of Tau are isoform-specific and determined by axon initial segment architecture.

PubMed

Zempel, Hans; Dennissen, Frank J A; Kumar, Yatender; Luedtke, Julia; Biernat, Jacek; Mandelkow, Eva-Maria; Mandelkow, Eckhard

2017-07-21

Subcellular mislocalization of the microtubule-associated protein Tau is a hallmark of Alzheimer disease (AD) and other tauopathies. Six Tau isoforms, differentiated by the presence or absence of a second repeat or of N-terminal inserts, exist in the human CNS, but their physiological and pathological differences have long remained elusive. Here, we investigated the properties and distributions of human and rodent Tau isoforms in primary forebrain rodent neurons. We found that the Tau diffusion barrier (TDB), located within the axon initial segment (AIS), controls retrograde (axon-to-soma) and anterograde (soma-to-axon) traffic of Tau. Tau isoforms without the N-terminal inserts were sorted efficiently into the axon. However, the longest isoform (2N4R-Tau) was partially retained in cell bodies and dendrites, where it accelerated spine and dendrite growth. The TDB (located within the AIS) was impaired when AIS components (ankyrin G, EB1) were knocked down or when glycogen synthase kinase-3β (GSK3β; an AD-associated kinase tethered to the AIS) was overexpressed. Using superresolution nanoscopy and live-cell imaging, we observed that microtubules within the AIS appeared highly dynamic, a feature essential for the TDB. Pathomechanistically, amyloid-β insult caused cofilin activation and F-actin remodeling and decreased microtubule dynamics in the AIS. Concomitantly with these amyloid-β-induced disruptions, the AIS/TDB sorting function failed, causing AD-like Tau missorting. In summary, we provide evidence that the human and rodent Tau isoforms differ in axodendritic sorting and amyloid-β-induced missorting and that the axodendritic distribution of Tau depends on AIS integrity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein kinase C isoforms in iris sphincter smooth muscle: differential effects of phorbol ester on contraction and cAMP accumulation are species specific.

PubMed

Husain, S; Abdel-Latif, A A

1996-03-01

Objectives were to identify PKC isoforms in iris sphincter isolated from rabbit, cat, dog and bovine irides, to determine their subcellular distribution, and to investigate the effects of the phorbol ester, PDBu, on contraction and cAMP accumulation in this tissue. Using six isoform (alpha, beta, gamma, epsilon, delta, zeta)-specific polyclonal antibodies, PKC alpha, beta, epsilon, delta, and zeta were detected in the four species, whereas PKC gamma was detected only in dog and bovine. PKC alpha and epsilon are the most abundant isoforms in this tissue. PKC alpha is mainly cytosolic in rabbit and bovine and membrane associated in cat and dog. PKC gamma is equally distributed in cytosol and membrane fractions of bovine, but mostly cytosolic in dog. PKC beta, delta and epsilon are mainly membraneous and PKC zeta is mainly cytosolic in all species. PDBu (100 nM) induced a contractile response in rabbit- and cat-, but not in dog and bovine, sphincters, and increased cAMP accumulation in rabbit, cat, dog and bovine by 111, 130, 458 and 294%, respectively. Therefore, the lack of effect of PDBu on contraction in dog and bovine, as compared to rabbit and cat, may be due: (a) to the presence of PKC gamma isoform, and (b) to the stronger stimulatory effects of the phorbol ester on cAMP production in the non-contracting species. In addition to demonstrating the presence of various PKC isoforms in the iris sphincter and the activation of adenylyl cyclase by this protein kinase, we have shown that the distribution of the PKC isoforms in this tissue is species specific. Furthermore, our data suggest that there may be specific physiological functions associated with each of the PKC isoforms and that PKC is involved in the contractile response of some but not all smooth muscles.

Regulation of cardiomyocyte autophagy by calcium

PubMed Central

Shaikh, Soni; Troncoso, Rodrigo; Criollo, Alfredo; Bravo-Sagua, Roberto; García, Lorena; Morselli, Eugenia; Cifuentes, Mariana; Quest, Andrew F. G.; Hill, Joseph A.

2016-01-01

Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy. PMID:26884385

Biosynthesis and subcellular distribution of hydrolyzable tannins.

PubMed

Grundhöfer, P; Niemetz, R; Schilling, G; Gross, G G

2001-07-01

Pathways to complex gallotannins have been elucidated by enzyme studies, indicating that beta-glucogallin is required as principal acyl donor. Evidence for the in vitro oxidation of pentagalloylglucose, the pivotal metabolite in this sequence, to ellagitannins, is presented. Immunohistochemical studies with antibodies raised against pentagalloylglucose and the galloyltransferase catalyzing the formation of this ester revealed that leaf mesophyll cell walls were a typical site of origin and deposition of hydrolyzable tannins. Seasonal changes of these compounds were studied with extracts from cell walls and intracellular space of oak leaves.

The Response of Prostate Smooth Muscle Cells to Testosterone Is Determined by the Subcellular Distribution of the Androgen Receptor.

PubMed

Peinetti, Nahuel; Scalerandi, María Victoria; Cuello Rubio, Mariana Micaela; Leimgruber, Carolina; Nicola, Juan Pablo; Torres, Alicia Ines; Quintar, Amado Alfredo; Maldonado, Cristina Alicia

2018-02-01

Androgen signaling in prostate smooth muscle cells (pSMCs) is critical for the maintenance of prostate homeostasis, the alterations of which are a central aspect in the development of pathological conditions. Testosterone can act through the classic androgen receptor (AR) in the cytoplasm, eliciting genomic signaling, or through different types of receptors located at the plasma membrane for nongenomic signaling. We aimed to find evidence of nongenomic testosterone-signaling mechanisms in pSMCs and their participation in cell proliferation, differentiation, and the modulation of the response to lipopolysaccharide. We demonstrated that pSMCs can respond to testosterone by a rapid activation of ERK1/2 and Akt. Furthermore, a pool of ARs localized at the cell surface of pSMCs is responsible for a nongenomic testosterone-induced increase in cell proliferation. Through membrane receptor stimulation, testosterone favors a muscle phenotype, indicated by an increase in smooth muscle markers. We also showed that the anti-inflammatory effects of testosterone, capable of attenuating lipopolysaccharide-induced proinflammatory actions, are promoted only by receptors located inside the cell. We postulate that testosterone might perform prohomeostatic effects through intracellular-initiated mechanisms by modulating cell proliferation and inflammation, whereas some pathological, hyperproliferative actions would be induced by membrane-initiated nongenomic signaling in pSMCs. Copyright © 2018 Endocrine Society.

Microscopy with spatial filtering for sorting particles and monitoring subcellular morphology

NASA Astrophysics Data System (ADS)

Zheng, Jing-Yi; Qian, Zhen; Pasternack, Robert M.; Boustany, Nada N.

2009-02-01

Optical scatter imaging (OSI) was developed to non-invasively track real-time changes in particle morphology with submicron sensitivity in situ without exogenous labeling, cell fixing, or organelle isolation. For spherical particles, the intensity ratio of wide-to-narrow angle scatter (OSIR, Optical Scatter Image Ratio) was shown to decrease monotonically with diameter and agree with Mie theory. In living cells, we recently reported this technique is able to detect mitochondrial morphological alterations, which were mediated by the Bcl-xL transmembrane domain, and could not be observed by fluorescence or differential interference contrast images. Here we further extend the ability of morphology assessment by adopting a digital micromirror device (DMD) for Fourier filtering. When placed in the Fourier plane the DMD can be used to select scattering intensities at desired combination of scattering angles. We designed an optical filter bank consisting of Gabor-like filters with various scales and rotations based on Gabor filters, which have been widely used for localization of spatial and frequency information in digital images and texture analysis. Using a model system consisting of mixtures of polystyrene spheres and bacteria, we show how this system can be used to sort particles on a microscopic slide based on their size, orientation and aspect ratio. We are currently applying this technique to characterize the morphology of subcellular organelles to help understand fundamental biological processes.

Temporal redistribution of inhibition over neuronal subcellular domains underlies state-dependent rhythmic change of excitability in the hippocampus

PubMed Central

Somogyi, Peter; Katona, Linda; Klausberger, Thomas; Lasztóczi, Bálint; Viney, Tim J.

2014-01-01

The behaviour-contingent rhythmic synchronization of neuronal activity is reported by local field potential oscillations in the theta, gamma and sharp wave-related ripple (SWR) frequency ranges. In the hippocampus, pyramidal cell assemblies representing temporal sequences are coordinated by GABAergic interneurons selectively innervating specific postsynaptic domains, and discharging phase locked to network oscillations. We compare the cellular network dynamics in the CA1 and CA3 areas recorded with or without anaesthesia. All parts of pyramidal cells, except the axon initial segment, receive GABA from multiple interneuron types, each with distinct firing dynamics. The axon initial segment is exclusively innervated by axo-axonic cells, preferentially firing after the peak of the pyramidal layer theta cycle, when pyramidal cells are least active. Axo-axonic cells are inhibited during SWRs, when many pyramidal cells fire synchronously. This dual inverse correlation demonstrates the key inhibitory role of axo-axonic cells. Parvalbumin-expressing basket cells fire phase locked to field gamma activity in both CA1 and CA3, and also strongly increase firing during SWRs, together with dendrite-innervating bistratified cells, phasing pyramidal cell discharge. Subcellular domain-specific GABAergic innervation probably developed for the coordination of multiple glutamatergic inputs on different parts of pyramidal cells through the temporally distinct activity of GABAergic interneurons, which differentially change their firing during different network states. PMID:24366131

The Expression Level of Septin12 Is Critical for Spermiogenesis

PubMed Central

Lin, Ying-Hung; Lin, Yung-Ming; Wang, Ya-Yun; Yu, I-Shing; Lin, Yi-Wen; Wang, Yun-Han; Wu, Ching-Ming; Pan, Hsien-An; Chao, Shin-Chih; Yen, Pauline H.; Lin, Shu-Wha; Kuo, Pao-Lin

2009-01-01

Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12 mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis. PMID:19359518

Melatonin biosynthesis in plants: multiple pathways catalyze tryptophan to melatonin in the cytoplasm or chloroplasts.

PubMed

Back, Kyoungwhan; Tan, Dun-Xian; Reiter, Russel J

2016-11-01

Melatonin is an animal hormone as well as a signaling molecule in plants. It was first identified in plants in 1995, and almost all enzymes responsible for melatonin biosynthesis had already been characterized in these species. Melatonin biosynthesis from tryptophan requires four-step reactions. However, six genes, that is, TDC, TPH, T5H, SNAT, ASMT, and COMT, have been implicated in the synthesis of melatonin in plants, suggesting the presence of multiple pathways. Two major pathways have been proposed based on the enzyme kinetics: One is the tryptophan/tryptamine/serotonin/N-acetylserotonin/melatonin pathway, which may occur under normal growth conditions; the other is the tryptophan/tryptamine/serotonin/5-methoxytryptamine/melatonin pathway, which may occur when plants produce large amounts of serotonin, for example, upon senescence. The melatonin biosynthetic capacity associated with conversion of tryptophan to serotonin is much higher than that associated with conversion of serotonin to melatonin, which yields a low level of melatonin synthesis in plants. Many melatonin intermediates are produced in various subcellular compartments, such as the cytoplasm, endoplasmic reticulum, and chloroplasts, which either facilitates or impedes the subsequent enzymatic steps. Depending on the pathways, the final subcellular sites of melatonin synthesis vary at either the cytoplasm or chloroplasts, which may differentially affect the mode of action of melatonin in plants. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The G protein-coupled estrogen receptor (GPER) is expressed in two different subcellular localizations reflecting distinct tumor properties in breast cancer.

PubMed

Samartzis, Eleftherios P; Noske, Aurelia; Meisel, Alexander; Varga, Zsuzsanna; Fink, Daniel; Imesch, Patrick

2014-01-01

The G protein-coupled estrogen receptor (GPER) is a novel estrogen receptor that mediates proliferative effects induced by estrogen but also by tamoxifen. The aim of our study was to analyze the frequency of GPER in a large collective of primary invasive breast carcinomas, with special emphasis on the subcellular expression and to evaluate the association with clinicopathological parameters and patient overall survival. The tissue microarrays from formalin-fixed, paraffin embedded samples of primary invasive breast carcinomas (n = 981) were analyzed for GPER expression using immunohistochemistry. Expression data were compared to the clinicopathological parameters and overall survival. GPER localization was also analyzed in two immortalized breast cancer cell lines T47D and MCF7 by confocal immunofluorescence microscopy. A predominantly cytoplasmic GPER expression was found in 189 carcinomas (19.3%), whereas a predominantly nuclear expression was observed in 529 cases (53.9%). A simultaneous comparable positive expression of both patterns was found in 32 of 981 cases (3.2%), and negative staining was detected in 295 cases (30%). Confocal microscopy confirmed the occurrence of cytoplasmic and nuclear GPER expression in T47D and MCF7. Cytoplasmic GPER expression was significantly associated with non-ductal histologic subtypes, low tumor stage, better histologic differentiation, as well as Luminal A and B subtypes. In contrast, nuclear GPER expression was significantly associated with poorly differentiated carcinomas and the triple-negative subtype. In univariate analysis, cytoplasmic GPER expression was associated with better overall survival (p = 0.012). Our data suggest that predominantly cytoplasmic and/or nuclear GPER expression are two distinct immunohistochemical patterns in breast carcinomas and may reflect different biological features, reason why these patterns should be clearly distinguished in histological evaluations. Prospective studies will be needed to assess whether the expression status of GPER in breast carcinomas should be routinely observed by clinicians, for instance, before implementing endocrine breast cancer treatment.

The G Protein-Coupled Estrogen Receptor (GPER) Is Expressed in Two Different Subcellular Localizations Reflecting Distinct Tumor Properties in Breast Cancer

PubMed Central

Samartzis, Eleftherios P.; Noske, Aurelia; Meisel, Alexander; Varga, Zsuzsanna; Fink, Daniel; Imesch, Patrick

2014-01-01

Introduction The G protein-coupled estrogen receptor (GPER) is a novel estrogen receptor that mediates proliferative effects induced by estrogen but also by tamoxifen. The aim of our study was to analyze the frequency of GPER in a large collective of primary invasive breast carcinomas, with special emphasis on the subcellular expression and to evaluate the association with clinicopathological parameters and patient overall survival. Methods The tissue microarrays from formalin-fixed, paraffin embedded samples of primary invasive breast carcinomas (n = 981) were analyzed for GPER expression using immunohistochemistry. Expression data were compared to the clinicopathological parameters and overall survival. GPER localization was also analyzed in two immortalized breast cancer cell lines T47D and MCF7 by confocal immunofluorescence microscopy. Results A predominantly cytoplasmic GPER expression was found in 189 carcinomas (19.3%), whereas a predominantly nuclear expression was observed in 529 cases (53.9%). A simultaneous comparable positive expression of both patterns was found in 32 of 981 cases (3.2%), and negative staining was detected in 295 cases (30%). Confocal microscopy confirmed the occurrence of cytoplasmic and nuclear GPER expression in T47D and MCF7. Cytoplasmic GPER expression was significantly associated with non-ductal histologic subtypes, low tumor stage, better histologic differentiation, as well as Luminal A and B subtypes. In contrast, nuclear GPER expression was significantly associated with poorly differentiated carcinomas and the triple-negative subtype. In univariate analysis, cytoplasmic GPER expression was associated with better overall survival (p = 0.012). Conclusion Our data suggest that predominantly cytoplasmic and/or nuclear GPER expression are two distinct immunohistochemical patterns in breast carcinomas and may reflect different biological features, reason why these patterns should be clearly distinguished in histological evaluations. Prospective studies will be needed to assess whether the expression status of GPER in breast carcinomas should be routinely observed by clinicians, for instance, before implementing endocrine breast cancer treatment. PMID:24421881

Protein subcellular localization assays using split fluorescent proteins

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2009-09-08

The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

Sub-cellular force microscopy in single normal and cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Babahosseini, H.; Carmichael, B.; Strobl, J.S.

2015-08-07

This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer andmore » significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. - Highlights: • The cells are modeled as a triple-layered structure using Generalized Maxwell model. • The sub-domains include membrane/cortex, cytoplasm/nucleus, and nuclear/integrin. • Biomechanics of corresponding sub-domains are compared among normal and cancer cells. • Viscoelasticity of sub-domains show a decreasing trend from normal to cancer cells. • The decreasing trend becomes most significant in the deeper sub-domain.« less

High Speed Size Sorting of Subcellular Organelles by Flow Field-Flow Fractionation.

PubMed

Yang, Joon Seon; Lee, Ju Yong; Moon, Myeong Hee

2015-06-16

Separation/isolation of subcellular species, such as mitochondria, lysosomes, peroxisomes, Golgi apparatus, and others, from cells is important for gaining an understanding of the cellular functions performed by specific organelles. This study introduces a high speed, semipreparative scale, biocompatible size sorting method for the isolation of subcellular organelle species from homogenate mixtures of HEK 293T cells using flow field-flow fractionation (FlFFF). Separation of organelles was achieved using asymmetrical FlFFF (AF4) channel system at the steric/hyperlayer mode in which nuclei, lysosomes, mitochondria, and peroxisomes were separated in a decreasing order of hydrodynamic diameter without complicated preprocessing steps. Fractions in which organelles were not clearly separated were reinjected to AF4 for a finer separation using the normal mode, in which smaller sized species can be well fractionated by an increasing order of diameter. The subcellular species contained in collected AF4 fractions were examined with scanning electron microscopy to evaluate their size and morphology, Western blot analysis using organelle specific markers was used for organelle confirmation, and proteomic analysis was performed with nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS). Since FlFFF operates with biocompatible buffer solutions, it offers great flexibility in handling subcellular components without relying on a high concentration sucrose solution for centrifugation or affinity- or fluorescence tag-based sorting methods. Consequently, the current study provides an alternative, competitive method for the isolation/purification of subcellular organelle species in their intact states.

Neuroglian and DE-cadherin activate independent cytoskeleton assembly pathways in Drosophila S2 cells.

PubMed

Dubreuil, R R; Grushko, T

1999-11-19

The cytoskeletal proteins spectrin and ankyrin colocalize with sites of E-cadherin-mediated cell-cell adhesion in mammalian cells. Here we examined the effects of Drosophila DE-cadherin expression on spectrin and ankyrin in Drosophila S2 tissue culture cells. DE-cadherin caused a dramatic change in the cytoplasmic concentration and distribution of armadillo, the Drosophila homolog of beta catenin. However, DE-cadherin expression had no detectable effect on the quantity or subcellular distribution of ankyrin or spectrin. In reciprocal experiments, recruitment of ankyrin and alphabeta spectrin to the plasma membrane by another cell adhesion molecule, neuroglian, had no effect on the quantity or distribution of armadillo. The results indicate that DE-cadherin-catenin complexes and neuroglian-spectrin/ankyrin complexes form by nonintersecting pathways. Recruitment of spectrin does not appear to be a conserved feature of DE-cadherin function. Copyright 1999 Academic Press.

Subcellular localization of full-length human myeloid leukemia factor 1 (MLF1) is independent of 14-3-3 proteins.

PubMed

Molzan, Manuela; Ottmann, Christian

2013-03-01

Myeloid leukemia factor 1 (MLF1) is associated with the development of leukemic diseases such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, information on the physiological function of MLF1 is limited and mostly derived from studies identifying MLF1 interaction partners like CSN3, MLF1IP, MADM, Manp and the 14-3-3 proteins. The 14-3-3-binding site surrounding S34 is one of the only known functional features of the MLF1 sequence, along with one nuclear export sequence (NES) and two nuclear localization sequences (NLS). It was recently shown that the subcellular localization of mouse MLF1 is dependent on 14-3-3 proteins. Based on these findings, we investigated whether the subcellular localization of human MLF1 was also directly 14-3-3-dependent. Live cell imaging with GFP-fused human MLF1 was used to study the effects of mutations and deletions on its subcellular localization. Surprisingly, we found that the subcellular localization of full-length human MLF1 is 14-3-3-independent, and is probably regulated by other as-yet-unknown proteins.

Predicting Human Protein Subcellular Locations by the Ensemble of Multiple Predictors via Protein-Protein Interaction Network with Edge Clustering Coefficients

PubMed Central

Du, Pufeng; Wang, Lusheng

2014-01-01

One of the fundamental tasks in biology is to identify the functions of all proteins to reveal the primary machinery of a cell. Knowledge of the subcellular locations of proteins will provide key hints to reveal their functions and to understand the intricate pathways that regulate biological processes at the cellular level. Protein subcellular location prediction has been extensively studied in the past two decades. A lot of methods have been developed based on protein primary sequences as well as protein-protein interaction network. In this paper, we propose to use the protein-protein interaction network as an infrastructure to integrate existing sequence based predictors. When predicting the subcellular locations of a given protein, not only the protein itself, but also all its interacting partners were considered. Unlike existing methods, our method requires neither the comprehensive knowledge of the protein-protein interaction network nor the experimentally annotated subcellular locations of most proteins in the protein-protein interaction network. Besides, our method can be used as a framework to integrate multiple predictors. Our method achieved 56% on human proteome in absolute-true rate, which is higher than the state-of-the-art methods. PMID:24466278

Deciphering the roles of acyl-CoA-binding proteins in plant cells.

PubMed

Lung, Shiu-Cheung; Chye, Mee-Len

2016-09-01

Lipid trafficking is vital for metabolite exchange and signal communications between organelles and endomembranes. Acyl-CoA-binding proteins (ACBPs) are involved in the intracellular transport, protection, and pool formation of acyl-CoA esters, which are important intermediates and regulators in lipid metabolism and cellular signaling. In this review, we highlight recent advances in our understanding of plant ACBP families from a cellular and developmental perspective. Plant ACBPs have been extensively studied in Arabidopsis thaliana (a dicot) and to a lesser extent in Oryza sativa (a monocot). Thus far, they have been detected in the plasma membrane, vesicles, endoplasmic reticulum, Golgi apparatus, apoplast, cytosol, nuclear periphery, and peroxisomes. In combination with biochemical and molecular genetic tools, the widespread subcellular distribution of respective ACBP members has been explicitly linked to their functions in lipid metabolism during development and in response to stresses. At the cellular level, strong expression of specific ACBP homologs in specialized cells, such as embryos, stem epidermis, guard cells, male gametophytes, and phloem sap, is of relevance to their corresponding distinct roles in organ development and stress responses. Other interesting patterns in their subcellular localization and spatial expression that prompt new directions in future investigations are discussed.

In vivo subcellular localization of Mal de Rio Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maroniche, Guillermo A.; Mongelli, Vanesa C.; Llauger, Gabriela

2012-09-01

The in vivo subcellular localization of Mal de Rio Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1more » viroplasm-like structures and co-localized with P7-2, P10 and {alpha}-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.« less

Parvovirus interference with intracellular signalling: mechanism of PKCeta activation in MVM-infected A9 fibroblasts.

PubMed

Lachmann, Sylvie; Bär, Severine; Rommelaere, Jean; Nüesch, Jürg P F

2008-03-01

Autonomous parvoviruses are strongly dependent on the phosphorylation of the major non-structural protein NS1 by members of the protein kinase C (PKC) family. Besides being accompanied with changes in the overall phosphorylation pattern of NS1 and acquiring new modifications at consensus PKC sites, ongoing minute virus of mice (MVM) infections lead to the appearance of new phosphorylated cellular protein species. This prompted us to investigate whether MVM actively interferes with phosphoinositol-dependent kinase (PDK)/PKC signalling. The activity, subcellular localization and phosphorylation status of the protein kinases PDK1, PKCeta and PKClambda were measured in A9 cells in the presence or absence of MVM infection. Parvovirus infection was found to result in activation of both PDK1 and PKCeta, as evidenced by changes in their subcellular distribution and overall (auto)phosphorylation. We show evidence that activation of PKCeta by PDK1 is driven by atypical PKClambda. By modifying the hydrophobic motif of PKCeta, PKClambda appeared to control docking and consecutive phosphorylation of PKCeta's activation-loop by PDK1, a process that was inhibited in vivo in the presence of a dominant-negative PKClambda mutant.

Localization and regulation of the N terminal splice variant of PGC-1α in adult skeletal muscle fibers.

PubMed

Shen, Tiansheng; Liu, Yewei; Schneider, Martin F

2012-01-01

The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) regulates expression of genes for metabolism and muscle fiber type. Recently, a novel splice variant of PGC-1α (NT-PGC-1α, amino acids 1-270) was cloned and found to be expressed in muscle. Here we use Flag-tagged NT-PGC-1α to examine the subcellular localization and regulation of NT-PGC-1α in skeletal muscle fibers. Flag-NT-PGC-1α is located predominantly in the myoplasm. Nuclear NT-PGC-1α can be increased by activation of protein kinase A. Activation of p38 MAPK by muscle activity or of AMPK had no effect on the subcellular distribution of NT-PGC-1α. Inhibition of CRM1-mediated export only caused relatively slow nuclear accumulation of NT-PGC-1α, indicating that nuclear export of NT-PGC-1α may be mediated by both CRM1-dependent and -independent pathways. Together these results suggest that the regulation of NT-PGC-1α in muscle fibers may be very different from that of the full-length PGC-1α, which is exclusively nuclear.

Copper and zinc contamination in oysters: subcellular distribution and detoxification.

PubMed

Wang, Wen-Xiong; Yang, Yubo; Guo, Xiaoyu; He, Mei; Guo, Feng; Ke, Caihuan

2011-08-01

Metal pollution levels in estuarine and coastal environments have been widely reported, but few documented reports exist of severe contamination in specific environments. Here, we report on a metal-contaminated estuary in Fujian Province, China, in which blue oysters (Crassostrea hongkongensis) and green oysters (Crassostrea angulata) were discovered to be contaminated with Cu and other metals. Extraordinarily high metal concentrations were found in the oysters collected from the estuary. Comparison with historical data suggests that the estuary has recently been contaminated with Cr, Cu, Ni, and Zn. Metal concentrations in blue oysters were as high as 1.4 and 2.4% of whole-body tissue dry wt for Cu and Zn, respectively. Cellular debris was the main subcellular fraction binding the metals, but metal-rich granules were important for Cr, Ni, and Pb. With increasing Cu accumulation, its partitioning into the cytosolic proteins decreased. In contrast, metallothionein-like proteins increased their importance in binding with Zn as tissue concentrations of Zn increased. In the most severely contaminated oysters, only a negligible fraction of their Cu and Zn was bound with the metal-sensitive fraction, which may explain the survival of oysters in such contaminated environments. Copyright © 2011 SETAC.

A Novel System for Visualizing Alphavirus Assembly

PubMed Central

Steel, J. Jordan; Geiss, Brian J.

2015-01-01

Alphaviruses are small, enveloped RNA viruses that form infectious particles by budding through the cellular plasma membrane. To help visualize and understand the intracellular assembly of alphavirus virions we have developed a bimolecular fluorescence complementation-based system (BiFC) that allows visualization of capsid and E2 subcellular localization and association in live cells. In this system, N- or C-terminal Venus fluorescent protein fragments (VN- and VC-) are fused to the N-terminus of the capsid protein on the Sindbis virus structural polyprotein, which results in the formation of fluorescent capsid-like structures in the absence of viral genomes that associate with the plasma membrane of cells. Mutation of the capsid autoprotease active site blocks structural polyprotein processing and alters the subcellular distribution of capsid fluorescence. Incorporating mCherry into the extracellular domain of the E2 glycoprotein allows the visualization of E2 glycoprotein localization and showed a close association of the E2 and capsid proteins at the plasma membrane as expected. These results suggest that this system is a useful new tool to study alphavirus assembly in live cells and may be useful in identifying molecules that inhibit alphavirus virion formation. PMID:26122073

Functional analysis of the Arabidopsis PHT4 family of intracellular phosphate transporters.

PubMed

Guo, B; Jin, Y; Wussler, C; Blancaflor, E B; Motes, C M; Versaw, W K

2008-01-01

The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic, biochemical and bioinformatics studies. Heterologous expression in yeast, and gene expression and localization in plants were used to characterize all six members of an Arabidopsis thaliana membrane transporter family designated here as PHT4. PHT4 proteins share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride. All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid envelope, and the sixth resides in the Golgi apparatus. PHT4 genes are expressed in both roots and leaves, although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.

Arginine Decarboxylase Is Localized in Chloroplasts.

PubMed Central

Borrell, A.; Culianez-Macia, F. A.; Altabella, T.; Besford, R. T.; Flores, D.; Tiburcio, A. F.

1995-01-01

Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC. PMID:12228631

Protein subcellular localization prediction using artificial intelligence technology.

PubMed

Nair, Rajesh; Rost, Burkhard

2008-01-01

Proteins perform many important tasks in living organisms, such as catalysis of biochemical reactions, transport of nutrients, and recognition and transmission of signals. The plethora of aspects of the role of any particular protein is referred to as its "function." One aspect of protein function that has been the target of intensive research by computational biologists is its subcellular localization. Proteins must be localized in the same subcellular compartment to cooperate toward a common physiological function. Aberrant subcellular localization of proteins can result in several diseases, including kidney stones, cancer, and Alzheimer's disease. To date, sequence homology remains the most widely used method for inferring the function of a protein. However, the application of advanced artificial intelligence (AI)-based techniques in recent years has resulted in significant improvements in our ability to predict the subcellular localization of a protein. The prediction accuracy has risen steadily over the years, in large part due to the application of AI-based methods such as hidden Markov models (HMMs), neural networks (NNs), and support vector machines (SVMs), although the availability of larger experimental datasets has also played a role. Automatic methods that mine textual information from the biological literature and molecular biology databases have considerably sped up the process of annotation for proteins for which some information regarding function is available in the literature. State-of-the-art methods based on NNs and HMMs can predict the presence of N-terminal sorting signals extremely accurately. Ab initio methods that predict subcellular localization for any protein sequence using only the native amino acid sequence and features predicted from the native sequence have shown the most remarkable improvements. The prediction accuracy of these methods has increased by over 30% in the past decade. The accuracy of these methods is now on par with high-throughput methods for predicting localization, and they are beginning to play an important role in directing experimental research. In this chapter, we review some of the most important methods for the prediction of subcellular localization.

pLoc_bal-mGpos: Predict subcellular localization of Gram-positive bacterial proteins by quasi-balancing training dataset and PseAAC.

PubMed

Xiao, Xuan; Cheng, Xiang; Chen, Genqiang; Mao, Qi; Chou, Kuo-Chen

2018-05-26

Knowledge of protein subcellular localization is vitally important for both basic research and drug development. With the avalanche of protein sequences emerging in the post-genomic age, it is highly desired to develop computational tools for timely and effectively identifying their subcellular localization purely based on the sequence information alone. Recently, a predictor called "pLoc-mGpos" was developed for identifying the subcellular localization of Gram-positive bacterial proteins. Its performance is overwhelmingly better than that of the other predictors for the same purpose, particularly in dealing with multi-label systems in which some proteins, called "multiplex proteins", may simultaneously occur in two or more subcellular locations. Although it is indeed a very powerful predictor, more efforts are definitely needed to further improve it. This is because pLoc-mGpos was trained by an extremely skewed dataset in which some subset (subcellular location) was over 11 times the size of the other subsets. Accordingly, it cannot avoid the bias consequence caused by such an uneven training dataset. To alleviate such bias consequence, we have developed a new and bias-reducing predictor called pLoc_bal-mGpos by quasi-balancing the training dataset. Rigorous target jackknife tests on exactly the same experiment-confirmed dataset have indicated that the proposed new predictor is remarkably superior to pLoc-mGpos, the existing state-of-the-art predictor in identifying the subcellular localization of Gram-positive bacterial proteins. To maximize the convenience for most experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc_bal-mGpos/, by which users can easily get their desired results without the need to go through the detailed mathematics. Copyright © 2018 Elsevier Inc. All rights reserved.

Compressed learning and its applications to subcellular localization.

PubMed

Zheng, Zhong-Long; Guo, Li; Jia, Jiong; Xie, Chen-Mao; Zeng, Wen-Cai; Yang, Jie

2011-09-01

One of the main challenges faced by biological applications is to predict protein subcellular localization in automatic fashion accurately. To achieve this in these applications, a wide variety of machine learning methods have been proposed in recent years. Most of them focus on finding the optimal classification scheme and less of them take the simplifying the complexity of biological systems into account. Traditionally, such bio-data are analyzed by first performing a feature selection before classification. Motivated by CS (Compressed Sensing) theory, we propose the methodology which performs compressed learning with a sparseness criterion such that feature selection and dimension reduction are merged into one analysis. The proposed methodology decreases the complexity of biological system, while increases protein subcellular localization accuracy. Experimental results are quite encouraging, indicating that the aforementioned sparse methods are quite promising in dealing with complicated biological problems, such as predicting the subcellular localization of Gram-negative bacterial proteins.

Geary autocorrelation and DCCA coefficient: Application to predict apoptosis protein subcellular localization via PSSM

NASA Astrophysics Data System (ADS)

Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

2017-02-01

Apoptosis is a fundamental process controlling normal tissue homeostasis by regulating a balance between cell proliferation and death. Predicting subcellular location of apoptosis proteins is very helpful for understanding its mechanism of programmed cell death. Prediction of apoptosis protein subcellular location is still a challenging and complicated task, and existing methods mainly based on protein primary sequences. In this paper, we propose a new position-specific scoring matrix (PSSM)-based model by using Geary autocorrelation function and detrended cross-correlation coefficient (DCCA coefficient). Then a 270-dimensional (270D) feature vector is constructed on three widely used datasets: ZD98, ZW225 and CL317, and support vector machine is adopted as classifier. The overall prediction accuracies are significantly improved by rigorous jackknife test. The results show that our model offers a reliable and effective PSSM-based tool for prediction of apoptosis protein subcellular localization.

Detrended cross-correlation coefficient: Application to predict apoptosis protein subcellular localization.

PubMed

Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

2016-12-01

Apoptosis, or programed cell death, plays a central role in the development and homeostasis of an organism. Obtaining information on subcellular location of apoptosis proteins is very helpful for understanding the apoptosis mechanism. The prediction of subcellular localization of an apoptosis protein is still a challenging task, and existing methods mainly based on protein primary sequences. In this paper, we introduce a new position-specific scoring matrix (PSSM)-based method by using detrended cross-correlation (DCCA) coefficient of non-overlapping windows. Then a 190-dimensional (190D) feature vector is constructed on two widely used datasets: CL317 and ZD98, and support vector machine is adopted as classifier. To evaluate the proposed method, objective and rigorous jackknife cross-validation tests are performed on the two datasets. The results show that our approach offers a novel and reliable PSSM-based tool for prediction of apoptosis protein subcellular localization. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of mycobacterial surface proteins released into subcellular compartments of infected macrophages.

PubMed

Beatty, W L; Russell, D G

2000-12-01

Considerable effort has focused on the identification of proteins secreted from Mycobacterium spp. that contribute to the development of protective immunity. Little is known, however, about the release of mycobacterial proteins from the bacterial phagosome and the potential role of these molecules in chronically infected macrophages. In the present study, the release of mycobacterial surface proteins from the bacterial phagosome into subcellular compartments of infected macrophages was analyzed. Mycobacterium bovis BCG was surface labeled with fluorescein-tagged succinimidyl ester, an amine-reactive probe. The fluorescein tag was then used as a marker for the release of bacterial proteins in infected macrophages. Fractionation studies revealed bacterial proteins within subcellular compartments distinct from mycobacteria and mycobacterial phagosomes. To identify these proteins, subcellular fractions free of bacteria were probed with mycobacterium-specific antibodies. The fibronectin attachment protein and proteins of the antigen 85-kDa complex were identified among the mycobacterial proteins released from the bacterial phagosome.

Power-law confusion: You say incremental, I say differential

NASA Technical Reports Server (NTRS)

Colwell, Joshua E.

1993-01-01

Power-law distributions are commonly used to describe the frequency of occurrences of crater diameters, stellar masses, ring particle sizes, planetesimal sizes, and meteoroid masses to name a few. The distributions are simple, and this simplicity has led to a number of misstatements in the literature about the kind of power-law that is being used: differential, cumulative, or incremental. Although differential and cumulative power-laws are mathematically trivial, it is a hybrid incremental distribution that is often used and the relationship between the incremental distribution and the differential or cumulative distributions is not trivial. In many cases the slope of an incremental power-law will be nearly identical to the slope of the cumulative power-law of the same distribution, not the differential slope. The discussion that follows argues for a consistent usage of these terms and against the oft-made implicit claim that incremental and differential distributions are indistinguishable.

Subcellular Distribution of S-Nitrosylated H-Ras in Differentiated and Undifferentiated PC12 Cells during Hypoxia.

PubMed

Barbakadze, Tamar; Goloshvili, Galina; Narmania, Nana; Zhuravliova, Elene; Mikeladze, David

2017-10-01

Hypoxia or exposure to excessive reactive oxygen or nitrogen species could induce S-nitrosylation of various target proteins, including GTPases of the Ras-superfamily. Under hypoxic conditions, the Ras-protein is translocated to the cytosol and interacts with the Golgi complex, endoplasmic reticulum, mitochondria. The mobility/translocation of Ras depend on the cells oxidative status. However, the importance of relocated Snitrosylated- H-Ras (NO-H-Ras) in proliferation/differentiation processes is not completely understood. We have determined the content of soluble- and membrane-bound-NO-HRas in differentiated (D) and undifferentiated (ND) rat pheochromocytoma (PC12) cells under hypoxic and normoxic conditions. In our experimental study, we analyzed NO-H-Ras levels under hypoxic/normoxic conditions in membrane and soluble fractions of ND and D PC12 cells with/without nitric oxide donor, sodium nitroprusside (SNP) treatment. Cells were analyzed by the S-nitrosylated kit, immunoprecipitation, and Western blot. We assessed the action of NO-H-Ras on oxidative metabolism of isolated mitochondria by determining mitochondrial hydrogen peroxide generation via the scopoletin oxidation method and ATPproduction as estimated by the luminometric method. Hypoxia did not influence nitrosylation of soluble H-Ras in ND PC12 cells. Under hypoxic conditions, the nitrosylation of soluble-H-Ras greatly decreased in D PC12 cells. SNP didn't change the levels of nitrosylation of soluble-H-Ras, in either hypoxic or normoxic conditions. On the other hand, hypoxia, per se, did not affect the nitrosylation of membrane-bound-H-Ras in D and ND PC12 cells. SNP-dependent nitrosylation of membrane-bound-H-Ras greatly increased in D PC12 cells. Both unmodified normal and mutated H-Ras enhanced the mitochondrial synthesis of ATP, whereas the stimulatory effects on ATP synthesis were eliminated after S-nitrosylation of H-Ras. According to the results, it may be proposed that hypoxia can decrease S-nitrosylation of soluble-H-Ras in D PC12 cells and abolish the inhibitory effect of NO-HRas in mitochondrial oxidative metabolism. Copyright© by Royan Institute. All rights reserved.

SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of ( sup 3 H)dextromethorphan and. sigma. ligands to guinea pig brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klein, M.; Canoll, P.D.; Musacchio, J.M.

1991-01-01

The DM{sub 1}/{sigma}{sub 1} site binds dextromethorphan (DM) and {sigma} receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of ({sup 3}H)dextromethorphan, ({sup 3}H)3- (3-Hydroxyphenyl) -N- (1-propyl) piperidine and (+)-({sup 3}H)1,3-Di-o-tolyl-guanidine (({sup 3}H)DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drugmore » has identical nM K{sub i} values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM{sub 1}{sigma}{sub 1} site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K{sub i} values of 9-13 and 3-4 {mu}M respectively against the three labeled ligands. These results, the broad specificity of the DM{sub 1}/{sigma}{sub 1} binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor.« less

Functional characterization of mutations in the myosin Vb gene associated with microvillus inclusion disease

PubMed Central

Szperl, Agata M.; Golachowska, Magdalena R.; Bruinenberg, Marcel; Prekeris, Rytis; Thunnissen, Andy-Mark W. H.; Karrenbeld, Arend; Dijkstra, Gerard; Hoekstra, Dick; Mercer, David; Ksiazyk, Janusz; Wijmenga, Cisca; Wapenaar, Martin C.; Rings, Edmond H. H. M.; van IJzendoorn, Sven C. D.

2010-01-01

Objectives Microvillus inclusion disease (MVID) is a rare autosomal recessive enteropathy characterized by intractable diarrhea and malabsorption. Recently, various MYO5B gene mutations have been identified in MVID patients. Interestingly, several MVID patients showed only a MYO5B mutation in one allele (heterozygous) or no mutations in the MYO5B gene, illustrating the need to further functionally characterize the cell biological effects of the MYO5B mutations. Methods The genomic DNA of nine patients diagnosed with microvillus inclusion disease was screened for MYO5B mutations, and qPCR and immunohistochemistry on the material of two patients was performed to investigate resultant cellular consequences. Results We demonstrate for the first time that MYO5B mutations can be correlated with altered myosin Vb mRNA expression and with an aberrant subcellular distribution of the myosin Vb protein. Moreover, we demonstrate that the typical and myosin Vb–controlled accumulation of rab11a-and FIP5-positive recycling endosomes in the apical cytoplasm of the cells is abolished in MVID enterocytes, which is indicative for altered myosin Vb function. Also, we report 8 novel MYO5B mutations in 9 MVID patients of various etnic backgrounds, including compound heterozygous mutations. Conclusions Our functional analysis indicate that MYO5B mutations can be correlated with an aberrant subcellular distribution of the myosin Vb protein and apical recycling endosomes which, together with the additional compound heterozygous mutations, significantly strengthen the link between MYO5B and MVID. PMID:21206382

Tissue distribution and subcellular localizations determine in vivo functional relationship among prostasin, matriptase, HAI-1, and HAI-2 in human skin.

PubMed

Lee, Shiao-Pieng; Kao, Chen-Yu; Chang, Shun-Cheng; Chiu, Yi-Lin; Chen, Yen-Ju; Chen, Ming-Hsing G; Chang, Chun-Chia; Lin, Yu-Wen; Chiang, Chien-Ping; Wang, Jehng-Kang; Lin, Chen-Yong; Johnson, Michael D

2018-01-01

The membrane-bound serine proteases prostasin and matriptase and the Kunitz-type protease inhibitors HAI-1 and HAI-2 are all expressed in human skin and may form a tightly regulated proteolysis network, contributing to skin pathophysiology. Evidence from other systems, however, suggests that the relationship between matriptase and prostasin and between the proteases and the inhibitors can be context-dependent. In this study the in vivo zymogen activation and protease inhibition status of matriptase and prostasin were investigated in the human skin. Immunohistochemistry detected high levels of activated prostasin in the granular layer, but only low levels of activated matriptase restricted to the basal layer. Immunoblot analysis of foreskin lysates confirmed this in vivo zymogen activation status and further revealed that HAI-1 but not HAI-2 is the prominent inhibitor for prostasin and matriptase in skin. The zymogen activation status and location of the proteases does not support a close functional relation between matriptase and prostasin in the human skin. The limited role for HAI-2 in the inhibition of matriptase and prostasin is the result of its primarily intracellular localization in basal and spinous layer keratinocytes, which probably prevents the Kunitz inhibitor from interacting with active prostasin or matriptase. In contrast, the cell surface expression of HAI-1 in all viable epidermal layers renders it an effective regulator for matriptase and prostasin. Collectively, our study suggests the importance of tissue distribution and subcellular localization in the functional relationship between proteases and protease inhibitors.

Nipah Virus V Protein Evades Alpha and Gamma Interferons by Preventing STAT1 and STAT2 Activation and Nuclear Accumulation

PubMed Central

Rodriguez, Jason J.; Parisien, Jean-Patrick; Horvath, Curt M.

2002-01-01

Characterization of recent outbreaks of fatal encephalitis in southeast Asia identified the causative agent to be a previously unrecognized enveloped negative-strand RNA virus of the Paramyxoviridae family, Nipah virus. One feature linking Nipah virus to this family is a conserved cysteine-rich domain that is the hallmark of paramyxovirus V proteins. The V proteins of other paramyxovirus species have been linked with evasion of host cell interferon (IFN) signal transduction and subsequent antiviral responses by inducing proteasomal degradation of the IFN-responsive transcription factors, STAT1 or STAT2. Here we demonstrate that Nipah virus V protein escapes IFN by a distinct mechanism involving direct inhibition of STAT protein function. Nipah virus V protein differs from other paramyxovirus V proteins in its subcellular distribution but not in its ability to inhibit cellular IFN responses. Nipah virus V protein does not induce STAT degradation but instead inhibits IFN responses by forming high-molecular-weight complexes with both STAT1 and STAT2. We demonstrate that Nipah virus V protein accumulates in the cytoplasm by a Crm1-dependent mechanism, alters the STAT protein subcellular distribution in the steady state, and prevents IFN-stimulated STAT redistribution. Consistent with the formation of complexes, STAT protein tyrosine phosphorylation is inhibited in cells expressing the Nipah virus V protein. As a result, Nipah virus V protein efficiently prevents STAT1 and STAT2 nuclear translocation in response to IFN, inhibiting cellular responses to both IFN-α and IFN-γ. PMID:12388709

From morphology to clinical pathophysiology: multiphoton fluorescence lifetime imaging at patients' bedside

NASA Astrophysics Data System (ADS)

Mess, Christian; Zens, Katharina; Gorzelanny, Christian; Metze, Dieter; Luger, Thomas A.; König, Karsten; Schneider, Stefan W.; Huck, Volker

2017-02-01

Application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of skin diseases. By means of multiphoton excitation, endogenous biomolecules like NADH, collagen or elastin show autofluorescence or second harmonic generation. Thus, these molecules provide information about the subcellular morphology, epidermal architecture and physiological condition of the skin. To gain a deeper understanding of the linkage between cellular structure and physiological processes, non-invasive multiphotonbased intravital tomography (MPT) and fluorescence lifetime imaging (FLIM) were combined within the scopes of inflammatory skin, chronic wounds and drug delivery in clinical application. The optical biopsies generated via MPT were morphologically analyzed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Independent morphometric algorithms reliably showed a perinuclear accumulation in lesional skin in contrast to an even distribution in healthy skin. Confirmatively, MPT-FLIM showed an obvious metabolic shift in lesions. Moreover, detection of the onset and progression of inflammatory processes could be achieved. The feasibility of primary in vivo tracking of applied therapeutic agents further broadened our scope: We examined the permeation and subsequent distribution of agents directly visualized in patientś skin in short-term repetitive measurements. Furthermore, we performed MPT-FLIM follow-up investigations in the long-term course of therapy. Therefore, clinical MPT-FLIM application offers new insights into the pathophysiology and the individual therapeutic course of skin diseases, facilitating a better understanding of the processes of inflammation and wound healing.

Analysis of sublethal arsenic toxicity to Ceratophyllum demersum: subcellular distribution of arsenic and inhibition of chlorophyll biosynthesis

PubMed Central

Mishra, Seema; Alfeld, Matthias; Sobotka, Roman; Andresen, Elisa; Falkenberg, Gerald; Küpper, Hendrik

2016-01-01

Arsenic (As) pollution is a serious concern worldwide. Recent studies under environmentally relevant conditions revealed that, in the aquatic plant Ceratophyllum demersum, pigments are the first observable target of toxicity, prior to any effect on photosynthetic parameters or to oxidative stress. Lethal toxicity was initiated by a change of As species and their distribution pattern in various tissues. Here, the localization of As was investigated at the subcellular level through X-ray fluorescence using a submicron beam and a Maia detector. Further, it was possible to obtain useful tissue structural information from the ratio of the tomogram of photon flux behind the sample to the tomogram of Compton scattering. The micro-X-ray fluorescence tomograms showed that As predominantly accumulated in the nucleus of the epidermal cells in young mature leaves exposed to sublethal 1 µM As. This suggests that As may exert toxic effects in the nucleus, for example, by interfering with nucleic acid synthesis by replacing phosphorous with As. At higher cellular concentrations, As was mainly stored in the vacuole, particularly in mature leaves. An analysis of precursors of chlorophyll and degradation metabolites revealed that the observed decrease in chlorophyll concentration was associated with hindered biosynthesis, and was not due to degradation. Coproporphyrinogen III could not be detected after exposure to only 0.5 µM As. Levels of subsequent precursors, for example, protoporphyrin IX, Mg-protoporphyrin, Mg-protoporphyrin methyl ester, and divinyl protochlorophyllide, were significantly decreased at this concentration as well, indicating that the pathway was blocked upstream of tetrapyrrole synthesis. PMID:27340233

Subcellular Localization of Rice Leaf Aryl Acylamidase Activity 1

PubMed Central

Gaynor, John J.; Still, Cecil C.

1983-01-01

The intracellular localization of aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13) in rice (Oryza sativa L. var Starbonnet) leaves was investigated. The enzyme hydrolyzes and detoxifies the herbicide propanil (3,4-dichloropropionanilide) thereby accounting for immunity of the rice plant to herbicidal action. Fractionation of mesophyll protoplasts by differential centrifugation yielded the highest specific activity of amidase in the crude mitochondrial fraction. Further separation of density gradients of the silica sol Percoll also indicated that this enzyme was mitochondrial. By the use of biochemical markers, the purified mitochondrial fraction was shown to be substantially free of contamination from nuclei, chloroplasts, golgi, and plasma membranes. Subfractionation of the purified mitochondria suggests that this enzyme is located on the outer membrane. PMID:16662987

[Immunohistochemical description of proliferative activity and apoptosis of lung squamous cell carcinoma (literature review)].

PubMed

Филенко, Борис Н; Ройко, Наталия В; Степанчук, Алла П; Проскурня, Сергей А

2016-01-01

The analysis of the publications are describe immunohistochemical study of proliferative activity and apoptosis of lung squamous cell carcinoma. Established that the imbalance between proliferation and cell death is a key process in the development of tumors. However, the value of tumor markers in histogenesis and morfogenesis of tumors and forecast their occurrence is not studied enough. Despite the significant amount of scientific literature devoted to this issue, has not yet established a clear link expression of immunohistochemical markers of proliferation and apoptosis with the degree of differentiation of squamous cell lung cancer. Analysis of the literature shows that the morphology of this histogenetics type lung cancer at the cellular, subcellular structural and functional levels are controversial and require detailed investigation.

Using the concept of Chou's pseudo amino acid composition to predict apoptosis proteins subcellular location: an approach by approximate entropy.

PubMed

Jiang, Xiaoying; Wei, Rong; Zhang, Tongliang; Gu, Quan

2008-01-01

The function of protein is closely correlated with it subcellular location. Prediction of subcellular location of apoptosis proteins is an important research area in post-genetic era because the knowledge of apoptosis proteins is useful to understand the mechanism of programmed cell death. Compared with the conventional amino acid composition (AAC), the Pseudo Amino Acid composition (PseAA) as originally introduced by Chou can incorporate much more information of a protein sequence so as to remarkably enhance the power of using a discrete model to predict various attributes of a protein. In this study, a novel approach is presented to predict apoptosis protein solely from sequence based on the concept of Chou's PseAA composition. The concept of approximate entropy (ApEn), which is a parameter denoting complexity of time series, is used to construct PseAA composition as additional features. Fuzzy K-nearest neighbor (FKNN) classifier is selected as prediction engine. Particle swarm optimization (PSO) algorithm is adopted for optimizing the weight factors which are important in PseAA composition. Two datasets are used to validate the performance of the proposed approach, which incorporate six subcellular location and four subcellular locations, respectively. The results obtained by jackknife test are quite encouraging. It indicates that the ApEn of protein sequence could represent effectively the information of apoptosis proteins subcellular locations. It can at least play a complimentary role to many of the existing methods, and might become potentially useful tool for protein function prediction. The software in Matlab is available freely by contacting the corresponding author.

SUBCELLULAR PHARMACOKINETICS AND ITS POTENTIAL FOR LIBRARY FOCUSING (R826652)

EPA Science Inventory

Abstract

Subcellular pharmacokinetics (SP) optimizes biology-related factors in the design of libraries for high throughput screening by defining comparatively narrow ranges of properties (lipophilicity, amphiphilicity, acidity, reactivity, 3D-structural features) of t...

Subcellular Redox Targeting: Bridging in Vitro and in Vivo Chemical Biology.

PubMed

Long, Marcus J C; Poganik, Jesse R; Ghosh, Souradyuti; Aye, Yimon

2017-03-17

Networks of redox sensor proteins within discrete microdomains regulate the flow of redox signaling. Yet, the inherent reactivity of redox signals complicates the study of specific redox events and pathways by traditional methods. Herein, we review designer chemistries capable of measuring flux and/or mimicking subcellular redox signaling at the cellular and organismal level. Such efforts have begun to decipher the logic underlying organelle-, site-, and target-specific redox signaling in vitro and in vivo. These data highlight chemical biology as a perfect gateway to interrogate how nature choreographs subcellular redox chemistry to drive precision redox biology.

FRET-based genetically-encoded sensors for quantitative monitoring of metabolites.

PubMed

Mohsin, Mohd; Ahmad, Altaf; Iqbal, Muhammad

2015-10-01

Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.

Visualization of living terminal hypertrophic chondrocytes of growth plate cartilage in situ by differential interference contrast microscopy and time-lapse cinematography.

PubMed

Farnum, C E; Turgai, J; Wilsman, N J

1990-09-01

The functional unit within the growth plate consists of a column of chondrocytes that passes through a sequence of phases including proliferation, hypertrophy, and death. It is important to our understanding of the biology of the growth plate to determine if distal hypertrophic cells are viable, highly differentiated cells with the potential of actively controlling terminal events of endochondral ossification prior to their death at the chondro-osseous junction. This study for the first time reports on the visualization of living hypertrophic chondrocytes in situ, including the terminal hypertrophic chondrocyte. Chondrocytes in growth plate explants are visualized using rectified differential interference contrast microscopy. We record and measure, using time-lapse cinematography, the rate of movement of subcellular organelles at the limit of resolution of this light microscopy system. Control experiments to assess viability of hypertrophic chondrocytes include coincubating organ cultures with the intravital dye fluorescein diacetate to assess the integrity of the plasma membrane and cytoplasmic esterases. In this system, all hypertrophic chondrocytes, including the very terminal chondrocyte, exist as rounded, fully hydrated cells. By the criteria of intravital dye staining and organelle movement, distal hypertrophic chondrocytes are identical to chondrocytes in the proliferative and early hypertrophic cell zones.

One new kind of phytohormonal signaling integrator: Up-and-coming GASA family genes.

PubMed

Zhang, Shengchun; Wang, Xiaojing

2017-02-01

GASA proteins are characterized by an N-terminal signal peptide and a C-terminal conserved GASA domain with 12 invariant cysteine residues. Despite being widely distributed among plant species, their functions are not completely elucidated and little is known about their mechanism of action. This review focuses on the current knowledge about the molecular structure, protein subcellular localization and phytohormones responses of this up-and-coming family of peptides. Furthermore, we discussed the roles of GASA proteins in plant growth and development, plant responses to biotic or abiotic stresses and their participation in phytohormonal signaling integration.

Met receptor inhibitor SU11274 localizes in the endoplasmic reticulum.

PubMed

Wiest, Edwin J; Smith, Heather Jensen; Hollingsworth, Michael A

2018-07-02

We discovered that SU11274, a class I c-Met inhibitor, fluoresces when excited by 488 nm laser light and showed rapid specific accumulation in distinct subcellular compartments. Given that SU11274 reduces cancer cell viability, we exploited these newly identified spectral properties to determine SU11274 intracellular distribution and accumulation in human pancreatic cancer cells. The aim of the studies reported here was to identify organelle(s) to which SU11274 is trafficked. We conclude that SU11274 rapidly and predominantly accumulates in the endoplasmic reticulum. Copyright © 2018. Published by Elsevier Inc.

[Roles of G protein-coupled estrogen receptor in the male reproductive system].

PubMed

Chen, Kai-hong; Zhang, Xian; Jiang, Xue-wu

2016-02-01

The G protein-coupled estrogen receptor (GPER), also known as G protein-coupled receptor 30 (GPR30), was identified in the recent years as a functional membrane receptor different from the classical nuclear estrogen receptors. This receptor is widely expressed in the cortex, cerebellum, hippocampus, heart, lung, liver, skeletal muscle, and the urogenital system. It is responsible for the mediation of nongenomic effects associated with estrogen and its derivatives, participating in the physiological activities of the body. The present study reviews the molecular structure, subcellular localization, signaling pathways, distribution, and function of GPER in the male reproductive system.

Regulation of cardiomyocyte autophagy by calcium.

PubMed

Shaikh, Soni; Troncoso, Rodrigo; Criollo, Alfredo; Bravo-Sagua, Roberto; García, Lorena; Morselli, Eugenia; Cifuentes, Mariana; Quest, Andrew F G; Hill, Joseph A; Lavandero, Sergio

2016-04-15

Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy. Copyright © 2016 the American Physiological Society.

Clinical coherent anti-Stokes Raman scattering and multiphoton tomography of human skin with a femtosecond laser and photonic crystal fiber

NASA Astrophysics Data System (ADS)

Breunig, Hans Georg; Weinigel, Martin; Bückle, Rainer; Kellner-Höfer, Marcel; Lademann, Jürgen; Darvin, Maxim E.; Sterry, Wolfram; König, Karsten

2013-02-01

We report on in vivo coherent anti-Stokes Raman scattering spectroscopy (CARS), two-photon fluorescence and second-harmonic-generation imaging on human skin with a novel multimodal clinical CARS/multiphoton tomograph. CARS imaging is realized by a combination of femtosecond pulses with broadband continuum pulses generated by a photonic crystal fiber. The images reveal the microscopic distribution of (i) non-fluorescent lipids, (ii) endogenous fluorophores and (iii) the collagen network inside the human skin in vivo with subcellular resolution. Examples of healthy as well as cancer-affected skin are presented.

Systems Imaging of the Immune Synapse.

PubMed

Ambler, Rachel; Ruan, Xiangtao; Murphy, Robert F; Wülfing, Christoph

2017-01-01

Three-dimensional live cell imaging of the interaction of T cells with antigen-presenting cells (APCs) visualizes the subcellular distributions of signaling intermediates during T cell activation at thousands of resolved positions within a cell. These information-rich maps of local protein concentrations are a valuable resource in understanding T cell signaling. Here, we describe a protocol for the efficient acquisition of such imaging data and their computational processing to create four-dimensional maps of local concentrations. This protocol allows quantitative analysis of T cell signaling as it occurs inside live cells with resolution in time and space across thousands of cells.

Localization of intracellular and plasma membrane Ca2+-ATPases in the cerebellum.

PubMed

Sepúlveda, M Rosario; Mata, Ana M

2005-01-01

The sarco-endoplasmic reticulum Ca2+-ATPase and the plasma membrane Ca2+-ATPase contribute to the regulation of the intracellular Ca2+ concentration. These proteins transport Ca2+ ions into the endoplasmic reticulum and to the extracellular medium, respectively. A different localization of the two families of Ca2+-ATPases has been shown in concrete subcellular areas of Purkinje cells and in other neuronal elements from cerebellum. In the light of the actual knowledge of Ca2+-ATPases, this strict distribution suggests the existence of different demands on Ca2+ homeostasis in these cerebellar and cellular subregions.

Understanding radiation damage on sub-cellular scale using RADAMOL simulation tool

NASA Astrophysics Data System (ADS)

Štěpán, Václav; Davídková, Marie

2016-11-01

We present an overview of the biophysical model RADAMOL developed as a Monte Carlo simulation tool for physical, physico-chemical and chemical stages of ionizing radiation action. Direct and indirect radiation damage by 10 keV electrons, and protons and alpha particles with energies from 1 MeV up to 30 MeV to a free DNA oligomer or DNA in the complex with lac repressor protein is analyzed. The role of radiation type and energy, oxygen concentration and DNA interaction with proteins on yields and distributions of primary biomolecular damage is demonstrated and discussed.