Determining the sub-cellular localization of proteins within Caenorhabditis elegans body wall muscle.

PubMed

Meissner, Barbara; Rogalski, Teresa; Viveiros, Ryan; Warner, Adam; Plastino, Lorena; Lorch, Adam; Granger, Laure; Segalat, Laurent; Moerman, Donald G

2011-01-01

Determining the sub-cellular localization of a protein within a cell is often an essential step towards understanding its function. In Caenorhabditis elegans, the relatively large size of the body wall muscle cells and the exquisite organization of their sarcomeres offer an opportunity to identify the precise position of proteins within cell substructures. Our goal in this study is to generate a comprehensive "localizome" for C. elegans body wall muscle by GFP-tagging proteins expressed in muscle and determining their location within the cell. For this project, we focused on proteins that we know are expressed in muscle and are orthologs or at least homologs of human proteins. To date we have analyzed the expression of about 227 GFP-tagged proteins that show localized expression in the body wall muscle of this nematode (e.g. dense bodies, M-lines, myofilaments, mitochondria, cell membrane, nucleus or nucleolus). For most proteins analyzed in this study no prior data on sub-cellular localization was available. In addition to discrete sub-cellular localization we observe overlapping patterns of localization including the presence of a protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 14 sub-cellular localization patterns within nematode body wall muscle. The localization of this large set of proteins within a muscle cell will serve as an invaluable resource in our investigation of muscle sarcomere assembly and function.

SubCellProt: predicting protein subcellular localization using machine learning approaches.

PubMed

Garg, Prabha; Sharma, Virag; Chaudhari, Pradeep; Roy, Nilanjan

2009-01-01

High-throughput genome sequencing projects continue to churn out enormous amounts of raw sequence data. However, most of this raw sequence data is unannotated and, hence, not very useful. Among the various approaches to decipher the function of a protein, one is to determine its localization. Experimental approaches for proteome annotation including determination of a protein's subcellular localizations are very costly and labor intensive. Besides the available experimental methods, in silico methods present alternative approaches to accomplish this task. Here, we present two machine learning approaches for prediction of the subcellular localization of a protein from the primary sequence information. Two machine learning algorithms, k Nearest Neighbor (k-NN) and Probabilistic Neural Network (PNN) were used to classify an unknown protein into one of the 11 subcellular localizations. The final prediction is made on the basis of a consensus of the predictions made by two algorithms and a probability is assigned to it. The results indicate that the primary sequence derived features like amino acid composition, sequence order and physicochemical properties can be used to assign subcellular localization with a fair degree of accuracy. Moreover, with the enhanced accuracy of our approach and the definition of a prediction domain, this method can be used for proteome annotation in a high throughput manner. SubCellProt is available at www.databases.niper.ac.in/SubCellProt.

Learning cellular sorting pathways using protein interactions and sequence motifs.

PubMed

Lin, Tien-Ho; Bar-Joseph, Ziv; Murphy, Robert F

2011-11-01

Proper subcellular localization is critical for proteins to perform their roles in cellular functions. Proteins are transported by different cellular sorting pathways, some of which take a protein through several intermediate locations until reaching its final destination. The pathway a protein is transported through is determined by carrier proteins that bind to specific sequence motifs. In this article, we present a new method that integrates protein interaction and sequence motif data to model how proteins are sorted through these sorting pathways. We use a hidden Markov model (HMM) to represent protein sorting pathways. The model is able to determine intermediate sorting states and to assign carrier proteins and motifs to the sorting pathways. In simulation studies, we show that the method can accurately recover an underlying sorting model. Using data for yeast, we show that our model leads to accurate prediction of subcellular localization. We also show that the pathways learned by our model recover many known sorting pathways and correctly assign proteins to the path they utilize. The learned model identified new pathways and their putative carriers and motifs and these may represent novel protein sorting mechanisms. Supplementary results and software implementation are available from http://murphylab.web.cmu.edu/software/2010_RECOMB_pathways/.

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).

Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants.

PubMed

Aung, Kyaw; Xin, Xiufang; Mecey, Christy; He, Sheng Yang

2017-01-01

Animal and plant pathogenic bacteria use type III secretion systems to translocate proteinaceous effectors to subvert innate immunity of their host organisms. Type III secretion/effector systems are a crucial pathogenicity factor in many bacterial pathogens of plants and animals. Pseudomonas syringae pv. tomato (Pst) DC3000 injects a total of 36 protein effectors that target a variety of host proteins. Studies of a subset of Pst DC3000 effectors demonstrated that bacterial effectors, once inside the host cell, are localized to different subcellular compartments, including plasma membrane, cytoplasm, mitochondria, chloroplast, and Trans-Golgi network, to carry out their virulence functions. Identifying the subcellular localization of bacterial effector proteins in host cells could provide substantial clues to understanding the molecular and cellular basis of the virulence activities of effector proteins. In this chapter, we present methods for transient or stable expression of bacterial effector proteins in tobacco and/or Arabidopsis thaliana for live cell imaging as well as confirming the subcellular localization in plants using fluorescent organelle markers or chemical treatment.

LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell

PubMed Central

Sperschneider, Jana; Catanzariti, Ann-Maree; DeBoer, Kathleen; Petre, Benjamin; Gardiner, Donald M.; Singh, Karam B.; Dodds, Peter N.; Taylor, Jennifer M.

2017-01-01

Pathogens secrete effector proteins and many operate inside plant cells to enable infection. Some effectors have been found to enter subcellular compartments by mimicking host targeting sequences. Although many computational methods exist to predict plant protein subcellular localization, they perform poorly for effectors. We introduce LOCALIZER for predicting plant and effector protein localization to chloroplasts, mitochondria, and nuclei. LOCALIZER shows greater prediction accuracy for chloroplast and mitochondrial targeting compared to other methods for 652 plant proteins. For 107 eukaryotic effectors, LOCALIZER outperforms other methods and predicts a previously unrecognized chloroplast transit peptide for the ToxA effector, which we show translocates into tobacco chloroplasts. Secretome-wide predictions and confocal microscopy reveal that rust fungi might have evolved multiple effectors that target chloroplasts or nuclei. LOCALIZER is the first method for predicting effector localisation in plants and is a valuable tool for prioritizing effector candidates for functional investigations. LOCALIZER is available at http://localizer.csiro.au/. PMID:28300209

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

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.

Semi-supervised protein subcellular localization.

PubMed

Xu, Qian; Hu, Derek Hao; Xue, Hong; Yu, Weichuan; Yang, Qiang

2009-01-30

Protein subcellular localization is concerned with predicting the location of a protein within a cell using computational method. The location information can indicate key functionalities of proteins. Accurate predictions of subcellular localizations of protein can aid the prediction of protein function and genome annotation, as well as the identification of drug targets. Computational methods based on machine learning, such as support vector machine approaches, have already been widely used in the prediction of protein subcellular localization. However, a major drawback of these machine learning-based approaches is that a large amount of data should be labeled in order to let the prediction system learn a classifier of good generalization ability. However, in real world cases, it is laborious, expensive and time-consuming to experimentally determine the subcellular localization of a protein and prepare instances of labeled data. In this paper, we present an approach based on a new learning framework, semi-supervised learning, which can use much fewer labeled instances to construct a high quality prediction model. We construct an initial classifier using a small set of labeled examples first, and then use unlabeled instances to refine the classifier for future predictions. Experimental results show that our methods can effectively reduce the workload for labeling data using the unlabeled data. Our method is shown to enhance the state-of-the-art prediction results of SVM classifiers by more than 10%.

Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

PubMed

Mas, Abraham; Amenós, Montse; Lois, L Maria

2016-01-01

Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

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.

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.

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.

A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana

PubMed Central

Mehlmer, Norbert; Parvin, Nargis; Hurst, Charlotte H.; Knight, Marc R.; Teige, Markus; Vothknecht, Ute C.

2014-01-01

Calcium has long been acknowledged as one of the most important signalling components in plants. Many abiotic and biotic stimuli are transduced into a cellular response by temporal and spatial changes in cellular calcium concentration and the calcium-sensitive protein aequorin has been exploited as a genetically encoded calcium indicator for the measurement of calcium in planta. The objective of this work was to generate a compatible set of aequorin expression plasmids for the generation of transgenic plant lines to measure changes in calcium levels in different cellular subcompartments. Aequorin was fused to different targeting peptides or organellar proteins as a means to localize it to the cytosol, the nucleus, the plasma membrane, and the mitochondria. Furthermore, constructs were designed to localize aequorin in the stroma as well as the inner and outer surface of the chloroplast envelope membranes. The modular set-up of the plasmids also allows the easy replacement of targeting sequences to include other compartments. An additional YFP-fusion was included to verify the correct subcellular localization of all constructs by laser scanning confocal microscopy. For each construct, pBin19-based binary expression vectors driven by the 35S or UBI10 promoter were made for Agrobacterium-mediated transformation. Stable Arabidopsis lines were generated and initial tests of several lines confirmed their feasibility to measure calcium signals in vivo. PMID:22213817

Analysis of the subcellular localization of the human histone methyltransferase SETDB1

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

Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp; Gotoh, Eiko; Kawamata, Natsuko

2015-10-02

SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclearmore » export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. - Highlights: • Endogenous human SETDB1 was localized mainly in the cytoplasm. • Combined treatment with LMB and MG132 led to accumulation of human SETDB1 in the nucleus. • HeLa cells expressing EFGP-hSETDB1 are useful for subcellular localization analyses.« less

The protein domains of the Dictyostelium microprocessor that are required for correct subcellular localization and for microRNA maturation.

PubMed

Kruse, Janis; Meier, Doreen; Zenk, Fides; Rehders, Maren; Nellen, Wolfgang; Hammann, Christian

2016-10-02

The maturation pathways of microRNAs (miRNAs) have been delineated for plants and several animals, belonging to the evolutionary supergroups of Archaeplastida and Opisthokonta, respectively. Recently, we reported the discovery of the microprocessor complex in Dictyostelium discoideum of the Amoebozoa supergroup. The complex is composed of the Dicer DrnB and the dsRBD (double-stranded RNA binding domain) containing protein RbdB. Both proteins localize at nucleoli, where they physically interact, and both are required for miRNA maturation. Here we show that the miRNA phenotype of a ΔdrnB gene deletion strain can be rescued by ectopic expression of a series of DrnB GFP fusion proteins, which consistently showed punctate perinucleolar localization in fluorescence microscopy. These punctate foci appear surprisingly stable, as they persist both disintegration of nucleoli and degradation of cellular nucleic acids. We observed that DrnB expression levels influence the number of microprocessor foci and alter RbdB accumulation. An investigation of DrnB variants revealed that its newly identified nuclear localization signal is necessary, but not sufficient for the perinucleolar localization. Biogenesis of miRNAs, which are RNA Pol II transcripts, is correlated with that localization. Besides its bidentate RNase III domains, DrnB contains only a dsRBD, which surprisingly is dispensable for miRNA maturation. This dsRBD can, however, functionally replace the homologous domain in RbdB. Based on the unique setup of the Dictyostelium microprocessor with a subcellular localization similar to plants, but a protein domain composition similar to animals, we propose a model for the evolutionary origin of RNase III proteins acting in miRNA maturation.

Prediction of protein subcellular localization by weighted gene ontology terms.

PubMed

Chi, Sang-Mun

2010-08-27

We develop a new weighting approach of gene ontology (GO) terms for predicting protein subcellular localization. The weights of individual GO terms, corresponding to their contribution to the prediction algorithm, are determined by the term-weighting methods used in text categorization. We evaluate several term-weighting methods, which are based on inverse document frequency, information gain, gain ratio, odds ratio, and chi-square and its variants. Additionally, we propose a new term-weighting method based on the logarithmic transformation of chi-square. The proposed term-weighting method performs better than other term-weighting methods, and also outperforms state-of-the-art subcellular prediction methods. Our proposed method achieves 98.1%, 99.3%, 98.1%, 98.1%, and 95.9% overall accuracies for the animal BaCelLo independent dataset (IDS), fungal BaCelLo IDS, animal Höglund IDS, fungal Höglund IDS, and PLOC dataset, respectively. Furthermore, the close correlation between high-weighted GO terms and subcellular localizations suggests that our proposed method appropriately weights GO terms according to their relevance to the localizations. Copyright 2010 Elsevier Inc. All rights reserved.

Subcellular localization of the Hpa RxLR effector repertoire identifies a tonoplast-associated protein HaRxL17 that confers enhanced plant susceptibility.

PubMed

Caillaud, Marie-Cécile; Piquerez, Sophie J M; Fabro, Georgina; Steinbrenner, Jens; Ishaque, Naveed; Beynon, Jim; Jones, Jonathan D G

2012-01-01

Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria in addition to their better-characterized role in suppressing plant defence. However, the specific mechanisms by which these effectors promote virulence remain unclear. To address this question, we examined changes in subcellular architecture using live-cell imaging during the compatible interaction between the oomycete Hyaloperonospora arabidopsidis (Hpa) and its host Arabidopsis. We monitored host-cell restructuring of subcellular compartments within plant mesophyll cells during haustoria ontogenesis. Live-cell imaging highlighted rearrangements in plant cell membranes upon infection, in particular to the tonoplast, which was located close to the extra-haustorial membrane surrounding the haustorium. We also investigated the subcellular localization patterns of Hpa RxLR effector candidates (HaRxLs) in planta. We identified two major classes of HaRxL effector based on localization: nuclear-localized effectors and membrane-localized effectors. Further, we identified a single effector, HaRxL17, that associated with the tonoplast in uninfected cells and with membranes around haustoria, probably the extra-haustorial membrane, in infected cells. Functional analysis of selected effector candidates in planta revealed that HaRxL17 enhances plant susceptibility. The roles of subcellular changes and effector localization, with specific reference to the potential role of HaRxL17 in plant cell membrane trafficking, are discussed with respect to Hpa virulence. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Learning Cellular Sorting Pathways Using Protein Interactions and Sequence Motifs

PubMed Central

Lin, Tien-Ho; Bar-Joseph, Ziv

2011-01-01

Abstract Proper subcellular localization is critical for proteins to perform their roles in cellular functions. Proteins are transported by different cellular sorting pathways, some of which take a protein through several intermediate locations until reaching its final destination. The pathway a protein is transported through is determined by carrier proteins that bind to specific sequence motifs. In this article, we present a new method that integrates protein interaction and sequence motif data to model how proteins are sorted through these sorting pathways. We use a hidden Markov model (HMM) to represent protein sorting pathways. The model is able to determine intermediate sorting states and to assign carrier proteins and motifs to the sorting pathways. In simulation studies, we show that the method can accurately recover an underlying sorting model. Using data for yeast, we show that our model leads to accurate prediction of subcellular localization. We also show that the pathways learned by our model recover many known sorting pathways and correctly assign proteins to the path they utilize. The learned model identified new pathways and their putative carriers and motifs and these may represent novel protein sorting mechanisms. Supplementary results and software implementation are available from http://murphylab.web.cmu.edu/software/2010_RECOMB_pathways/. PMID:21999284

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.

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.

A set of GFP-based organelle marker lines combined with DsRed-based gateway vectors for subcellular localization study in rice (Oryza sativa L.).

PubMed

Wu, Tsung-Meng; Lin, Ke-Chun; Liau, Wei-Shiang; Chao, Yun-Yang; Yang, Ling-Hung; Chen, Szu-Yun; Lu, Chung-An; Hong, Chwan-Yang

2016-01-01

In the post-genomic era, many useful tools have been developed to accelerate the investigation of gene functions. Fluorescent proteins have been widely used as protein tags for studying the subcellular localization of proteins in plants. Several fluorescent organelle marker lines have been generated in dicot plants; however, useful and reliable fluorescent organelle marker lines are lacking in the monocot model rice. Here, we developed eight different GFP-based organelle markers in transgenic rice and created a set of DsRed-based gateway vectors for combining with the marker lines. Two mitochondrial-localized rice ascorbate peroxidase genes fused to DsRed and successfully co-localized with mitochondrial-targeted marker lines verified the practical use of this system. The co-localization of GFP-fusion marker lines and DsRed-fusion proteins provide a convenient platform for in vivo or in vitro analysis of subcellular localization of rice proteins.

DeepLoc: prediction of protein subcellular localization using deep learning.

PubMed

Almagro Armenteros, José Juan; Sønderby, Casper Kaae; Sønderby, Søren Kaae; Nielsen, Henrik; Winther, Ole

2017-11-01

The prediction of eukaryotic protein subcellular localization is a well-studied topic in bioinformatics due to its relevance in proteomics research. Many machine learning methods have been successfully applied in this task, but in most of them, predictions rely on annotation of homologues from knowledge databases. For novel proteins where no annotated homologues exist, and for predicting the effects of sequence variants, it is desirable to have methods for predicting protein properties from sequence information only. Here, we present a prediction algorithm using deep neural networks to predict protein subcellular localization relying only on sequence information. At its core, the prediction model uses a recurrent neural network that processes the entire protein sequence and an attention mechanism identifying protein regions important for the subcellular localization. The model was trained and tested on a protein dataset extracted from one of the latest UniProt releases, in which experimentally annotated proteins follow more stringent criteria than previously. We demonstrate that our model achieves a good accuracy (78% for 10 categories; 92% for membrane-bound or soluble), outperforming current state-of-the-art algorithms, including those relying on homology information. The method is available as a web server at http://www.cbs.dtu.dk/services/DeepLoc. Example code is available at https://github.com/JJAlmagro/subcellular_localization. The dataset is available at http://www.cbs.dtu.dk/services/DeepLoc/data.php. jjalma@dtu.dk. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Remote Control of Gene Function by Local Translation

PubMed Central

Jung, Hosung; Gkogkas, Christos G.; Sonenberg, Nahum; Holt, Christine E.

2014-01-01

The subcellular position of a protein is a key determinant of its function. Mounting evidence indicates that RNA localization, where specific mRNAs are transported subcellularly and subsequently translated in response to localized signals, is an evolutionarily conserved mechanism to control protein localization. On-site synthesis confers novel signaling properties to a protein and helps to maintain local proteome homeostasis. Local translation plays particularly important roles in distal neuronal compartments, and dysregulated RNA localization and translation cause defects in neuronal wiring and survival. Here, we discuss key findings in this area and possible implications of this adaptable and swift mechanism for spatial control of gene function. PMID:24679524

Distinct domains within the NITROGEN LIMITATION ADAPTATION protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway

USDA-ARS?s Scientific Manuscript database

The NITROGEN LIMITATION ADAPTATION (NLA) protein is a RING-type E3 ubiquitin ligase that plays an essential role in the regulation of nitrogen and phosphate homeostasis. NLA is localized to two distinct subcellular sites, the plasma membrane and nucleus, and contains four distinct domains: i) a RING...

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.

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

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.

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.

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.

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

Determination of the Subcellular Distribution of Liposomes Using Confocal Microscopy.

PubMed

Solomon, Melani A

2017-01-01

It is being increasingly recognized that therapeutics need to be delivered to specific organelle targets within cells. Liposomes are versatile lipid-based drug delivery vehicles that can be surface-modified to deliver the loaded cargo to specific subcellular locations within the cell. Hence, the development of such technology requires a means of measuring the subcellular distribution possibly by utilizing imaging techniques that can visualize and quantitate the extent of this subcellular localization. The apparent increase of resolution along the Z-axis offered by confocal microscopy makes this technique suitable for such studies. In this chapter, we describe the application of confocal laser scanning microscopy (CLSM) to determine the subcellular distribution of fluorescently labeled mitochondriotropic liposomes.

Cdc2/cyclin B1 regulates centrosomal Nlp proteolysis and subcellular localization.

PubMed

Zhao, Xuelian; Jin, Shunqian; Song, Yongmei; Zhan, Qimin

2010-11-01

The formation of proper mitotic spindles is required for appropriate chromosome segregation during cell division. Aberrant spindle formation often causes aneuploidy and results in tumorigenesis. However, the underlying mechanism of regulating spindle formation and chromosome separation remains to be further defined. Centrosomal Nlp (ninein-like protein) is a recently characterized BRCA1-regulated centrosomal protein and plays an important role in centrosome maturation and spindle formation. In this study, we show that Nlp can be phosphorylated by cell cycle protein kinase Cdc2/cyclin B1. The phosphorylation sites of Nlp are mapped at Ser185 and Ser589. Interestingly, the Cdc2/cyclin B1 phosphorylation site Ser185 of Nlp is required for its recognition by PLK1, which enable Nlp depart from centrosomes to allow the establishment of a mitotic scaffold at the onset of mitosis . PLK1 fails to dissociate the Nlp mutant lacking Ser185 from centrosome, suggesting that Cdc2/cyclin B1 might serve as a primary kinase of PLK1 in regulating Nlp subcellular localization. However, the phosphorylation at the site Ser589 by Cdc2/cyclin B1 plays an important role in Nlp protein stability probably due to its effect on protein degradation. Furthermore, we show that deregulated expression or subcellular localization of Nlp lead to multinuclei in cells, indicating that scheduled levels of Nlp and proper subcellular localization of Nlp are critical for successful completion of normal cell mitosis, These findings demonstrate that Cdc2/cyclin B1 is a key regulator in maintaining appropriate degradation and subcellular localization of Nlp, providing novel insights into understanding on the role of Cdc2/cyclin B1 in mitotic progression.

Sparse regressions for predicting and interpreting subcellular localization of multi-label proteins.

PubMed

Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

2016-02-24

Predicting protein subcellular localization is indispensable for inferring protein functions. Recent studies have been focusing on predicting not only single-location proteins, but also multi-location proteins. Almost all of the high performing predictors proposed recently use gene ontology (GO) terms to construct feature vectors for classification. Despite their high performance, their prediction decisions are difficult to interpret because of the large number of GO terms involved. This paper proposes using sparse regressions to exploit GO information for both predicting and interpreting subcellular localization of single- and multi-location proteins. Specifically, we compared two multi-label sparse regression algorithms, namely multi-label LASSO (mLASSO) and multi-label elastic net (mEN), for large-scale predictions of protein subcellular localization. Both algorithms can yield sparse and interpretable solutions. By using the one-vs-rest strategy, mLASSO and mEN identified 87 and 429 out of more than 8,000 GO terms, respectively, which play essential roles in determining subcellular localization. More interestingly, many of the GO terms selected by mEN are from the biological process and molecular function categories, suggesting that the GO terms of these categories also play vital roles in the prediction. With these essential GO terms, not only where a protein locates can be decided, but also why it resides there can be revealed. Experimental results show that the output of both mEN and mLASSO are interpretable and they perform significantly better than existing state-of-the-art predictors. Moreover, mEN selects more features and performs better than mLASSO on a stringent human benchmark dataset. For readers' convenience, an online server called SpaPredictor for both mLASSO and mEN is available at http://bioinfo.eie.polyu.edu.hk/SpaPredictorServer/.

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.

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.

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.

mLASSO-Hum: A LASSO-based interpretable human-protein subcellular localization predictor.

PubMed

Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

2015-10-07

Knowing the subcellular compartments of human proteins is essential to shed light on the mechanisms of a broad range of human diseases. In computational methods for protein subcellular localization, knowledge-based methods (especially gene ontology (GO) based methods) are known to perform better than sequence-based methods. However, existing GO-based predictors often lack interpretability and suffer from overfitting due to the high dimensionality of feature vectors. To address these problems, this paper proposes an interpretable multi-label predictor, namely mLASSO-Hum, which can yield sparse and interpretable solutions for large-scale prediction of human protein subcellular localization. By using the one-vs-rest LASSO-based classifiers, 87 out of more than 8000 GO terms are found to play more significant roles in determining the subcellular localization. Based on these 87 essential GO terms, we can decide not only where a protein resides within a cell, but also why it is located there. To further exploit information from the remaining GO terms, a method based on the GO hierarchical information derived from the depth distance of GO terms is proposed. Experimental results show that mLASSO-Hum performs significantly better than state-of-the-art predictors. We also found that in addition to the GO terms from the cellular component category, GO terms from the other two categories also play important roles in the final classification decisions. For readers' convenience, the mLASSO-Hum server is available online at http://bioinfo.eie.polyu.edu.hk/mLASSOHumServer/. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biochemical localization of a protein involved in Gluconacetobacter hansenii cellulose synthesis

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

Iyer, Prashanti R; Catchmark, Jeffrey M; Brown, Nicole Robitaille

2011-02-08

Using subcellular fractionation and Western blot methods, we have shown that AcsD, one of the proteins encoded by the Acetobacter cellulose synthase (acs) operon, is localized in the periplasmic region of the cell. AcsD protein was heterologously expressed in Escherichia coli and purified using histidine tag affinity methods. The purified protein was used to obtain rabbit polyclonal antibodies. The purity of the subcellular fractions was assessed by marker enzyme assays.

RNA deep sequencing as a tool for selection of cell lines for systematic subcellular localization of all human proteins.

PubMed

Danielsson, Frida; Wiking, Mikaela; Mahdessian, Diana; Skogs, Marie; Ait Blal, Hammou; Hjelmare, Martin; Stadler, Charlotte; Uhlén, Mathias; Lundberg, Emma

2013-01-04

One of the major challenges of a chromosome-centric proteome project is to explore in a systematic manner the potential proteins identified from the chromosomal genome sequence, but not yet characterized on a protein level. Here, we describe the use of RNA deep sequencing to screen human cell lines for RNA profiles and to use this information to select cell lines suitable for characterization of the corresponding gene product. In this manner, the subcellular localization of proteins can be analyzed systematically using antibody-based confocal microscopy. We demonstrate the usefulness of selecting cell lines with high expression levels of RNA transcripts to increase the likelihood of high quality immunofluorescence staining and subsequent successful subcellular localization of the corresponding protein. The results show a path to combine transcriptomics with affinity proteomics to characterize the proteins in a gene- or chromosome-centric manner.

Subcellular localization of Mitf in monocytic cells.

PubMed

Lu, Ssu-Yi; Wan, Hsiao-Ching; Li, Mengtao; Lin, Yi-Ling

2010-06-01

Microphthalmia-associated transcription factor (Mitf) is a transcription factor that plays an important role in regulating the development of several cell lineages. The subcellular localization of Mitf is dynamic and is associated with its transcription activity. In this study, we examined factors that affect its subcellular localization in cells derived from the monocytic lineage since Mitf is present abundantly in these cells. We identified a domain encoded by Mitf exon 1B1b to be important for Mitf to commute between the cytoplasm and the nucleus. Deletion of this domain disrupts the shuttling of Mitf to the cytoplasm and results in its retention in the nucleus. M-CSF and RANKL both induce nuclear translocation of Mitf. We showed that Mitf nuclear transport is greatly influenced by ratio of M-CSF/Mitf protein expression. In addition, cell attachment to a solid surface also is needed for the nuclear transport of Mitf.

Enhancing membrane protein subcellular localization prediction by parallel fusion of multi-view features.

PubMed

Yu, Dongjun; Wu, Xiaowei; Shen, Hongbin; Yang, Jian; Tang, Zhenmin; Qi, Yong; Yang, Jingyu

2012-12-01

Membrane proteins are encoded by ~ 30% in the genome and function importantly in the living organisms. Previous studies have revealed that membrane proteins' structures and functions show obvious cell organelle-specific properties. Hence, it is highly desired to predict membrane protein's subcellular location from the primary sequence considering the extreme difficulties of membrane protein wet-lab studies. Although many models have been developed for predicting protein subcellular locations, only a few are specific to membrane proteins. Existing prediction approaches were constructed based on statistical machine learning algorithms with serial combination of multi-view features, i.e., different feature vectors are simply serially combined to form a super feature vector. However, such simple combination of features will simultaneously increase the information redundancy that could, in turn, deteriorate the final prediction accuracy. That's why it was often found that prediction success rates in the serial super space were even lower than those in a single-view space. The purpose of this paper is investigation of a proper method for fusing multiple multi-view protein sequential features for subcellular location predictions. Instead of serial strategy, we propose a novel parallel framework for fusing multiple membrane protein multi-view attributes that will represent protein samples in complex spaces. We also proposed generalized principle component analysis (GPCA) for feature reduction purpose in the complex geometry. All the experimental results through different machine learning algorithms on benchmark membrane protein subcellular localization datasets demonstrate that the newly proposed parallel strategy outperforms the traditional serial approach. We also demonstrate the efficacy of the parallel strategy on a soluble protein subcellular localization dataset indicating the parallel technique is flexible to suite for other computational biology problems. The software and datasets are available at: http://www.csbio.sjtu.edu.cn/bioinf/mpsp.

Structural requirements of oleosin domains for subcellular targeting to the oil body.

PubMed Central

van Rooijen, G J; Moloney, M M

1995-01-01

We have investigated the protein domains responsible for the correct subcellular targeting of plant seed oleosins. We have attempted to study this targeting in vivo using "tagged" oleosins in transgenic plants. Different constructs were prepared lacking gene sequences encoding one of three structural domains of natural oleosins. Each was fused in frame to the Escherichia coli uid A gene encoding beta-glucuronidase (GUS). These constructs were introduced into Brassica napus using Agrobacterium-mediated transformation. GUS activity was measured in washed oil bodies and in the soluble protein fraction of the transgenic seeds. It was found that complete Arabidopsis oleosin-GUS fusions undergo correct subcellular targeting in transgenic Brassica seeds. Removal of the C-terminal domain of the Arabidopsis oleosin comprising the last 48 amino acids had no effect on overall subcellular targeting. In contrast, loss of the first 47 amino acids (N terminus) or amino acids 48 to 113 (which make up a lipophilic core) resulted in impaired targeting of the fusion protein to the oil bodies and greatly reduced accumulation of the fusion protein. Northern blotting revealed that this reduction is not due to differences in mRNA accumulation. Results from these measurements indicated that both the N-terminal and central oleosin domain are important for targeting to the oil body and show that there is a direct correlation between the inability to target to the oil body and protein stability. PMID:8539295

The microtubule-associated protein EB1 maintains cell polarity through activation of protein kinase C.

PubMed

Schober, Joseph M; Kwon, Guim; Jayne, Debbie; Cain, Jeanine M

2012-01-06

The plus-ends of microtubules target the cell cortex to modulate actin protrusion dynamics and polarity, but little is known of the molecular mechanism that couples the interaction. EB1 protein associates with the plus-ends of microtubules, placing EB1 in an ideal spatial position to mediate microtubule-actin cross talk. The objective of the current study was to further understand intracellular signaling involved in EB1-dependent cell polarity and motility. B16F10 mouse melanoma cells were depleted of EB1 protein using short hair-pin RNA interference. Correlative live cell-immunofluorescence microscopy was performed to determine localization of WAVE2 and IQGAP1 to protruding versus retracting edges. EB1 knock down caused poor subcellular separation of WAVE2 and IQGAP1, and overall decreased localization. Activation of PKC corrected defects in WAVE2 and IQGAP1 localization, cell spreading and cell shape to levels observed in control cells, but did not correct defects in cell migration. Consistent with these findings, decreased PKC phosphorylation was observed in EB1 knock down cells. These findings support a model where EB1 protein links microtubules to actin protrusion and cell polarity through signaling pathways involving PKC. Copyright © 2011 Elsevier Inc. All rights reserved.

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.

Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis.

PubMed

Kim, Hyun-Jun; Kwon, Hye-Rim; Bae, Chang-Dae; Park, Joobae; Hong, Kyung U

2010-05-15

During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 ((625) RRSRRL (630)) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.

LocSigDB: a database of protein localization signals

PubMed Central

Negi, Simarjeet; Pandey, Sanjit; Srinivasan, Satish M.; Mohammed, Akram; Guda, Chittibabu

2015-01-01

LocSigDB (http://genome.unmc.edu/LocSigDB/) is a manually curated database of experimental protein localization signals for eight distinct subcellular locations; primarily in a eukaryotic cell with brief coverage of bacterial proteins. Proteins must be localized at their appropriate subcellular compartment to perform their desired function. Mislocalization of proteins to unintended locations is a causative factor for many human diseases; therefore, collection of known sorting signals will help support many important areas of biomedical research. By performing an extensive literature study, we compiled a collection of 533 experimentally determined localization signals, along with the proteins that harbor such signals. Each signal in the LocSigDB is annotated with its localization, source, PubMed references and is linked to the proteins in UniProt database along with the organism information that contain the same amino acid pattern as the given signal. From LocSigDB webserver, users can download the whole database or browse/search for data using an intuitive query interface. To date, LocSigDB is the most comprehensive compendium of protein localization signals for eight distinct subcellular locations. Database URL: http://genome.unmc.edu/LocSigDB/ PMID:25725059

Subcellular localization of rice acyl-CoA-binding proteins (ACBPs) indicates that OsACBP6::GFP is targeted to the peroxisomes.

PubMed

Meng, Wei; Hsiao, An-Shan; Gao, Caiji; Jiang, Liwen; Chye, Mee-Len

2014-07-01

Acyl-CoA-binding proteins (ACBPs) show conservation at the acyl-CoA-binding (ACB) domain which facilitates binding to acyl-CoA esters. In Arabidopsis thaliana, six ACBPs participate in development and stress responses. Rice (Oryza sativa) also contains six genes encoding ACBPs. We investigated differences in subcellular localization between monocot rice and eudicot A. thaliana ACBPs. The subcellular localization of the six OsACBPs was achieved via transient expression of green fluorescence protein (GFP) fusions in tobacco (Nicotiana tabacum) epidermal cells, and stable transformation of A. thaliana. As plant ACBPs had not been reported in the peroxisomes, OsACBP6::GFP localization was confirmed by transient expression in rice sheath cells. The function of OsACBP6 was investigated by overexpressing 35S::OsACBP6 in the peroxisomal abc transporter1 (pxa1) mutant defective in peroxisomal fatty acid β-oxidation. As predicted, OsACBP1::GFP and OsACBP2::GFP were localized to the cytosol, and OsACBP4::GFP and OsACBP5::GFP to the endoplasmic reticulum (ER). However, OsACBP3::GFP displayed subcellular multi-localization while OsACBP6::GFP was localized to the peroxisomes. 35S::OsACBP6-OE/pxa1 lines showed recovery in indole-3-butyric acid (IBA) peroxisomal β-oxidation, wound-induced VEGETATIVE STORAGE PROTEIN1 (VSP1) expression and jasmonic acid (JA) accumulation. These findings indicate a role for OsACBP6 in peroxisomal β-oxidation, and suggest that rice ACBPs are involved in lipid degradation in addition to lipid biosynthesis. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

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

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.

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.

Identifying essential proteins based on sub-network partition and prioritization by integrating subcellular localization information.

PubMed

Li, Min; Li, Wenkai; Wu, Fang-Xiang; Pan, Yi; Wang, Jianxin

2018-06-14

Essential proteins are important participants in various life activities and play a vital role in the survival and reproduction of living organisms. Identification of essential proteins from protein-protein interaction (PPI) networks has great significance to facilitate the study of human complex diseases, the design of drugs and the development of bioinformatics and computational science. Studies have shown that highly connected proteins in a PPI network tend to be essential. A series of computational methods have been proposed to identify essential proteins by analyzing topological structures of PPI networks. However, the high noise in the PPI data can degrade the accuracy of essential protein prediction. Moreover, proteins must be located in the appropriate subcellular localization to perform their functions, and only when the proteins are located in the same subcellular localization, it is possible that they can interact with each other. In this paper, we propose a new network-based essential protein discovery method based on sub-network partition and prioritization by integrating subcellular localization information, named SPP. The proposed method SPP was tested on two different yeast PPI networks obtained from DIP database and BioGRID database. The experimental results show that SPP can effectively reduce the effect of false positives in PPI networks and predict essential proteins more accurately compared with other existing computational methods DC, BC, CC, SC, EC, IC, NC. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

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.

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

Unsupervised Clustering of Subcellular Protein Expression Patterns in High-Throughput Microscopy Images Reveals Protein Complexes and Functional Relationships between Proteins

PubMed Central

Handfield, Louis-François; Chong, Yolanda T.; Simmons, Jibril; Andrews, Brenda J.; Moses, Alan M.

2013-01-01

Protein subcellular localization has been systematically characterized in budding yeast using fluorescently tagged proteins. Based on the fluorescence microscopy images, subcellular localization of many proteins can be classified automatically using supervised machine learning approaches that have been trained to recognize predefined image classes based on statistical features. Here, we present an unsupervised analysis of protein expression patterns in a set of high-resolution, high-throughput microscope images. Our analysis is based on 7 biologically interpretable features which are evaluated on automatically identified cells, and whose cell-stage dependency is captured by a continuous model for cell growth. We show that it is possible to identify most previously identified localization patterns in a cluster analysis based on these features and that similarities between the inferred expression patterns contain more information about protein function than can be explained by a previous manual categorization of subcellular localization. Furthermore, the inferred cell-stage associated to each fluorescence measurement allows us to visualize large groups of proteins entering the bud at specific stages of bud growth. These correspond to proteins localized to organelles, revealing that the organelles must be entering the bud in a stereotypical order. We also identify and organize a smaller group of proteins that show subtle differences in the way they move around the bud during growth. Our results suggest that biologically interpretable features based on explicit models of cell morphology will yield unprecedented power for pattern discovery in high-resolution, high-throughput microscopy images. PMID:23785265

Biochemical properties and subcellular localization of tyrosine aminotransferases in Arabidopsis thaliana.

PubMed

Wang, Minmin; Toda, Kyoko; Maeda, Hiroshi A

2016-12-01

Plants produce various L-tyrosine (Tyr)-derived compounds that are of pharmaceutical or nutritional importance to humans. Tyr aminotransferase (TAT) catalyzes the reversible transamination between Tyr and 4-hydroxyphenylpyruvate (HPP), the initial step in the biosynthesis of many Tyr-derived plant natural products. Herein reported is the biochemical characterization and subcellular localization of TAT enzymes from the model plant Arabidopsis thaliana. Phylogenetic analysis showed that Arabidopsis has at least two homologous TAT genes, At5g53970 (AtTAT1) and At5g36160 (AtTAT2). Their recombinant enzymes showed distinct biochemical properties: AtTAT1 had the highest activity towards Tyr, while AtTAT2 exhibited a broad substrate specificity for both amino and keto acid substrates. Also, AtTAT1 favored the direction of Tyr deamination to HPP, whereas AtTAT2 preferred transamination of HPP to Tyr. Subcellular localization analysis using GFP-fusion proteins and confocal microscopy showed that AtTAT1, AtTAT2, and HPP dioxygenase (HPPD), which catalyzes the subsequent step of TAT, are localized in the cytosol, unlike plastid-localized Tyr and tocopherol biosynthetic enzymes. Furthermore, subcellular fractionation indicated that, while HPPD activity is restricted to the cytosol, TAT activity is detected in both cytosolic and plastidic fractions of Arabidopsis leaf tissue, suggesting that an unknown aminotransferase(s) having TAT activity is also present in the plastids. Biochemical and cellular analyses of Arabidopsis TATs provide a fundamental basis for future in vivo studies and metabolic engineering for enhanced production of Tyr-derived phytochemicals in plants. Copyright © 2016 Elsevier Ltd. All rights reserved.

The SubCons webserver: A user friendly web interface for state-of-the-art subcellular localization prediction.

PubMed

Salvatore, M; Shu, N; Elofsson, A

2018-01-01

SubCons is a recently developed method that predicts the subcellular localization of a protein. It combines predictions from four predictors using a Random Forest classifier. Here, we present the user-friendly web-interface implementation of SubCons. Starting from a protein sequence, the server rapidly predicts the subcellular localizations of an individual protein. In addition, the server accepts the submission of sets of proteins either by uploading the files or programmatically by using command line WSDL API scripts. This makes SubCons ideal for proteome wide analyses allowing the user to scan a whole proteome in few days. From the web page, it is also possible to download precalculated predictions for several eukaryotic organisms. To evaluate the performance of SubCons we present a benchmark of LocTree3 and SubCons using two recent mass-spectrometry based datasets of mouse and drosophila proteins. The server is available at http://subcons.bioinfo.se/. © 2017 The Protein Society.

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.

pLoc-mGneg: Predict subcellular localization of Gram-negative bacterial proteins by deep gene ontology learning via general PseAAC.

PubMed

Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

2017-10-06

Information of the proteins' subcellular localization is crucially important for revealing their biological functions in a cell, the basic unit of life. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to develop computational tools for timely identifying their subcellular locations based on the sequence information alone. The current study is focused on the Gram-negative bacterial proteins. Although considerable efforts have been made in protein subcellular prediction, the problem is far from being solved yet. This is because mounting evidences have indicated that many Gram-negative bacterial proteins exist in two or more location sites. Unfortunately, most existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions important for both basic research and drug design. In this study, by using the multi-label theory, we developed a new predictor called "pLoc-mGneg" for predicting the subcellular localization of Gram-negative bacterial proteins with both single and multiple locations. Rigorous cross-validation on a high quality benchmark dataset indicated that the proposed predictor is remarkably superior to "iLoc-Gneg", the state-of-the-art predictor for the same purpose. For the convenience of most experimental scientists, a user-friendly web-server for the novel predictor has been established at http://www.jci-bioinfo.cn/pLoc-mGneg/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative Multispectral Analysis Of Discrete Subcellular Particles By Digital Imaging Fluorescence Microscopy (DIFM)

NASA Astrophysics Data System (ADS)

Dorey, C. K.; Ebenstein, David B.

1988-10-01

Subcellular localization of multiple biochemical markers is readily achieved through their characteristic autofluorescence or through use of appropriately labelled antibodies. Recent development of specific probes has permitted elegant studies in calcium and pH in living cells. However, each of these methods measured fluorescence at one wavelength; precise quantitation of multiple fluorophores at individual sites within a cell has not been possible. Using DIFM, we have achieved spectral analysis of discrete subcellular particles 1-2 gm in diameter. The fluorescence emission is broken into narrow bands by an interference monochromator and visualized through the combined use of a silicon intensified target (SIT) camera, a microcomputer based framegrabber with 8 bit resolution, and a color video monitor. Image acquisition, processing, analysis and display are under software control. The digitized image can be corrected for the spectral distortions induced by the wavelength dependent sensitivity of the camera, and the displayed image can be enhanced or presented in pseudocolor to facilitate discrimination of variation in pixel intensity of individual particles. For rapid comparison of the fluorophore composition of granules, a ratio image is produced by dividing the image captured at one wavelength by that captured at another. In the resultant ratio image, a granule which has a fluorophore composition different from the majority is selectively colored. This powerful system has been utilized to obtain spectra of endogenous autofluorescent compounds in discrete cellular organelles of human retinal pigment epithelium, and to measure immunohistochemically labelled components of the extracellular matrix associated with the human optic nerve.

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

CellMap visualizes protein-protein interactions and subcellular localization

PubMed Central

Dallago, Christian; Goldberg, Tatyana; Andrade-Navarro, Miguel Angel; Alanis-Lobato, Gregorio; Rost, Burkhard

2018-01-01

Many tools visualize protein-protein interaction (PPI) networks. The tool introduced here, CellMap, adds one crucial novelty by visualizing PPI networks in the context of subcellular localization, i.e. the location in the cell or cellular component in which a PPI happens. Users can upload images of cells and define areas of interest against which PPIs for selected proteins are displayed (by default on a cartoon of a cell). Annotations of localization are provided by the user or through our in-house database. The visualizer and server are written in JavaScript, making CellMap easy to customize and to extend by researchers and developers. PMID:29497493

Correlation of N-myc downstream-regulated gene 1 subcellular localization and lymph node metastases of colorectal neoplasms

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

Song, Yan; Lv, Liyang; Du, Juan

2013-09-20

Highlights: •We clarified NDRG1 subcellular location in colorectal cancer. •We found the changes of NDRG1 distribution during colorectal cancer progression. •We clarified the correlation between NDRG1 distribution and lymph node metastasis. •It is possible that NDRG1 subcellular localization may determine its function. •Maybe NDRG1 is valuable early diagnostic markers for metastasis. -- Abstract: In colorectal neoplasms, N-myc downstream-regulated gene 1 (NDRG1) is a primarily cytoplasmic protein, but it is also expressed on the cell membrane and in the nucleus. NDRG1 is involved in various stages of tumor development in colorectal cancer, and it is possible that the different subcellular localizationsmore » may determine the function of NDRG1 protein. Here, we attempt to clarify the characteristics of NDRG1 protein subcellular localization during the progression of colorectal cancer. We examined NDRG1 expression in 49 colorectal cancer patients in cancerous, non-cancerous, and corresponding lymph node tissues. Cytoplasmic and membrane NDRG1 expression was higher in the lymph nodes with metastases than in those without metastases (P < 0.01). Nuclear NDRG1 expression in colorectal neoplasms was significantly higher than in the normal colorectal mucosa, and yet the normal colorectal mucosa showed no nuclear expression. Furthermore, our results showed higher cytoplasmic NDRG1 expression was better for differentiation, and higher membrane NDRG1 expression resulted in a greater possibility of lymph node metastasis. These data indicate that a certain relationship between the cytoplasmic and membrane expression of NDRG1 in lymph nodes exists with lymph node metastasis. NDRG1 expression may translocate from the membrane of the colorectal cancer cells to the nucleus, where it is involved in lymph node metastasis. Combination analysis of NDRG1 subcellular expression and clinical variables will help predict the incidence of lymph node metastasis.« less

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

Prognostic Subcellular Notch2, Notch3 and Jagged1 Localization Patterns in Early Triple-negative Breast Cancer.

PubMed

Strati, Titika-Marina; Kotoula, Vassiliki; Kostopoulos, Ioannis; Manousou, Kyriaki; Papadimitriou, Christos; Lazaridis, Georgios; Lakis, Sotiris; Pentheroudakis, George; Pectasides, Dimitrios; Pazarli, Elissavet; Christodoulou, Christos; Razis, Evangelia; Pavlakis, Kitty; Magkou, Christina; Chrisafi, Sofia; Aravantinos, Gerasimos; Bafaloukos, Dimitrios; Papakostas, Pavlos; Gogas, Helen; Kalogeras, Konstantine T; Fountzilas, George

2017-05-01

The Notch pathway has been implicated in triple-negative breast cancer (TNBC). Herein, we studied the subcellular localization of the less investigated Notch2 and Notch3 and that of the Jagged1 (Jag1) ligand in patients with operable TNBC. We applied immunohistochemistry for Notch2, Notch3 and Jag1 in 333 tumors from TNBC patients treated with adjuvant anthracycline-based chemotherapy. We evaluated cytoplasmic (c), membranous (m) and nuclear (n) protein localization. c-Notch2 (35% positive tumors), c-Notch3 (63%), c-Jag1 (43%), m-Notch3 (23%) and n-Jag1 (17%) were analyzed individually and by using hierarchical clustering for prognostic evaluation. Upon multivariate analysis, compared to high m-Notch3 in the absence of n-Jag1 (cluster 4), all other marker combinations (clusters 1, 2, 3) conferred significantly higher risk for relapse (p<0.05). Specific Notch3 and Jag1 subcellular localization patterns may provide clues for the behavior of the tumors and potentially for Jag1 targeting in TNBC patients. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Identification of two internal signal peptide sequences: critical for classical swine fever virus non-structural protein 2 to trans-localize to the endoplasmic reticulum.

PubMed

Guo, Kang-kang; Tang, Qing-hai; Zhang, Yan-ming; Kang, Kai; He, Lei

2011-05-18

The membrane topology and molecular mechanisms for endoplasmic reticulum (ER) localization of classical swine fever virus (CSFV) non-structural 2 (NS2) protien is unclear. We attempted to elucidate the subcellular localization, and the molecular mechanisms responsible for the localization of this protein in our study. The NS2 gene was amplified by reverse transcription polymerase chain reaction, with the transmembrane region and hydrophilicity of the NS2 protein was predicted by bioinformatics analysis. Twelve cDNAs of the NS2 gene were amplified by the PCR deletion method and cloned into a eukaryotic expression vector, which was transfected into a swine umbilical vein endothelial cell line (SUVEC). Subcellular localization of the NS2 protein was characterized by confocal microscopy, and western blots were carried out to analyze protein expression. Our results showed that the -NH2 terminal of the CSFV NS2 protein was highly hydrophobic and the protein localized in the ER. At least four transmembrane regions and two internal signal peptide sequences (amino acids103-138 and 220-262) were identified and thought to be critical for its trans-localization to the ER. This is the first study to identify the internal signal peptide sequences of the CSFV NS2 protein and its subcellular localization, providing the foundation for further exploration of this protein's function of this protein and its role in CSFV pathogenesis.

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

pLoc-mHum: predict subcellular localization of multi-location human proteins via general PseAAC to winnow out the crucial GO information.

PubMed

Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

2018-05-01

For in-depth understanding the functions of proteins in a cell, the knowledge of their subcellular localization is indispensable. The current study is focused on human protein subcellular location prediction based on the sequence information alone. Although considerable efforts have been made in this regard, the problem is far from being solved yet. Most existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions that are particularly important for both basic research and drug design. Using the multi-label theory, we present a new predictor called 'pLoc-mHum' by extracting the crucial GO (Gene Ontology) information into the general PseAAC (Pseudo Amino Acid Composition). Rigorous cross-validations on a same stringent benchmark dataset have indicated that the proposed pLoc-mHum predictor is remarkably superior to iLoc-Hum, the state-of-the-art method in predicting the human protein subcellular localization. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc-mHum/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. xcheng@gordonlifescience.org. Supplementary data are available at Bioinformatics online.

L-Ilf3 and L-NF90 Traffic to the Nucleolus Granular Component: Alternatively-Spliced Exon 3 Encodes a Nucleolar Localization Motif

PubMed Central

Viranaicken, Wildriss; Gasmi, Laila; Chaumet, Alexandre; Durieux, Christiane; Georget, Virginie; Denoulet, Philippe; Larcher, Jean-Christophe

2011-01-01

Ilf3 and NF90, two proteins containing double-stranded RNA-binding domains, are generated by alternative splicing and involved in several functions. Their heterogeneity results from posttranscriptional and posttranslational modifications. Alternative splicing of exon 3, coding for a 13 aa N-terminal motif, generates for each protein a long and short isoforms. Subcellular fractionation and localization of recombinant proteins showed that this motif acts as a nucleolar localization signal. Deletion and substitution mutants identified four arginines, essential for nucleolar targeting, and three histidines to stabilize the proteins within the nucleolus. The short isoforms are never found in the nucleoli, whereas the long isoforms are present in the nucleoplasm and the nucleoli. For Ilf3, only the posttranslationally-unmodified long isoform is nucleolar, suggesting that this nucleolar targeting is abrogated by posttranslational modifications. Confocal microscopy and FRAP experiments have shown that the long Ilf3 isoform localizes to the granular component of the nucleolus, and that L-Ilf3 and L-NF90 exchange rapidly between nucleoli. The presence of this 13 aminoacid motif, combined with posttranslational modifications, is responsible for the differences in Ilf3 and NF90 isoforms subcellular localizations. The protein polymorphism of Ilf3/NF90 and the various subcellular localizations of their isoforms may partially explain the various functions previously reported for these proteins. PMID:21811582

Nanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging

PubMed Central

Zurbuchen, Mark A.; Lake, Michael P.; Kohan, Sirus A.; Leung, Belinda; Bouchard, Louis-S.

2013-01-01

There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could enable targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here we demonstrate a sub-cellular multi-modal imaging methodology that enables localization of inert particulate probes, consisting of nanodiamonds having fluorescent nitrogen-vacancy centers. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Nanodiamonds targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to enable “zooming-in” to regions of interest for detailed structural investigations. Optical microscopies reveal nanodiamonds for in-vitro tracking or uptake-confirmation. The approach is general, works down to the single nanodiamond level, and can leverage the unique capabilities of nanodiamonds, such as biocompatibility, sensitive magnetometry, and gene and drug delivery. PMID:24036840

Characterization of karyopherins in androgen receptor intracellular trafficking in the yeast model

PubMed Central

Nguyen, Minh M; Harmon, Robert M; Wang, Zhou

2014-01-01

Background: Mechanisms regulating androgen receptor (AR) subcellular localization represent an essential component of AR signaling. Karyopherins are a family of nucleocytoplasmic trafficking factors. In this paper, we used the yeast model to study the effects of karyopherins on the subcellular localization of the AR. Methods: Yeast mutants deficient in different nuclear transport factors were transformed with various AR based, GFP tagged constructs and their localization was monitored using microscopy. Results: We showed that yeast can mediate androgen-induced AR nuclear localization and that in addition to the import factor, Importinα/β, this process required the import karyopherin Sxm1. We also showed that a previously identified nuclear export sequence (NESAR) in the ligand binding domain of AR does not appear to rely on karyopherins for cytoplasmic localization. Conclusions: These results suggest that while AR nuclear import relies on karyopherin activity, AR nuclear export and/or cytoplasmic localization may require other undefined mechanisms. PMID:25031696

Identification of novel nuclear localization signals of Drosophila myeloid leukemia factor.

PubMed

Sugano, Wakana; Yamaguchi, Masamitsu

2007-01-01

Myeloid leukemia factor 1 (MLF1) was first identified as part of a leukemic fusion protein produced by a chromosomal translocation, and MLF family proteins are present in many animals. In mammalian cells, MLF1 has been described as mainly cytoplasmic, but in Drosophila, one of the dMLF isoforms (dMLFA) localized mainly in the nucleus while the other isoform (dMLFB), that appears to be produced by the alternative splicing, displays both nuclear and cytoplasmic localization. To investigate the difference in subcellular localization between MLF family members, we examined the subcellular localization of deletion mutants of dMLFA isoform. The analyses showed that the C-terminal 40 amino acid region of dMLFA is necessary and sufficient for nuclear localization. Based on amino acid sequences, we hypothesized that two nuclear localization signals (NLSs) are present within the region. Site-directed mutagenesis of critical residues within the two putative NLSs leads to loss of nuclear localization, suggesting that both NLS motifs are necessary for nuclear localization.

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.

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.

Trafficking of plant plasma membrane aquaporins: multiple regulation levels and complex sorting signals.

PubMed

Chevalier, Adrien S; Chaumont, François

2015-05-01

Aquaporins are small channel proteins which facilitate the diffusion of water and small neutral molecules across biological membranes. Compared with animals, plant genomes encode numerous aquaporins, which display a large variety of subcellular localization patterns. More specifically, plant aquaporins of the plasma membrane intrinsic protein (PIP) subfamily were first described as plasma membrane (PM)-resident proteins, but recent research has demonstrated that the trafficking and subcellular localization of these proteins are complex and highly regulated. In the past few years, PIPs emerged as new model proteins to study subcellular sorting and membrane dynamics in plant cells. At least two distinct sorting motifs (one cytosolic, the other buried in the membrane) are required to direct PIPs to the PM. Hetero-oligomerization and interaction with SNAREs (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptors) also influence the subcellular trafficking of PIPs. In addition to these constitutive processes, both the progression of PIPs through the secretory pathway and their dynamics at the PM are responsive to changing environmental conditions. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Quantitative imaging with fluorescent biosensors.

PubMed

Okumoto, Sakiko; Jones, Alexander; Frommer, Wolf B

2012-01-01

Molecular activities are highly dynamic and can occur locally in subcellular domains or compartments. Neighboring cells in the same tissue can exist in different states. Therefore, quantitative information on the cellular and subcellular dynamics of ions, signaling molecules, and metabolites is critical for functional understanding of organisms. Mass spectrometry is generally used for monitoring ions and metabolites; however, its temporal and spatial resolution are limited. Fluorescent proteins have revolutionized many areas of biology-e.g., fluorescent proteins can report on gene expression or protein localization in real time-yet promoter-based reporters are often slow to report physiologically relevant changes such as calcium oscillations. Therefore, novel tools are required that can be deployed in specific cells and targeted to subcellular compartments in order to quantify target molecule dynamics directly. We require tools that can measure enzyme activities, protein dynamics, and biophysical processes (e.g., membrane potential or molecular tension) with subcellular resolution. Today, we have an extensive suite of tools at our disposal to address these challenges, including translocation sensors, fluorescence-intensity sensors, and Förster resonance energy transfer sensors. This review summarizes sensor design principles, provides a database of sensors for more than 70 different analytes/processes, and gives examples of applications in quantitative live cell imaging.

A Comprehensive Subcellular Proteomic Survey of Salmonella Grown under Phagosome-Mimicking versus Standard Laboratory Conditions

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

Brown, Roslyn N.; Sanford, James A.; Park, Jea H.

Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and infection-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of over 30% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators ofmore » two-component regulatory systems (e.g., ArcB, PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein location for Salmonella and a framework for further investigations using computational modeling.« less

Internalization and Subcellular Trafficking of Poly-l-lysine Dendrimers Are Impacted by the Site of Fluorophore Conjugation.

PubMed

Avaritt, Brittany R; Swaan, Peter W

2015-06-01

Internalization and intracellular trafficking of dendrimer-drug conjugates play an important role in achieving successful drug delivery. In this study, we aimed to elucidate the endocytosis mechanisms and subcellular localization of poly-l-lysine (PLL) dendrimers in Caco-2 cells. We also investigated the impact of fluorophore conjugation on cytotoxicity, uptake, and transepithelial transport. Oregon green 514 (OG) was conjugated to PLL G3 at either the dendrimer periphery or the core. Chemical inhibitors of clathrin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis pathways and macropinocytosis were employed to establish internalization mechanisms, while colocalization with subcellular markers was used to determine dendrimer trafficking. Cell viability, internalization, and uptake were all influenced by the site of fluorophore conjugation. Uptake was found to be highly dependent on cholesterol- and dynamin-mediated endocytosis as well as macropinocytosis. Dendrimers were trafficked to endosomes and lysosomes, and subcellular localization was impacted by the fluorophore conjugation site. The results of this study indicate that PLL dendrimers exploit multiple pathways for cellular entry, and internalization and trafficking can be impacted by conjugation. Therefore, design of dendrimer-drug conjugates requires careful consideration to achieve successful drug delivery.

Coherence and diffraction limited resolution in microscopic OCT by a unified approach for the correction of dispersion and aberrations

NASA Astrophysics Data System (ADS)

Schulz-Hildebrandt, H.; Münter, Michael; Ahrens, M.; Spahr, H.; Hillmann, D.; König, P.; Hüttmann, G.

2018-03-01

Optical coherence tomography (OCT) images scattering tissues with 5 to 15 μm resolution. This is usually not sufficient for a distinction of cellular and subcellular structures. Increasing axial and lateral resolution and compensation of artifacts caused by dispersion and aberrations is required to achieve cellular and subcellular resolution. This includes defocus which limit the usable depth of field at high lateral resolution. OCT gives access the phase of the scattered light and hence correction of dispersion and aberrations is possible by numerical algorithms. Here we present a unified dispersion/aberration correction which is based on a polynomial parameterization of the phase error and an optimization of the image quality using Shannon's entropy. For validation, a supercontinuum light sources and a costume-made spectrometer with 400 nm bandwidth were combined with a high NA microscope objective in a setup for tissue and small animal imaging. Using this setup and computation corrections, volumetric imaging at 1.5 μm resolution is possible. Cellular and near cellular resolution is demonstrated in porcine cornea and the drosophila larva, when computational correction of dispersion and aberrations is used. Due to the excellent correction of the used microscope objective, defocus was the main contribution to the aberrations. In addition, higher aberrations caused by the sample itself were successfully corrected. Dispersion and aberrations are closely related artifacts in microscopic OCT imaging. Hence they can be corrected in the same way by optimization of the image quality. This way microscopic resolution is easily achieved in OCT imaging of static biological tissues.

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

Rice DB: an Oryza Information Portal linking annotation, subcellular location, function, expression, regulation, and evolutionary information for rice and Arabidopsis

PubMed Central

Narsai, Reena; Devenish, James; Castleden, Ian; Narsai, Kabir; Xu, Lin; Shou, Huixia; Whelan, James

2013-01-01

Omics research in Oryza sativa (rice) relies on the use of multiple databases to obtain different types of information to define gene function. We present Rice DB, an Oryza information portal that is a functional genomics database, linking gene loci to comprehensive annotations, expression data and the subcellular location of encoded proteins. Rice DB has been designed to integrate the direct comparison of rice with Arabidopsis (Arabidopsis thaliana), based on orthology or ‘expressology’, thus using and combining available information from two pre-eminent plant models. To establish Rice DB, gene identifiers (more than 40 types) and annotations from a variety of sources were compiled, functional information based on large-scale and individual studies was manually collated, hundreds of microarrays were analysed to generate expression annotations, and the occurrences of potential functional regulatory motifs in promoter regions were calculated. A range of computational subcellular localization predictions were also run for all putative proteins encoded in the rice genome, and experimentally confirmed protein localizations have been collated, curated and linked to functional studies in rice. A single search box allows anything from gene identifiers (for rice and/or Arabidopsis), motif sequences, subcellular location, to keyword searches to be entered, with the capability of Boolean searches (such as AND/OR). To demonstrate the utility of Rice DB, several examples are presented including a rice mitochondrial proteome, which draws on a variety of sources for subcellular location data within Rice DB. Comparisons of subcellular location, functional annotations, as well as transcript expression in parallel with Arabidopsis reveals examples of conservation between rice and Arabidopsis, using Rice DB (http://ricedb.plantenergy.uwa.edu.au). PMID:24147765

Rice DB: an Oryza Information Portal linking annotation, subcellular location, function, expression, regulation, and evolutionary information for rice and Arabidopsis.

PubMed

Narsai, Reena; Devenish, James; Castleden, Ian; Narsai, Kabir; Xu, Lin; Shou, Huixia; Whelan, James

2013-12-01

Omics research in Oryza sativa (rice) relies on the use of multiple databases to obtain different types of information to define gene function. We present Rice DB, an Oryza information portal that is a functional genomics database, linking gene loci to comprehensive annotations, expression data and the subcellular location of encoded proteins. Rice DB has been designed to integrate the direct comparison of rice with Arabidopsis (Arabidopsis thaliana), based on orthology or 'expressology', thus using and combining available information from two pre-eminent plant models. To establish Rice DB, gene identifiers (more than 40 types) and annotations from a variety of sources were compiled, functional information based on large-scale and individual studies was manually collated, hundreds of microarrays were analysed to generate expression annotations, and the occurrences of potential functional regulatory motifs in promoter regions were calculated. A range of computational subcellular localization predictions were also run for all putative proteins encoded in the rice genome, and experimentally confirmed protein localizations have been collated, curated and linked to functional studies in rice. A single search box allows anything from gene identifiers (for rice and/or Arabidopsis), motif sequences, subcellular location, to keyword searches to be entered, with the capability of Boolean searches (such as AND/OR). To demonstrate the utility of Rice DB, several examples are presented including a rice mitochondrial proteome, which draws on a variety of sources for subcellular location data within Rice DB. Comparisons of subcellular location, functional annotations, as well as transcript expression in parallel with Arabidopsis reveals examples of conservation between rice and Arabidopsis, using Rice DB (http://ricedb.plantenergy.uwa.edu.au). © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

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.

Modulatory compartments in cortex and local regulation of cholinergic tone.

PubMed

Coppola, Jennifer J; Ward, Nicholas J; Jadi, Monika P; Disney, Anita A

2016-09-01

Neuromodulatory signaling is generally considered broad in its impact across cortex. However, variations in the characteristics of cortical circuits may introduce regionally-specific responses to diffuse modulatory signals. Features such as patterns of axonal innervation, tissue tortuosity and molecular diffusion, effectiveness of degradation pathways, subcellular receptor localization, and patterns of receptor expression can lead to local modification of modulatory inputs. We propose that modulatory compartments exist in cortex and can be defined by variation in structural features of local circuits. Further, we argue that these compartments are responsible for local regulation of neuromodulatory tone. For the cholinergic system, these modulatory compartments are regions of cortical tissue within which signaling conditions for acetylcholine are relatively uniform, but between which signaling can vary profoundly. In the visual system, evidence for the existence of compartments indicates that cholinergic modulation likely differs across the visual pathway. We argue that the existence of these compartments calls for thinking about cholinergic modulation in terms of finer-grained control of local cortical circuits than is implied by the traditional view of this system as a diffuse modulator. Further, an understanding of modulatory compartments provides an opportunity to better understand and perhaps correct signal modifications that lead to pathological states. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

The in vitro sub-cellular localization and in vivo efficacy of novel chitosan/GMO nanostructures containing paclitaxel.

PubMed

Trickler, W J; Nagvekar, A A; Dash, A K

2009-08-01

To determine the in vitro sub-cellular localization and in vivo efficacy of chitosan/GMO nanostructures containing paclitaxel (PTX) compared to a conventional PTX treatment (Taxol). The sub-cellular localization of coumarin-6 labeled chitosan/GMO nanostructures was determined by confocal microscopy in MDA-MB-231 cells. The antitumor efficacy was evaluated in two separate studies using FOX-Chase (CB17) SCID Female-Mice MDA-MB-231 xenograph model. Treatments consisted of intravenous Taxol or chitosan/GMO nanostructures with or without PTX, local intra-tumor bolus of Taxol or chitosan/GMO nanostructures with or without PTX. The tumor diameter and animal weight was monitored at various intervals. Histopathological changes were evaluated in end-point tumors. The tumor diameter increased at a constant rate for all the groups between days 7-14. After a single intratumoral bolus dose of chitosan/GMO containing PTX showed significant reduction in tumor diameter on day 15 when compared to control, placebo and intravenous PTX administration. The tumor diameter reached a maximal decrease (4-fold) by day 18, and the difference was reduced to approximately 2-fold by day 21. Qualitatively similar results were observed in a separate study containing PTX when administered intravenously. Chitosan/GMO nanostructures containing PTX are safe and effective administered locally or intravenously. Partially supported by DOD Award BC045664.

Acetylation of the RhoA GEF Net1A controls its subcellular localization and activity

PubMed Central

Song, Eun Hyeon; Oh, Wonkyung; Ulu, Arzu; Carr, Heather S.; Zuo, Yan; Frost, Jeffrey A.

2015-01-01

ABSTRACT Net1 isoform A (Net1A) is a RhoA GEF that is required for cell motility and invasion in multiple cancers. Nuclear localization of Net1A negatively regulates its activity, and we have recently shown that Rac1 stimulates Net1A relocalization to the plasma membrane to promote RhoA activation and cytoskeletal reorganization. However, mechanisms controlling the subcellular localization of Net1A are not well understood. Here, we show that Net1A contains two nuclear localization signal (NLS) sequences within its N-terminus and that residues surrounding the second NLS sequence are acetylated. Treatment of cells with deacetylase inhibitors or expression of active Rac1 promotes Net1A acetylation. Deacetylase inhibition is sufficient for Net1A relocalization outside the nucleus, and replacement of the N-terminal acetylation sites with arginine residues prevents cytoplasmic accumulation of Net1A caused by deacetylase inhibition or EGF stimulation. By contrast, replacement of these sites with glutamine residues is sufficient for Net1A relocalization, RhoA activation and downstream signaling. Moreover, the N-terminal acetylation sites are required for rescue of F-actin accumulation and focal adhesion maturation in Net1 knockout MEFs. These data indicate that Net1A acetylation regulates its subcellular localization to impact on RhoA activity and actin cytoskeletal organization. PMID:25588829

Stochastic theory of large-scale enzyme-reaction networks: Finite copy number corrections to rate equation models

NASA Astrophysics Data System (ADS)

Thomas, Philipp; Straube, Arthur V.; Grima, Ramon

2010-11-01

Chemical reactions inside cells occur in compartment volumes in the range of atto- to femtoliters. Physiological concentrations realized in such small volumes imply low copy numbers of interacting molecules with the consequence of considerable fluctuations in the concentrations. In contrast, rate equation models are based on the implicit assumption of infinitely large numbers of interacting molecules, or equivalently, that reactions occur in infinite volumes at constant macroscopic concentrations. In this article we compute the finite-volume corrections (or equivalently the finite copy number corrections) to the solutions of the rate equations for chemical reaction networks composed of arbitrarily large numbers of enzyme-catalyzed reactions which are confined inside a small subcellular compartment. This is achieved by applying a mesoscopic version of the quasisteady-state assumption to the exact Fokker-Planck equation associated with the Poisson representation of the chemical master equation. The procedure yields impressively simple and compact expressions for the finite-volume corrections. We prove that the predictions of the rate equations will always underestimate the actual steady-state substrate concentrations for an enzyme-reaction network confined in a small volume. In particular we show that the finite-volume corrections increase with decreasing subcellular volume, decreasing Michaelis-Menten constants, and increasing enzyme saturation. The magnitude of the corrections depends sensitively on the topology of the network. The predictions of the theory are shown to be in excellent agreement with stochastic simulations for two types of networks typically associated with protein methylation and metabolism.

Free Radicals Generated by Ionizing Radiation Signal Nuclear Translocation of p53

NASA Technical Reports Server (NTRS)

Martinez, J. D.; Pennington, M. E.; Craven, M. T.; Warters, R. L.

1997-01-01

The p53 tumor suppressor is a transcription factor that regulates several pathways, which function collectively to maintain the integrity of the genome. Nuclear localization is critical for wild-type function. However, the signals that regulate subcellular localization of p53 have not been identified. Here, we examine the effect of ionizing radiation on the subcellular localization of p53 in two cell lines in which p63 is normally sequestered in the cytoplasm and found that ionizing radiation caused a biphasic translocation response. p53 entered the nucleus 1-2 hours postirradiation (early response), subsequently emerged from the nucleus, and then again entered the nucleus 12-24 hours after the cells had been irradiated (delayed response). These changes in subcellular localization could be completely blocked by the free radical scavenger, WR1065. By comparison, two DNA-damaging agents that do not generate free radicals, mitomycin C and doxorubicin, caused translocation only after 12-24 h of exposure to the drugs, and this effect could not be inhibited by WR1065. Hence, although all three DNA-damaging agents induced relocalization of p53 to the nucleus, only the translocation caused by radiation was sensitive to free radical scavenging. We suggest that the free radicals generated by ionizing radiation can signal p53 translocation to the nucleus.

A Variable Polyglutamine Repeat Affects Subcellular Localization and Regulatory Activity of a Populus ANGUSTIFOLIA Protein.

PubMed

Bryan, Anthony C; Zhang, Jin; Guo, Jianjun; Ranjan, Priya; Singan, Vasanth; Barry, Kerrie; Schmutz, Jeremy; Weighill, Deborah; Jacobson, Daniel; Jawdy, Sara; Tuskan, Gerald A; Chen, Jin-Gui; Muchero, Wellington

2018-06-08

Polyglutamine (polyQ) stretches have been reported to occur in proteins across many organisms including animals, fungi and plants. Expansion of these repeats has attracted much attention due their associations with numerous human diseases including Huntington's and other neurological maladies. This suggests that the relative length of polyQ stretches is an important modulator of their function. Here, we report the identification of a Populus C-terminus binding protein (CtBP) ANGUSTIFOLIA ( PtAN1 ) which contains a polyQ stretch whose functional relevance had not been established. Analysis of 917 resequenced Populus trichocarpa genotypes revealed three allelic variants at this locus encoding 11-, 13- and 15-glutamine residues. Transient expression assays using Populus leaf mesophyll protoplasts revealed that the 11Q variant exhibited strong nuclear localization whereas the 15Q variant was only found in the cytosol, with the 13Q variant exhibiting localization in both subcellular compartments. We assessed functional implications by evaluating expression changes of putative PtAN1 targets in response to overexpression of the three allelic variants and observed allele-specific differences in expression levels of putative targets. Our results provide evidence that variation in polyQ length modulates PtAN1 function by altering subcellular localization. Copyright © 2018, G3: Genes, Genomes, Genetics.

Quantitative Proteomic Profiling of Low-Dose Ionizing Radiation Effects in a Human Skin Model

PubMed Central

Hengel, Shawna M.; Aldrich, Joshua T.; Waters, Katrina M.; Pasa-Tolic, Ljiljana; Stenoien, David L.

2014-01-01

To assess responses to low-dose ionizing radiation (LD-IR) exposures potentially encountered during medical diagnostic procedures, nuclear accidents or terrorist acts, a quantitative proteomic approach was used to identify changes in protein abundance in a reconstituted human skin tissue model treated with 0.1 Gy of ionizing radiation. To improve the dynamic range of the assay, subcellular fractionation was employed to remove highly abundant structural proteins and to provide insight into radiation-induced alterations in protein localization. Relative peptide quantification across cellular fractions, control and irradiated samples was performing using 8-plex iTRAQ labeling followed by online two-dimensional nano-scale liquid chromatography and high resolution MS/MS analysis. A total of 107 proteins were detected with statistically significant radiation-induced change in abundance (>1.5 fold) and/or subcellular localization compared to controls. The top biological pathways identified using bioinformatics include organ development, anatomical structure formation and the regulation of actin cytoskeleton. From the proteomic data, a change in proteolytic processing and subcellular localization of the skin barrier protein, filaggrin, was identified, and the results were confirmed by western blotting. This data indicate post-transcriptional regulation of protein abundance, localization and proteolytic processing playing an important role in regulating radiation response in human tissues. PMID:28250387

Mono- and Dinuclear Phosphorescent Rhenium(I) Complexes: Impact of Subcellular Localization on Anticancer Mechanisms.

PubMed

Ye, Rui-Rong; Tan, Cai-Ping; Chen, Mu-He; Hao, Liang; Ji, Liang-Nian; Mao, Zong-Wan

2016-06-01

Elucidation of relationship among chemical structure, cellular uptake, localization, and biological activity of anticancer metal complexes is important for the understanding of their mechanisms of action. Organometallic rhenium(I) tricarbonyl compounds have emerged as potential multifunctional anticancer drug candidates that can integrate therapeutic and imaging capabilities in a single molecule. Herein, two mononuclear phosphorescent rhenium(I) complexes (Re1 and Re2), along with their corresponding dinuclear complexes (Re3 and Re4), were designed and synthesized as potent anticancer agents. The subcellular accumulation of Re1-Re4 was conveniently analyzed by confocal microscopy in situ in live cells by utilizing their intrinsic phosphorescence. We found that increased lipophilicity of the bidentate ligands could enhance their cellular uptake, leading to improved anticancer efficacy. The dinuclear complexes were more potent than the mononuclear counterparts. The molecular anticancer mechanisms of action evoked by Re3 and Re4 were explored in detail. Re3 with a lower lipophilicity localizes to lysosomes and induces caspase-independent apoptosis, whereas Re4 with higher lipophilicity specially accumulates in mitochondria and induces caspase-independent paraptosis in cancer cells. Our study demonstrates that subcellular localization is crucial for the anticancer mechanisms of these phosphorescent rhenium(I) complexes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HybridGO-Loc: mining hybrid features on gene ontology for predicting subcellular localization of multi-location proteins.

PubMed

Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

2014-01-01

Protein subcellular localization prediction, as an essential step to elucidate the functions in vivo of proteins and identify drugs targets, has been extensively studied in previous decades. Instead of only determining subcellular localization of single-label proteins, recent studies have focused on predicting both single- and multi-location proteins. Computational methods based on Gene Ontology (GO) have been demonstrated to be superior to methods based on other features. However, existing GO-based methods focus on the occurrences of GO terms and disregard their relationships. This paper proposes a multi-label subcellular-localization predictor, namely HybridGO-Loc, that leverages not only the GO term occurrences but also the inter-term relationships. This is achieved by hybridizing the GO frequencies of occurrences and the semantic similarity between GO terms. Given a protein, a set of GO terms are retrieved by searching against the gene ontology database, using the accession numbers of homologous proteins obtained via BLAST search as the keys. The frequency of GO occurrences and semantic similarity (SS) between GO terms are used to formulate frequency vectors and semantic similarity vectors, respectively, which are subsequently hybridized to construct fusion vectors. An adaptive-decision based multi-label support vector machine (SVM) classifier is proposed to classify the fusion vectors. Experimental results based on recent benchmark datasets and a new dataset containing novel proteins show that the proposed hybrid-feature predictor significantly outperforms predictors based on individual GO features as well as other state-of-the-art predictors. For readers' convenience, the HybridGO-Loc server, which is for predicting virus or plant proteins, is available online at http://bioinfo.eie.polyu.edu.hk/HybridGoServer/.

Dynamic subcellular localization of a respiratory complex controls bacterial respiration

PubMed Central

Alberge, François; Espinosa, Leon; Seduk, Farida; Sylvi, Léa; Toci, René; Walburger, Anne; Magalon, Axel

2015-01-01

Respiration, an essential process for most organisms, has to optimally respond to changes in the metabolic demand or the environmental conditions. The branched character of their respiratory chains allows bacteria to do so by providing a great metabolic and regulatory flexibility. Here, we show that the native localization of the nitrate reductase, a major respiratory complex under anaerobiosis in Escherichia coli, is submitted to tight spatiotemporal regulation in response to metabolic conditions via a mechanism using the transmembrane proton gradient as a cue for polar localization. These dynamics are critical for controlling the activity of nitrate reductase, as the formation of polar assemblies potentiates the electron flux through the complex. Thus, dynamic subcellular localization emerges as a critical factor in the control of respiration in bacteria. DOI: http://dx.doi.org/10.7554/eLife.05357.001 PMID:26077726

Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds

PubMed Central

Ibeas, Miguel A.; Grant-Grant, Susana; Navarro, Nathalia; Perez, M. F.; Roschzttardtz, Hannetz

2017-01-01

Iron is an essential micronutrient for plants. Little is know about how iron is loaded in embryo during seed development. In this article we used Perls/DAB staining in order to reveal iron localization at the cellular and subcellular levels in different Brassicaceae seed species. In dry seeds of Brassica napus, Nasturtium officinale, Lepidium sativum, Camelina sativa, and Brassica oleracea iron localizes in vacuoles of cells surrounding provasculature in cotyledons and hypocotyl. Using B. napus and N. officinale as model plants we determined where iron localizes during seed development. Our results indicate that iron is not detectable by Perls/DAB staining in heart stage embryo cells. Interestingly, at torpedo development stage iron localizes in nuclei of different cells type, including integument, free cell endosperm and almost all embryo cells. Later, iron is detected in cytoplasmic structures in different embryo cell types. Our results indicate that iron accumulates in nuclei in specific stages of embryo maturation before to be localized in vacuoles of cells surrounding provasculature in mature seeds. PMID:29312417

Multiple marker abundance profiling: combining selected reaction monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples

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

Hooper, Cornelia M.; Stevens, Tim J.; Saukkonen, Anna

Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combinedmore » with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live).« less

Multiple marker abundance profiling: combining selected reaction monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples

DOE PAGES

Hooper, Cornelia M.; Stevens, Tim J.; Saukkonen, Anna; ...

2017-10-12

Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combinedmore » with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live).« less

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

Protein Subcellular Localization with Gaussian Kernel Discriminant Analysis and Its Kernel Parameter Selection.

PubMed

Wang, Shunfang; Nie, Bing; Yue, Kun; Fei, Yu; Li, Wenjia; Xu, Dongshu

2017-12-15

Kernel discriminant analysis (KDA) is a dimension reduction and classification algorithm based on nonlinear kernel trick, which can be novelly used to treat high-dimensional and complex biological data before undergoing classification processes such as protein subcellular localization. Kernel parameters make a great impact on the performance of the KDA model. Specifically, for KDA with the popular Gaussian kernel, to select the scale parameter is still a challenging problem. Thus, this paper introduces the KDA method and proposes a new method for Gaussian kernel parameter selection depending on the fact that the differences between reconstruction errors of edge normal samples and those of interior normal samples should be maximized for certain suitable kernel parameters. Experiments with various standard data sets of protein subcellular localization show that the overall accuracy of protein classification prediction with KDA is much higher than that without KDA. Meanwhile, the kernel parameter of KDA has a great impact on the efficiency, and the proposed method can produce an optimum parameter, which makes the new algorithm not only perform as effectively as the traditional ones, but also reduce the computational time and thus improve efficiency.

Prediction of rat protein subcellular localization with pseudo amino acid composition based on multiple sequential features.

PubMed

Shi, Ruijia; Xu, Cunshuan

2011-06-01

The study of rat proteins is an indispensable task in experimental medicine and drug development. The function of a rat protein is closely related to its subcellular location. Based on the above concept, we construct the benchmark rat proteins dataset and develop a combined approach for predicting the subcellular localization of rat proteins. From protein primary sequence, the multiple sequential features are obtained by using of discrete Fourier analysis, position conservation scoring function and increment of diversity, and these sequential features are selected as input parameters of the support vector machine. By the jackknife test, the overall success rate of prediction is 95.6% on the rat proteins dataset. Our method are performed on the apoptosis proteins dataset and the Gram-negative bacterial proteins dataset with the jackknife test, the overall success rates are 89.9% and 96.4%, respectively. The above results indicate that our proposed method is quite promising and may play a complementary role to the existing predictors in this area.

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.

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

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

Virus-PLoc: a fusion classifier for predicting the subcellular localization of viral proteins within host and virus-infected cells.

PubMed

Shen, Hong-Bin; Chou, Kuo-Chen

2007-02-15

Viruses can reproduce their progenies only within a host cell, and their actions depend both on its destructive tendencies toward a specific host cell and on environmental conditions. Therefore, knowledge of the subcellular localization of viral proteins in a host cell or virus-infected cell is very useful for in-depth studying of their functions and mechanisms as well as designing antiviral drugs. An analysis on the Swiss-Prot database (version 50.0, released on May 30, 2006) indicates that only 23.5% of viral protein entries are annotated for their subcellular locations in this regard. As for the gene ontology database, the corresponding percentage is 23.8%. Such a gap calls for the development of high throughput tools for timely annotating the localization of viral proteins within host and virus-infected cells. In this article, a predictor called "Virus-PLoc" has been developed that is featured by fusing many basic classifiers with each engineered according to the K-nearest neighbor rule. The overall jackknife success rate obtained by Virus-PLoc in identifying the subcellular compartments of viral proteins was 80% for a benchmark dataset in which none of proteins has more than 25% sequence identity to any other in a same location site. Virus-PLoc will be freely available as a web-server at http://202.120.37.186/bioinf/virus for the public usage. Furthermore, Virus-PLoc has been used to provide large-scale predictions of all viral protein entries in Swiss-Prot database that do not have subcellular location annotations or are annotated as being uncertain. The results thus obtained have been deposited in a downloadable file prepared with Microsoft Excel and named "Tab_Virus-PLoc.xls." This file is available at the same website and will be updated twice a year to include the new entries of viral proteins and reflect the continuous development of Virus-PLoc. 2006 Wiley Periodicals, Inc.

Subcellular localization of pituitary enzymes

NASA Technical Reports Server (NTRS)

Smith, R. E.

1970-01-01

A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

PubMed

Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M; Zhou, Xinchun

2014-10-01

Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Student's t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC scores for any S1PR (cytoplasmic or nuclear) between benign and malignant changes. This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue. Copyright © 2014 Elsevier Inc. All rights reserved.

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues

PubMed Central

Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M.; Zhou, Xinchun

2014-01-01

Aims Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. Methods We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data were then compared in benign and malignant tissues from the same organ/tissue using the student t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. Results We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (either in nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC score of any S1PR (cytoplasmic or nuclear) between benign and malignant changes. Conclusion This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue. PMID:25084322

Global, quantitative and dynamic mapping of protein subcellular localization.

PubMed

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg Hh

2016-06-09

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.

Bioimaging techniques for subcellular localization of plant hemoglobins and measurement of hemoglobin-dependent nitric oxide scavenging in planta.

PubMed

Hebelstrup, Kim H; Østergaard-Jensen, Erik; Hill, Robert D

2008-01-01

Plant hemoglobins are ubiquitous in all plant families. They are expressed at low levels in specific tissues. Several studies have established that plant hemoglobins are scavengers of nitric oxide (NO) and that varying the endogenous level of hemoglobin in plant cells negatively modulates bioactivity of NO generated under hypoxic conditions or during cellular signaling. Earlier methods for determination of hemoglobin-dependent scavenging in planta were based on measuring activity in whole plants or organs. Plant hemoglobins do not contain specific organelle localization signals; however, earlier reports on plant hemoglobin have demonstrated either cytosolic or nuclear localization, depending on the method or cell type investigated. We have developed two bioimaging techniques: one for visualization of hemoglobin-catalyzed scavenging of NO in specific cells and another for visualization of subcellular localization of green fluorescent protein-tagged plant hemoglobins in transformed Arabidopsis thaliana plants.

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

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

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

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

Synthesis, characterization, and subcellular localization studies of amino acid-substituted porphyrinic pigments

NASA Astrophysics Data System (ADS)

van Diggelen, Lisa; Khin, Hnin; Conner, Kip; Shao, Jenny; Sweezy, Margaretta; Jung, Anna H.; Isaac, Meden; Simonis, Ursula

2009-06-01

Stopping cancer in its path occurs when photosensitizers (PSs) induce apoptotic cell death after their exposure to light and the subsequent formation of reactive oxygen species. In pursuit of our hypothesis that mitochondrial localizing PSs will enhance the efficacy of the photosensitizing process in photodynamic therapy, since they provoke cell death by inducing apoptosis, we synthesized and characterized tetraphenylporphyrins (TPPs) that are substituted at the paraphenyl positions by two amino acids and two fluoro or hydroxyl groups, respectively. They were prepared according to the Lindsey-modified Adler-Longo methodology using trifluoromethanesulfonylchloride (CF3SO2Cl) as a catalyst instead of trifluoroacetic acid. The use of CF3SO2Cl yielded cleaner products in significantly higher yields. During the synthesis, not only the yields and work-up procedure of the TPPs were improved by using CF3SO2Cl as a catalyst, but also a better means of synthesizing the precursor dipyrromethanes was tested by using indium(III) chloride. Column chromatography, HPLC, and NMR spectroscopy were used to separate and characterize the di-amino acid-dihydroxy, or difluoro-substituted porphyrins and to ascertain their purity before subcellular localization studies were carried out. Studies using androgen-sensitive human prostate adenocarcinoma cells LNCaP revealed that certain amino acid substituted porphyrins that are positively charged in the slightly acidic medium of cancer cells are very useful in shedding light on the targets of TPPs in subcellular organelles of cancer cells. Although some of these compounds have properties of promising photosensitizers by revealing increased water solubility, acidic properties, and innate ability to provoke cell death by apoptosis, the cell killing efficacy of these TPPs is low. This correlates with their subcellular localization. The di-amino acid, di-hydroxy substituted TPPs localize mainly to the lysosomes, whereas the di-fluoro-substituted TPPs are trapped in the plasma membrane. Only a pheophorbide derivative recently synthesized in our laboratory localized to the mitochondria of LNCaP cells, which are at the center of cell death as is reflected in their key role during apoptosis, thus reassuring our attempts toward rational drug design.

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.

Mass Spectrometry Analysis of Spatial Protein Networks by Colocalization Analysis (COLA).

PubMed

Mardakheh, Faraz K

2017-01-01

A major challenge in systems biology is comprehensive mapping of protein interaction networks. Crucially, such interactions are often dynamic in nature, necessitating methods that can rapidly mine the interactome across varied conditions and treatments to reveal change in the interaction networks. Recently, we described a fast mass spectrometry-based method to reveal functional interactions in mammalian cells on a global scale, by revealing spatial colocalizations between proteins (COLA) (Mardakheh et al., Mol Biosyst 13:92-105, 2017). As protein localization and function are inherently linked, significant colocalization between two proteins is a strong indication for their functional interaction. COLA uses rapid complete subcellular fractionation, coupled with quantitative proteomics to generate a subcellular localization profile for each protein quantified by the mass spectrometer. Robust clustering is then applied to reveal significant similarities in protein localization profiles, indicative of colocalization.

CerebralWeb: a Cytoscape.js plug-in to visualize networks stratified by subcellular localization.

PubMed

Frias, Silvia; Bryan, Kenneth; Brinkman, Fiona S L; Lynn, David J

2015-01-01

CerebralWeb is a light-weight JavaScript plug-in that extends Cytoscape.js to enable fast and interactive visualization of molecular interaction networks stratified based on subcellular localization or other user-supplied annotation. The application is designed to be easily integrated into any website and is configurable to support customized network visualization. CerebralWeb also supports the automatic retrieval of Cerebral-compatible localizations for human, mouse and bovine genes via a web service and enables the automated parsing of Cytoscape compatible XGMML network files. CerebralWeb currently supports embedded network visualization on the InnateDB (www.innatedb.com) and Allergy and Asthma Portal (allergen.innatedb.com) database and analysis resources. Database tool URL: http://www.innatedb.com/CerebralWeb © The Author(s) 2015. Published by Oxford University Press.

Targeting mechanisms of high voltage-activated Ca2+ channels.

PubMed

Herlitze, Stefan; Xie, Mian; Han, Jing; Hümmer, Alexander; Melnik-Martinez, Katya V; Moreno, Rosa L; Mark, Melanie D

2003-12-01

Functional voltage-dependent Ca2+ channel complexes are assembled by three to four subunits: alpha1, beta, alpha2delta subunits (C. Leveque et al., 1994, J. Biol Chem. 269, 6306-6312; M. W. McEnery et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88, 11095-11099) and at least in muscle cells also y subunits (B. M. Curtis and W. A. Catterall, 1984, Biochemistry 23, 2113-2118). Ca2+ channels mediate the voltage-dependent Ca2+ influx in subcellular compartments, triggering such diverse processes as neurotransmitter release, dendritic action potentials, excitation-contraction, and excitation-transcription coupling. The targeting of biophysically defined Ca2+ channel complexes to the correct subcellular structures is, thus, critical to proper cell and physiological functioning. Despite their importance, surprisingly little is known about the targeting mechanisms by which Ca2+ channel complexes are transported to their site of function. Here we summarize what we know about the targeting of Ca2+ channel complexes through the cell to the plasma membrane and subcellular structures.

Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

PubMed

de Jong, Arjan S; Wessels, Els; Dijkman, Henri B P M; Galama, Jochem M D; Melchers, Willem J G; Willems, Peter H G M; van Kuppeveld, Frank J M

2003-01-10

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

Comparison of intracellular water content measurements by dark-field imaging and EELS in medium voltage TEM

NASA Astrophysics Data System (ADS)

Terryn, C.; Michel, J.; Kilian, L.; Bonhomme, P.; Balossier, G.

2000-09-01

Knowledge of the water content at the subcellular level is important to evaluate the intracellular concentration of either diffusible or non-diffusible elements in the physiological state measured by the electron microprobe methods. Water content variations in subcellular compartments are directly related to secretion phenomena and to transmembrane exchange processes, which could be attributed to pathophysiological states. In this paper we will describe in details and compare two local water measurement methods using analytical electron microscopy. The first one is based on darkfield imaging. It is applied on freeze-dried biological cryosections; it allows indirect measurement of the water content at the subcellular level from recorded maps of darkfield intensity. The second method uses electron energy loss spectroscopy. It is applied to hydrated biological cryosections. It is based on the differences that appear in the electron energy loss spectra of macromolecular assemblies and vitrified ice in the 0-30 eV range. By a multiple least squares (MLS) fit between an experimental energy loss spectrum and reference spectra of both frozen-hydrated ice and macromolecular assemblies we can deduce directly the local water concentration in biological cryosections at the subcellular level. These two methods are applied to two test specimens: human erythrocytes in plasma, and baker's yeast (Saccharomyses Cerevisiae) cryosections. We compare the water content measurements obtained by these two methods and discuss their advantages and drawbacks.

Oncogenic role of p21 in hepatocarcinogenesis suggests a new treatment strategy.

PubMed

Ohkoshi, Shogo; Yano, Masahiko; Matsuda, Yasunobu

2015-11-14

A well-known tumor suppressor, p21, acts paradoxically by promoting tumor growth in some cellular conditions. These conflicting functions have been demonstrated in association with the HBx gene and in hepatocarcinogenesis. The molecular behavior of p21 depends on its subcellular localization. Nuclear p21 may inhibit cell proliferation and be proapoptotic, while cytoplasmic p21 may have oncogenic and anti-apoptotic functions. Because most typical tumor suppressive proteins also have different effects according to subcellular localization, elucidating the regulatory mechanisms underlying nucleo-cytoplasmic transport of these proteins would be significant and may lead to a new strategy for anti-hepatocellular carcinoma (HCC) therapy. Chromosome region maintenance 1 (CRM1) is a major nuclear export receptor involved in transport of tumor suppressors from nucleus to cytoplasm. Expression of CRM1 is enhanced in a variety of malignancies and in vitro studies have shown the efficacy of specific inhibition of CRM1 against cancer cell lines. Interestingly, interferon may keep p21 in the nucleus; this is one of the mechanisms of its anti-hepatocarcinogenic function. Here we review the oncogenic property of p21, which depends on its subcellular localization, and discuss the rationale underlying a new strategy for HCC treatment and prevention.

Oncogenic role of p21 in hepatocarcinogenesis suggests a new treatment strategy

PubMed Central

Ohkoshi, Shogo; Yano, Masahiko; Matsuda, Yasunobu

2015-01-01

A well-known tumor suppressor, p21, acts paradoxically by promoting tumor growth in some cellular conditions. These conflicting functions have been demonstrated in association with the HBx gene and in hepatocarcinogenesis. The molecular behavior of p21 depends on its subcellular localization. Nuclear p21 may inhibit cell proliferation and be proapoptotic, while cytoplasmic p21 may have oncogenic and anti-apoptotic functions. Because most typical tumor suppressive proteins also have different effects according to subcellular localization, elucidating the regulatory mechanisms underlying nucleo-cytoplasmic transport of these proteins would be significant and may lead to a new strategy for anti-hepatocellular carcinoma (HCC) therapy. Chromosome region maintenance 1 (CRM1) is a major nuclear export receptor involved in transport of tumor suppressors from nucleus to cytoplasm. Expression of CRM1 is enhanced in a variety of malignancies and in vitro studies have shown the efficacy of specific inhibition of CRM1 against cancer cell lines. Interestingly, interferon may keep p21 in the nucleus; this is one of the mechanisms of its anti-hepatocarcinogenic function. Here we review the oncogenic property of p21, which depends on its subcellular localization, and discuss the rationale underlying a new strategy for HCC treatment and prevention. PMID:26576099

Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes.

PubMed

Rojas, Joselyn; Teran-Angel, Guillermo; Barbosa, Luisa; Peterson, Darrell L; Berrueta, Lisbeth; Salmen, Siham

2016-05-01

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 shuttling 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. Copyright © 2016 Elsevier Inc. All rights reserved.

Hum-mPLoc 3.0: prediction enhancement of human protein subcellular localization through modeling the hidden correlations of gene ontology and functional domain features.

PubMed

Zhou, Hang; Yang, Yang; Shen, Hong-Bin

2017-03-15

Protein subcellular localization prediction has been an important research topic in computational biology over the last decade. Various automatic methods have been proposed to predict locations for large scale protein datasets, where statistical machine learning algorithms are widely used for model construction. A key step in these predictors is encoding the amino acid sequences into feature vectors. Many studies have shown that features extracted from biological domains, such as gene ontology and functional domains, can be very useful for improving the prediction accuracy. However, domain knowledge usually results in redundant features and high-dimensional feature spaces, which may degenerate the performance of machine learning models. In this paper, we propose a new amino acid sequence-based human protein subcellular location prediction approach Hum-mPLoc 3.0, which covers 12 human subcellular localizations. The sequences are represented by multi-view complementary features, i.e. context vocabulary annotation-based gene ontology (GO) terms, peptide-based functional domains, and residue-based statistical features. To systematically reflect the structural hierarchy of the domain knowledge bases, we propose a novel feature representation protocol denoted as HCM (Hidden Correlation Modeling), which will create more compact and discriminative feature vectors by modeling the hidden correlations between annotation terms. Experimental results on four benchmark datasets show that HCM improves prediction accuracy by 5-11% and F 1 by 8-19% compared with conventional GO-based methods. A large-scale application of Hum-mPLoc 3.0 on the whole human proteome reveals proteins co-localization preferences in the cell. www.csbio.sjtu.edu.cn/bioinf/Hum-mPLoc3/. hbshen@sjtu.edu.cn. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

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.

Imbalanced multi-modal multi-label learning for subcellular localization prediction of human proteins with both single and multiple sites.

PubMed

He, Jianjun; Gu, Hong; Liu, Wenqi

2012-01-01

It is well known that an important step toward understanding the functions of a protein is to determine its subcellular location. Although numerous prediction algorithms have been developed, most of them typically focused on the proteins with only one location. In recent years, researchers have begun to pay attention to the subcellular localization prediction of the proteins with multiple sites. However, almost all the existing approaches have failed to take into account the correlations among the locations caused by the proteins with multiple sites, which may be the important information for improving the prediction accuracy of the proteins with multiple sites. In this paper, a new algorithm which can effectively exploit the correlations among the locations is proposed by using gaussian process model. Besides, the algorithm also can realize optimal linear combination of various feature extraction technologies and could be robust to the imbalanced data set. Experimental results on a human protein data set show that the proposed algorithm is valid and can achieve better performance than the existing approaches.

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.

Concepts of Protein Sorting or Targeting Signals and Membrane Topology in Undergraduate Teaching

ERIC Educational Resources Information Center

Tang, Bor Luen; Teng, Felicia Yu Hsuan

2005-01-01

The process of protein biogenesis culminates in its correct targeting to specific subcellular locations where it serves a function. Contemporary molecular and cell biology investigations often involve the exogenous expression of epitope- or fluorescent protein-tagged recombinant molecules as well as subsequent analysis of protein-protein…

Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3

PubMed Central

Riquelme, Denise; Silva, Ian; Philp, Ashleigh M.; Huidobro-Toro, Juan P.; Cerda, Oscar; Trimmer, James S.; Leiva-Salcedo, Elias

2018-01-01

TRPM4 is a Ca2+-activated non-selective cationic channel that conducts monovalent cations. TRPM4 has been proposed to contribute to burst firing and sustained activity in several brain regions, however, the cellular and subcellular pattern of TRPM4 expression in medial prefrontal cortex (mPFC) during postnatal development has not been elucidated. Here, we use multiplex immunofluorescence labeling of brain sections to characterize the postnatal developmental expression of TRPM4 in the mouse mPFC. We also performed electrophysiological recordings to correlate the expression of TRPM4 immunoreactivity with the presence of TRPM4-like currents. We found that TRPM4 is expressed from the first postnatal day, with expression increasing up to postnatal day 35. Additionally, in perforated patch clamp experiments, we found that TRPM4-like currents were active at resting membrane potentials at all postnatal ages studied. Moreover, TRPM4 is expressed in both pyramidal neurons and interneurons. TRPM4 expression is localized in the soma and proximal dendrites, but not in the axon initial segment of pyramidal neurons. This subcellular localization is consistent with a reduction in the basal current only when we locally perfused 9-Phenanthrol in the soma, but not upon perfusion in the medial or distal dendrites. Our results show a specific localization of TRPM4 expression in neurons in the mPFC and that a 9-Phenanthrol sensitive current is active at resting membrane potential, suggesting specific functional roles in mPFC neurons during postnatal development and in adulthood. PMID:29440991

Role of NH{sub 2}-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: Common features in eukaryotic organisms

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

Lee, Asaka; Asahina, Kota; Okamoto, Takumi

Highlights: • ABCD proteins classifies based on with or without NH{sub 2}-terminal hydrophobic segment. • The ABCD proteins with the segment are targeted peroxisomes. • The ABCD proteins without the segment are targeted to the endoplasmic reticulum. • The role of the segment in organelle targeting is conserved in eukaryotic organisms. - Abstract: In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1–3 possesses the NH{sub 2}-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that severalmore » eukaryotes have ABCD protein homologs lacking the NH{sub 2}-terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH{sub 2}-terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH{sub 2}-terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH{sub 2}-terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH{sub 2}-terminal H0 motif in organelle targeting is widely conserved in living organisms.« less

Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3.

PubMed

Riquelme, Denise; Silva, Ian; Philp, Ashleigh M; Huidobro-Toro, Juan P; Cerda, Oscar; Trimmer, James S; Leiva-Salcedo, Elias

2018-01-01

TRPM4 is a Ca 2+ -activated non-selective cationic channel that conducts monovalent cations. TRPM4 has been proposed to contribute to burst firing and sustained activity in several brain regions, however, the cellular and subcellular pattern of TRPM4 expression in medial prefrontal cortex (mPFC) during postnatal development has not been elucidated. Here, we use multiplex immunofluorescence labeling of brain sections to characterize the postnatal developmental expression of TRPM4 in the mouse mPFC. We also performed electrophysiological recordings to correlate the expression of TRPM4 immunoreactivity with the presence of TRPM4-like currents. We found that TRPM4 is expressed from the first postnatal day, with expression increasing up to postnatal day 35. Additionally, in perforated patch clamp experiments, we found that TRPM4-like currents were active at resting membrane potentials at all postnatal ages studied. Moreover, TRPM4 is expressed in both pyramidal neurons and interneurons. TRPM4 expression is localized in the soma and proximal dendrites, but not in the axon initial segment of pyramidal neurons. This subcellular localization is consistent with a reduction in the basal current only when we locally perfused 9-Phenanthrol in the soma, but not upon perfusion in the medial or distal dendrites. Our results show a specific localization of TRPM4 expression in neurons in the mPFC and that a 9-Phenanthrol sensitive current is active at resting membrane potential, suggesting specific functional roles in mPFC neurons during postnatal development and in adulthood.

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

Localization and function of GABA transporters in the globus pallidus of parkinsonian monkeys

PubMed Central

Galvan, Adriana; Hu, Xing; Smith, Yoland; Wichmann, Thomas

2010-01-01

The GABA transporters GAT-1 and GAT-3 are abundant in the external and internal segments of the globus pallidus (GPe and GPi, respectively). We have shown that pharmacological blockade of either of these transporters results in decreased neuronal firing, and in elevated levels of extracellular GABA in normal monkeys. We now studied whether the electrophysiologic and biochemical effects of local intra-pallidal injections of GAT-1 and GAT-3 blockers, or the subcellular localization of these transporters, are altered in monkeys rendered parkinsonian by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The subcellular localization of the transporters in GPe and GPi, studied with electron microscopy immunoperoxidase, was similar to that found in normal animals: i.e., GAT-3 immunoreactivity was mostly confined to glial processes, while GAT-1 labeling was expressed in unmyelinated axons and glial processes. A combined injection/recording device was used to record extracellular activity of single neurons in GPe and GPi, before, during and after administration of small volumes (1 μl) of either the GAT-1 inhibitor, SKF-89976A hydrochloride (720 ng), or the GAT-3 inhibitor, (S)-SNAP-5114 (500 ng). In GPe, the effects of GAT-1 or GAT-3 blockade were similar to those seen in normal monkeys. However, unlike the findings in the normal state, the firing of most neurons was not affected by blockade of either transporter in GPi. These results suggest that, after dopaminergic depletion, the functions of GABA transporters are altered in GPi; without major changes in their subcellular localization. PMID:20138865

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

Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniques--subcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis.

PubMed

Leonardo, T; Farhi, E; Boisson, A-M; Vial, J; Cloetens, P; Bohic, S; Rivasseau, C

2014-02-01

The newly discovered unicellular micro-alga Coccomyxa actinabiotis proves to be highly radio-tolerant and strongly concentrates radionuclides, as well as large amounts of toxic metals. This study helps in the understanding of the mechanisms involved in the accumulation and detoxification of silver and cobalt. Elemental distribution inside Coccomyxa actinabiotis cells was determined using synchrotron nano X-ray fluorescence spectroscopy at the ID22 nano fluorescence imaging beamline of the European Synchrotron Radiation Facility. The high resolution and high sensitivity of this technique enabled the assessment of elemental associations and exclusions in subcellular micro-algae compartments. A quantitative treatment of the scans was implemented to yield absolute concentrations of each endogenous and exogenous element with a spatial resolution of 100 nm and compared to the macroscopic content in cobalt and silver determined using inductively coupled plasma-mass spectrometry. The nano X-ray fluorescence imaging was complemented by transmission electron microscopy coupled to X-ray microanalysis (TEM-EDS), yielding differential silver distribution in the cell wall, cytosol, nucleus, chloroplast and mitochondria with unique resolution. The analysis of endogenous elements in control cells revealed that iron had a unique distribution; zinc, potassium, manganese, molybdenum, and phosphate had their maxima co-localized in the same area; and sulfur, copper and chlorine were almost homogeneously distributed among the whole cell. The subcellular distribution and quantification of cobalt and silver in micro-alga, assessed after controlled exposure to various concentrations, revealed that exogenous metals were mainly sequestered inside the cell rather than on mucilage or the cell wall, with preferential compartmentalization. Cobalt was homogeneously distributed outside of the chloroplast. Silver was localized in the cytosol at low concentration and in the whole cell excluding the nucleus at high concentration. Exposure to low concentrations of cobalt or silver did not alter the localization nor the concentration of endogenous elements within the cells. To our knowledge, this is the first report on element co-localization and segregation at the sub-cellular level in micro-algae by means of synchrotron nano X-ray fluorescence spectroscopy.

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.

TRPC6-mediated ERK1/2 Activation Regulates Neuronal Excitability via Subcellular Kv4.3 Localization in the Rat Hippocampus

PubMed Central

Kim, Ji-Eun; Park, Jin-Young; Kang, Tae-Cheon

2017-01-01

Recently, we have reported that transient receptor potential channel-6 (TRPC6) plays an important role in the regulation of neuronal excitability and synchronization of spiking activity in the dentate granule cells (DGC). However, the underlying mechanisms of TRPC6 in these phenomena have been still unclear. In the present study, we investigated the role of TRPC6 in subcellular localization of Kv4.3 and its relevance to neuronal excitability in the rat hippocampus. TRPC6 knockdown increased excitability and inhibitory transmission in the DGC and the CA1 neurons in response to a paired-pulse stimulus. However, TRPC6 knockdown impaired γ-aminobutyric acid (GABA)ergic inhibition in the hippocampus during and after high-frequency stimulation (HFS). TRPC6 knockdown reduced the Kv4.3 clusters in membrane fractions and its dendritic localization on DGC and GABAergic interneurons. TRPC6 knockdown also decreased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and the efficacy of 4-aminopyridine (4-AP) in neuronal excitability. An ERK1/2 inhibitor generated multiple population spikes in response to a paired-pulse stimulus, concomitant with reduced membrane Kv4.3 translocation. A TRPC6 activator (hyperforin) reversed the effects of TRPC knockdown, except paired-pulse inhibition. These findings provide valuable clues indicating that TRPC6-mediated ERK1/2 activation may regulate subcellular Kv4.3 localization in DGC and interneurons, which is cause-effect relationship between neuronal excitability and seizure susceptibility. PMID:29326557

Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

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

Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha

2014-07-18

Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated thatmore » TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations.« less

Global, quantitative and dynamic mapping of protein subcellular localization

PubMed Central

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

2016-01-01

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

The role of endomembrane-localized VHA-c in plant growth.

PubMed

Zhou, Aimin; Takano, Tetsuo; Liu, Shenkui

2018-01-02

In plant cells, the vacuolar-type H + -ATPase (V-ATPase), a large multis`ubunit endomembrane proton pump, plays an important role in acidification of subcellular organelles, pH and ion homeostasis, and endocytic and secretory trafficking. V-ATPase subunit c (VHA-c) is essential for V-ATPase assembly, and is directly responsible for binding and transmembrane transport of protons. In previous studies, we identified a PutVHA-c gene from Puccinellia tenuiflora, and investigated its function in plant growth. Subcellular localization revealed that PutVHA-c is mainly localized in endosomal compartments. Overexpression of PutVHA-c enhanced V-ATPase activity and promoted plant growth in transgenic Arabidopsis. Furthermore, the activity of V-ATPase affected intracellular transport of the Golgi-derived endosomes. Our results showed that endomembrane localized-VHA-c contributes to plant growth by influencing V-ATPase-dependent endosomal trafficking. Here, we discuss these recent findings and speculate on the VHA-c mediated molecular mechanisms involved in plant growth, providing a better understanding of the functions of VHA-c and V-ATPase.

Nucleolar localization of cirhin, the protein mutated in North American Indian childhood cirrhosis

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

Yu, Bin; Mitchell, Grant A.; Richter, Andrea

2005-12-10

Cirhin (NP{sub 1}16219), the product of the CIRH1A gene is mutated in North American Indian childhood cirrhosis (NAIC/CIRH1A, OMIM 604901), a severe autosomal recessive intrahepatic cholestasis. It is a 686-amino-acid WD40-repeat containing protein of unknown function that is predicted to contain multiple targeting signals, including an N-terminal mitochondrial targeting signal, a C-terminal monopartite nuclear localization signal (NLS) and a bipartite nuclear localization signal (BNLS). We performed the direct determination of subcellular localization of cirhin as a crucial first step in unraveling its biological function. Using EGFP and His-tagged cirhin fusion proteins expressed in HeLa and HepG2, cells we show thatmore » cirhin is a nucleolar protein and that the R565W mutation, for which all NAIC patients are homozygous, has no effect on subcellular localization. Cirhin has an active C-terminal monopartite nuclear localization signal (NLS) and a unique nucleolar localization signal (NrLS) between residues 315 and 432. The nucleolus is not known to be important specifically for intrahepatic cholestasis. These observations provide a new dimension in the study of hereditary cholestasis.« less

Differential subcellular distribution of ion channels and the diversity of neuronal function.

PubMed

Nusser, Zoltan

2012-06-01

Following the astonishing molecular diversity of voltage-gated ion channels that was revealed in the past few decades, the ion channel repertoire expressed by neurons has been implicated as the major factor governing their functional heterogeneity. Although the molecular structure of ion channels is a key determinant of their biophysical properties, their subcellular distribution and densities on the surface of nerve cells are just as important for fulfilling functional requirements. Recent results obtained with high resolution quantitative localization techniques revealed complex, subcellular compartment-specific distribution patterns of distinct ion channels. Here I suggest that within a given neuron type every ion channel has a unique cell surface distribution pattern, with the functional consequence that this dramatically increases the computational power of nerve cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

PubMed

Okuhira, Keiichiro; Shoda, Takuji; Omura, Risa; Ohoka, Nobumichi; Hattori, Takayuki; Shibata, Norihito; Demizu, Yosuke; Sugihara, Ryo; Ichino, Asato; Kawahara, Haruka; Itoh, Yukihiro; Ishikawa, Minoru; Hashimoto, Yuichi; Kurihara, Masaaki; Itoh, Susumu; Saito, Hiroyuki; Naito, Mikihiko

2017-03-01

Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress.

PubMed

Messaoudi, Lydia; Yang, Yun-Gui; Kinomura, Aiko; Stavreva, Diana A; Yan, Gonghong; Bortolin-Cavaillé, Marie-Line; Arakawa, Hiroshi; Buerstedde, Jean-Marie; Hainaut, Pierre; Cavaillé, Jérome; Takata, Minoru; Van Dyck, Eric

2007-01-01

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1alpha), and other RDM1 isoforms. We show that RDM1alpha undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response.

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

pLoc-mPlant: predict subcellular localization of multi-location plant proteins by incorporating the optimal GO information into general PseAAC.

PubMed

Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

2017-08-22

One of the fundamental goals in cellular biochemistry is to identify the functions of proteins in the context of compartments that organize them in the cellular environment. To realize this, it is indispensable to develop an automated method for fast and accurate identification of the subcellular locations of uncharacterized proteins. The current study is focused on plant protein subcellular location prediction based on the sequence information alone. Although considerable efforts have been made in this regard, the problem is far from being solved yet. Most of the existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions. This kind of multiplex protein is particularly important for both basic research and drug design. Using the multi-label theory, we present a new predictor called "pLoc-mPlant" by extracting the optimal GO (Gene Ontology) information into the Chou's general PseAAC (Pseudo Amino Acid Composition). Rigorous cross-validation on the same stringent benchmark dataset indicated that the proposed pLoc-mPlant predictor is remarkably superior to iLoc-Plant, the state-of-the-art method for predicting plant protein subcellular localization. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at , by which users can easily get their desired results without the need to go through the complicated mathematics involved.

pLoc-mVirus: Predict subcellular localization of multi-location virus proteins via incorporating the optimal GO information into general PseAAC.

PubMed

Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

2017-09-10

Knowledge of subcellular locations of proteins is crucially important for in-depth understanding their functions in a cell. With the explosive growth of protein sequences generated in the postgenomic age, it is highly demanded to develop computational tools for timely annotating their subcellular locations based on the sequence information alone. The current study is focused on virus proteins. Although considerable efforts have been made in this regard, the problem is far from being solved yet. Most existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions. This kind of multiplex proteins is particularly important for both basic research and drug design. Using the multi-label theory, we present a new predictor called "pLoc-mVirus" by extracting the optimal GO (Gene Ontology) information into the general PseAAC (Pseudo Amino Acid Composition). Rigorous cross-validation on a same stringent benchmark dataset indicated that the proposed pLoc-mVirus predictor is remarkably superior to iLoc-Virus, the state-of-the-art method in predicting virus protein subcellular localization. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc-mVirus/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome-wide identification of the subcellular localization of the Escherichia coli B proteome using experimental and computational methods.

PubMed

Han, Mee-Jung; Yun, Hongseok; Lee, Jeong Wook; Lee, Yu Hyun; Lee, Sang Yup; Yoo, Jong-Shin; Kim, Jin Young; Kim, Jihyun F; Hur, Cheol-Goo

2011-04-01

Escherichia coli K-12 and B strains have most widely been employed for scientific studies as well as industrial applications. Recently, the complete genome sequences of two representative descendants of E. coli B strains, REL606 and BL21(DE3), have been determined. Here, we report the subproteome reference maps of E. coli B REL606 by analyzing cytoplasmic, periplasmic, inner and outer membrane, and extracellular proteomes based on the genome information using experimental and computational approaches. Among the total of 3487 spots, 651 proteins including 410 non-redundant proteins were identified and characterized by 2-DE and LC-MS/MS; they include 440 cytoplasmic, 45 periplasmic, 50 inner membrane, 61 outer membrane, and 55 extracellular proteins. In addition, subcellular localizations of all 4205 ORFs of E. coli B were predicted by combined computational prediction methods. The subcellular localizations of 1812 (43.09%) proteins of currently unknown function were newly assigned. The results of computational prediction were also compared with the experimental results, showing that overall precision and recall were 92.16 and 92.16%, respectively. This work represents the most comprehensive analyses of the subproteomes of E. coli B, and will be useful as a reference for proteome profiling studies under various conditions. The complete proteome data are available online (http://ecolib.kaist.ac.kr). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Multi-location gram-positive and gram-negative bacterial protein subcellular localization using gene ontology and multi-label classifier ensemble.

PubMed

Wang, Xiao; Zhang, Jun; Li, Guo-Zheng

2015-01-01

It has become a very important and full of challenge task to predict bacterial protein subcellular locations using computational methods. Although there exist a lot of prediction methods for bacterial proteins, the majority of these methods can only deal with single-location proteins. But unfortunately many multi-location proteins are located in the bacterial cells. Moreover, multi-location proteins have special biological functions capable of helping the development of new drugs. So it is necessary to develop new computational methods for accurately predicting subcellular locations of multi-location bacterial proteins. In this article, two efficient multi-label predictors, Gpos-ECC-mPLoc and Gneg-ECC-mPLoc, are developed to predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. The two multi-label predictors construct the GO vectors by using the GO terms of homologous proteins of query proteins and then adopt a powerful multi-label ensemble classifier to make the final multi-label prediction. The two multi-label predictors have the following advantages: (1) they improve the prediction performance of multi-label proteins by taking the correlations among different labels into account; (2) they ensemble multiple CC classifiers and further generate better prediction results by ensemble learning; and (3) they construct the GO vectors by using the frequency of occurrences of GO terms in the typical homologous set instead of using 0/1 values. Experimental results show that Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently improve prediction accuracy of subcellular localization of multi-location gram-positive and gram-negative bacterial proteins respectively. The online web servers for Gpos-ECC-mPLoc and Gneg-ECC-mPLoc predictors are freely accessible at http://biomed.zzuli.edu.cn/bioinfo/gpos-ecc-mploc/ and http://biomed.zzuli.edu.cn/bioinfo/gneg-ecc-mploc/ respectively.

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

An intrinsic DFF40/CAD endonuclease deficiency impairs oligonucleosomal DNA hydrolysis during caspase-dependent cell death: a common trait in human glioblastoma cells

PubMed Central

Sánchez-Osuna, María; Martínez-Escardó, Laura; Granados-Colomina, Carla; Martínez-Soler, Fina; Pascual-Guiral, Sònia; Iglesias-Guimarais, Victoria; Velasco, Roser; Plans, Gerard; Vidal, Noemi; Tortosa, Avelina; Barcia, Carlos; Bruna, Jordi; Yuste, Victor J.

2016-01-01

Background Glioblastoma (GBM) or grade IV astrocytoma is one of the most devastating human cancers. The loss of DFF40/CAD, the key endonuclease that triggers oligonucleosomal DNA fragmentation during apoptosis, has been linked to genomic instability and cell survival after radiation. Despite the near inevitability of GBM tumor recurrence after treatment, the relationship between DFF40/CAD and GBM remains unexplored. Methods We studied the apoptotic behavior of human GBM-derived cells after apoptotic insult. We analyzed caspase activation and the protein levels and subcellular localization of DFF40/CAD apoptotic endonuclease. DFF40/CAD was also evaluated in histological sections from astrocytic tumors and nontumoral human brain. Results We showed that GBM cells undergo incomplete apoptosis without generating oligonucleosomal DNA degradation despite the correct activation of executioner caspases. The major defect of GBM cells relied on the improper accumulation of DFF40/CAD at the nucleoplasmic subcellular compartment. Supporting this finding, DFF40/CAD overexpression allowed GBM cells to display oligonucleosomal DNA degradation after apoptotic challenge. Moreover, the analysis of histological slices from astrocytic tumors showed that DFF40/CAD immunoreactivity in tumoral GFAP-positive cells was markedly reduced when compared with nontumoral samples. Conclusions Our data highlight the low expression levels of DFF40/CAD and the absence of DNA laddering as common molecular traits in GBM. These findings could be of major importance for understanding the malignant behavior of remaining tumor cells after radiochemotherapy. PMID:26755073

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.

Prognostic value of loss of heterozygosity and sub-cellular localization of SMAD4 varies with tumor stage in colorectal cancer.

PubMed

Jia, Xu; Shanmugam, Chandrakumar; Paluri, Ravi K; Jhala, Nirag C; Behring, Michael P; Katkoori, Venkat R; Sugandha, Shajan P; Bae, Sejong; Samuel, Temesgen; Manne, Upender

2017-03-21

Although loss of heterozygosity (LOH) at chromosome location 18q21 and decreased expression of SMAD4 in invasive colorectal cancers (CRCs) correlate with poor patient survival, the prognostic value of LOH at 18q21 and sub-cellular localization of SMAD4 have not been evaluated in relation to tumor stage. Genomic DNA samples from 209 formalin-fixed, paraffin-embedded sporadic CRC tissues and their matching controls were analyzed for 18q21 LOH, and corresponding tissue sections were evaluated by immunohistochemistry for expression of SMAD4 and assessed for its sub-cellular localization (nuclear vs. cytoplasmic). In addition, 53 frozen CRCs and their matching control tissues were analyzed for their mutational status and mRNA expression of SMAD4. The phenotypic expression pattern and LOH status were evaluated for correlation with patient survival by the use of Kaplan-Meier and Cox regression models. LOH of 18q21 was detected in 61% of the informative cases. In 8% of the cases, missense point mutations were detected in Smad4. In CRCs, relative to controls, there was increased SMAD4 staining in the cytoplasm (74%) and decreased staining in the nuclei (37%). LOH of 18q21 and high cytoplasmic localization of SMAD4 were associated with shortened overall survival of Stage II patients, whereas low nuclear expression of SMAD4 was associated with worse survival, but only for patients with Stage III CRCs. LOH of 18q21 and high cytoplasmic localization of SMAD4 in Stage II CRCs and low nuclear SMAD4 in Stage III CRCs are predictors of shortened patient survival.

Prognostic value of loss of heterozygosity and sub-cellular localization of SMAD4 varies with tumor stage in colorectal cancer

PubMed Central

Jia, Xu; Shanmugam, Chandrakumar; Paluri, Ravi K.; Jhala, Nirag C.; Behring, Michael P.; Katkoori, Venkat R.; Sugandha, Shajan P.; Bae, Sejong; Samuel, Temesgen; Manne, Upender

2017-01-01

Background Although loss of heterozygosity (LOH) at chromosome location 18q21 and decreased expression of SMAD4 in invasive colorectal cancers (CRCs) correlate with poor patient survival, the prognostic value of LOH at 18q21 and sub-cellular localization of SMAD4 have not been evaluated in relation to tumor stage. Methods Genomic DNA samples from 209 formalin-fixed, paraffin-embedded sporadic CRC tissues and their matching controls were analyzed for 18q21 LOH, and corresponding tissue sections were evaluated by immunohistochemistry for expression of SMAD4 and assessed for its sub-cellular localization (nuclear vs. cytoplasmic). In addition, 53 frozen CRCs and their matching control tissues were analyzed for their mutational status and mRNA expression of SMAD4. The phenotypic expression pattern and LOH status were evaluated for correlation with patient survival by the use of Kaplan-Meier and Cox regression models. Results LOH of 18q21 was detected in 61% of the informative cases. In 8% of the cases, missense point mutations were detected in Smad4. In CRCs, relative to controls, there was increased SMAD4 staining in the cytoplasm (74%) and decreased staining in the nuclei (37%). LOH of 18q21 and high cytoplasmic localization of SMAD4 were associated with shortened overall survival of Stage II patients, whereas low nuclear expression of SMAD4 was associated with worse survival, but only for patients with Stage III CRCs. Conclusions LOH of 18q21 and high cytoplasmic localization of SMAD4 in Stage II CRCs and low nuclear SMAD4 in Stage III CRCs are predictors of shortened patient survival. PMID:28423626

Saccharomyces cerevisiae Mob1p Is Required for Cytokinesis and Mitotic Exit

PubMed Central

Luca, Francis C.; Mody, Manali; Kurischko, Cornelia; Roof, David M.; Giddings, Thomas H.; Winey, Mark

2001-01-01

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G1 by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G1 transition to control cyclin-CDK inactivation and cytokinesis. PMID:11564880

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

The intricacies of p21 phosphorylation: protein/protein interactions, subcellular localization and stability.

PubMed

Child, Emma S; Mann, David J

2006-06-01

p21 was originally described as functioning as a cell cycle regulator via inhibition of both cyclin-dependent kinases and processive DNA replication. Nowadays it is recognized to play other fundamental roles including transcriptional regulation and the modulation of apoptosis. Each of these functions of p21 is achieved through direct p21/protein interactions and the subcellular localization of p21 plays an important part in dictating the binding partners to which p21 is exposed. Over recent years, a number of phosphorylation sites in p21 have been identified, these being targeted by several important intracellular signalling protein kinases. Here we review the state of our knowledge of p21 phosphorylation with respect to the kinases involved and the molecular biological effects of each phosphorylation event.

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.

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.

[Correcting influence of vitamin E short chain derivatives on lipid peroxidation, liver cell membrane, and chromatin structure when rats are exposed to embichin].

PubMed

Kovalenko, V M; Byshovets', T F; Hubs'kyĭ, Iu I; Levyts'kyĭ, Ie L; Shaiakhmetova, H M; Marchenko, O M; Voloshyna, O S; Saĭfetdinova, H A; Okhrimenko, V O; Donchenko, H V

2000-01-01

Embikhin causes activation of LPO processes in endoplasmic reticulum and in nuclear chromatine fractions of rat liver cells. The latter is accompanied by the impairment of repressive and active nuclear chromatine fractions structure. Derivate of vitamin E in these conditions renders correcting action on parameters of lipid peroxidation in the investigated subcellular structures, testifying its positive influence on the cell heredity apparatus state. The normalizing action of tocopherol derivative on cytochromes P450 and b5 levels is shown.

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.

Human cell structure-driven model construction for predicting protein subcellular location from biological images.

PubMed

Shao, Wei; Liu, Mingxia; Zhang, Daoqiang

2016-01-01

The systematic study of subcellular location pattern is very important for fully characterizing the human proteome. Nowadays, with the great advances in automated microscopic imaging, accurate bioimage-based classification methods to predict protein subcellular locations are highly desired. All existing models were constructed on the independent parallel hypothesis, where the cellular component classes are positioned independently in a multi-class classification engine. The important structural information of cellular compartments is missed. To deal with this problem for developing more accurate models, we proposed a novel cell structure-driven classifier construction approach (SC-PSorter) by employing the prior biological structural information in the learning model. Specifically, the structural relationship among the cellular components is reflected by a new codeword matrix under the error correcting output coding framework. Then, we construct multiple SC-PSorter-based classifiers corresponding to the columns of the error correcting output coding codeword matrix using a multi-kernel support vector machine classification approach. Finally, we perform the classifier ensemble by combining those multiple SC-PSorter-based classifiers via majority voting. We evaluate our method on a collection of 1636 immunohistochemistry images from the Human Protein Atlas database. The experimental results show that our method achieves an overall accuracy of 89.0%, which is 6.4% higher than the state-of-the-art method. The dataset and code can be downloaded from https://github.com/shaoweinuaa/. dqzhang@nuaa.edu.cn Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Subcellular localization of rat CYP2E1 impacts metabolic efficiency toward common substrates.

PubMed

Hartman, Jessica H; Martin, H Cass; Caro, Andres A; Pearce, Amy R; Miller, Grover P

2015-12-02

Cytochrome P450 2E1 (CYP2E1) detoxifies or bioactivates many low molecular-weight compounds. Most knowledge about CYP2E1 activity relies on studies of the enzyme localized to endoplasmic reticulum (erCYP2E1); however, CYP2E1 undergoes transport to mitochondria (mtCYP2E1) and becomes metabolically active. We report the first comparison of in vitro steady-state kinetic profiles for erCYP2E1 and mtCYP2E1 oxidation of probe substrate 4-nitrophenol and pollutants styrene and aniline using subcellular fractions from rat liver. For all substrates, metabolic efficiency changed with substrate concentration for erCYP2E1 reflected in non-hyperbolic kinetic profiles but not for mtCYP2E1. Hyperbolic kinetic profiles for the mitochondrial enzyme were consistent with Michaelis-Menten mechanism in which metabolic efficiency was constant. By contrast, erCYP2E1 metabolism of 4-nitrophenol led to a loss of enzyme efficiency at high substrate concentrations when substrate inhibited the reaction. Similarly, aniline metabolism by erCYP2E1 demonstrated negative cooperativity as metabolic efficiency decreased with increasing substrate concentration. The opposite was observed for erCYP2E1 oxidation of styrene; the sigmoidal kinetic profile indicated increased efficiency at higher substrate concentrations. These mechanisms and CYP2E1 levels in mitochondria and endoplasmic reticulum were used to estimate the impact of CYP2E1 subcellular localization on metabolic flux of pollutants. Those models showed that erCYP2E1 mainly carries out aniline metabolism at all aniline concentrations. Conversely, mtCYP2E1 dominates styrene oxidation at low styrene concentrations and erCYP2E1 at higher concentrations. Taken together, subcellular localization of CYP2E1 results in distinctly different enzyme activities that could impact overall metabolic clearance and/or activation of substrates and thus impact the interpretation and prediction of toxicological outcomes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

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.

Expression of Tocopherol-Associated Protein in Mast Cells

PubMed Central

Ikeda, Teruo; Murakami, Masaru; Funaba, Masayuki

2004-01-01

Tocopherol-associated protein (TAP) was expressed in mouse mast cells. TAP was predominantly localized in the cytoplasm, and the subcellular localization was not changed by α-tocopherol. The results suggest that the physiological role of TAP in mast cells is not regulation of tocopherol function but an as-yet-unidentified activity. PMID:15539527

A catechol oxidase AcPPO from cherimoya (Annona cherimola Mill.) is localized to the Golgi apparatus.

PubMed

Olmedo, Patricio; Moreno, Adrián A; Sanhueza, Dayan; Balic, Iván; Silva-Sanzana, Christian; Zepeda, Baltasar; Verdonk, Julian C; Arriagada, César; Meneses, Claudio; Campos-Vargas, Reinaldo

2018-01-01

Cherimoya (Annona cherimola) is an exotic fruit with attractive organoleptic characteristics. However, it is highly perishable and susceptible to postharvest browning. In fresh fruit, browning is primarily caused by the polyphenol oxidase (PPO) enzyme catalyzing the oxidation of o-diphenols to quinones, which polymerize to form brown melanin pigment. There is no consensus in the literature regarding a specific role of PPO, and its subcellular localization in different plant species is mainly described within plastids. The present work determined the subcellular localization of a PPO protein from cherimoya (AcPPO). The obtained results revealed that the AcPPO- green fluorescent protein co-localized with a Golgi apparatus marker, and AcPPO activity was present in Golgi apparatus-enriched fractions. Likewise, transient expression assays revealed that AcPPO remained active in Golgi apparatus-enriched fractions obtained from tobacco leaves. These results suggest a putative function of AcPPO in the Golgi apparatus of cherimoya, providing new perspectives on PPO functionality in the secretory pathway, its effects on cherimoya physiology, and the evolution of this enzyme. Copyright © 2017. Published by Elsevier B.V.

Analyses of expression and localization of two mammalian-type transglutaminases in Physarum polycephalum, an acellular slime mold.

PubMed

Wada, Fumitaka; Ogawa, Atsuko; Hanai, Yuko; Nakamura, Akio; Maki, Masatoshi; Hitomi, Kiyotaka

2004-11-01

Transglutaminase (TGase) is an enzyme that modifies proteins by crosslinking or polyamination. Physarum polycephalum, an acellular slime mold, is the evolutionally lowest organism that has a mammalian-type transglutaminase. We have cloned a cDNA for Physarum polycephalum TGase (PpTGB), homologous to a previously identified TGase (PpTGA), whose sequence is similar to that of mammalian TGases. PpTGB encodes a primary sequence identical to that of PpTGA except for 11 amino acid residues at the N-terminus. Reverse transcription-PCR and Western blotting analyses showed that both PpTGA and PpTGB are expressed in microplasmodia and macroplasmodia during their life cycle, except for in sporangia. For biochemical characterization, we carried out the ectopical expressions of PpTGA and PpTGB in Dictyostelium discoideum. Subcellular fractionation of these Dictyostelium cells showed that the expressed PpTGA, but not PpTGB, localizes to the membrane fraction. Furthermore, in Physarum, subcellular fractionation and immunostaining indicated specific localization at the plasma membrane in macroplasmodia, while the localization was entirely cytoplasmic in microplasmodia.

A maize database resource that captures tissue-specific and subcellular-localized gene expression, via fluorescent tags and confocal imaging (Maize Cell Genomics Database).

PubMed

Krishnakumar, Vivek; Choi, Yongwook; Beck, Erin; Wu, Qingyu; Luo, Anding; Sylvester, Anne; Jackson, David; Chan, Agnes P

2015-01-01

Maize is a global crop and a powerful system among grain crops for genetic and genomic studies. However, the development of novel biological tools and resources to aid in the functional identification of gene sequences is greatly needed. Towards this goal, we have developed a collection of maize marker lines for studying native gene expression in specific cell types and subcellular compartments using fluorescent proteins (FPs). To catalog FP expression, we have developed a public repository, the Maize Cell Genomics (MCG) Database, (http://maize.jcvi.org/cellgenomics), to organize a large data set of confocal images generated from the maize marker lines. To date, the collection represents major subcellular structures and also developmentally important progenitor cell populations. The resource is available to the research community, for example to study protein localization or interactions under various experimental conditions or mutant backgrounds. A subset of the marker lines can also be used to induce misexpression of target genes through a transactivation system. For future directions, the image repository can be expanded to accept new image submissions from the research community, and to perform customized large-scale computational image analysis. This community resource will provide a suite of new tools for gaining biological insights by following the dynamics of protein expression at the subcellular, cellular and tissue levels. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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.

Measurement and correction of transverse chromatic offsets for multi-wavelength retinal microscopy in the living eye.

PubMed

Harmening, Wolf M; Tiruveedhula, Pavan; Roorda, Austin; Sincich, Lawrence C

2012-09-01

A special challenge arises when pursuing multi-wavelength imaging of retinal tissue in vivo, because the eye's optics must be used as the main focusing elements, and they introduce significant chromatic dispersion. Here we present an image-based method to measure and correct for the eye's transverse chromatic aberrations rapidly, non-invasively, and with high precision. We validate the technique against hyperacute psychophysical performance and the standard chromatic human eye model. In vivo correction of chromatic dispersion will enable confocal multi-wavelength images of the living retina to be aligned, and allow targeted chromatic stimulation of the photoreceptor mosaic to be performed accurately with sub-cellular resolution.

Measurement and correction of transverse chromatic offsets for multi-wavelength retinal microscopy in the living eye

PubMed Central

Harmening, Wolf M.; Tiruveedhula, Pavan; Roorda, Austin; Sincich, Lawrence C.

2012-01-01

A special challenge arises when pursuing multi-wavelength imaging of retinal tissue in vivo, because the eye’s optics must be used as the main focusing elements, and they introduce significant chromatic dispersion. Here we present an image-based method to measure and correct for the eye’s transverse chromatic aberrations rapidly, non-invasively, and with high precision. We validate the technique against hyperacute psychophysical performance and the standard chromatic human eye model. In vivo correction of chromatic dispersion will enable confocal multi-wavelength images of the living retina to be aligned, and allow targeted chromatic stimulation of the photoreceptor mosaic to be performed accurately with sub-cellular resolution. PMID:23024901

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

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.

Subcellular localization of the five members of the human steroid 5α-reductase family.

PubMed

Scaglione, Antonella; Montemiglio, Linda Celeste; Parisi, Giacomo; Asteriti, Italia Anna; Bruni, Renato; Cerutti, Gabriele; Testi, Claudia; Savino, Carmelinda; Mancia, Filippo; Lavia, Patrizia; Vallone, Beatrice

2017-06-01

In humans the steroid 5alpha-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in lipidic substrates: Δ 4 -3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum localization. We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.

Euk-mPLoc: a fusion classifier for large-scale eukaryotic protein subcellular location prediction by incorporating multiple sites.

PubMed

Chou, Kuo-Chen; Shen, Hong-Bin

2007-05-01

One of the critical challenges in predicting protein subcellular localization is how to deal with the case of multiple location sites. Unfortunately, so far, no efforts have been made in this regard except for the one focused on the proteins in budding yeast only. For most existing predictors, the multiple-site proteins are either excluded from consideration or assumed even not existing. Actually, proteins may simultaneously exist at, or move between, two or more different subcellular locations. For instance, according to the Swiss-Prot database (version 50.7, released 19-Sept-2006), among the 33,925 eukaryotic protein entries that have experimentally observed subcellular location annotations, 2715 have multiple location sites, meaning about 8% bearing the multiplex feature. Proteins with multiple locations or dynamic feature of this kind are particularly interesting because they may have some very special biological functions intriguing to investigators in both basic research and drug discovery. Meanwhile, according to the same Swiss-Prot database, the number of total eukaryotic protein entries (except those annotated with "fragment" or those with less than 50 amino acids) is 90,909, meaning a gap of (90,909-33,925) = 56,984 entries for which no knowledge is available about their subcellular locations. Although one can use the computational approach to predict the desired information for the blank, so far, all the existing methods for predicting eukaryotic protein subcellular localization are limited in the case of single location site only. To overcome such a barrier, a new ensemble classifier, named Euk-mPLoc, was developed that can be used to deal with the case of multiple location sites as well. Euk-mPLoc is freely accessible to the public as a Web server at http://202.120.37.186/bioinf/euk-multi. Meanwhile, to support the people working in the relevant areas, Euk-mPLoc has been used to identify all eukaryotic protein entries in the Swiss-Prot database that do not have subcellular location annotations or are annotated as being uncertain. The large-scale results thus obtained have been deposited at the same Web site via a downloadable file prepared with Microsoft Excel and named "Tab_Euk-mPLoc.xls". Furthermore, to include new entries of eukaryotic proteins and reflect the continuous development of Euk-mPLoc in both the coverage scope and prediction accuracy, we will timely update the downloadable file as well as the predictor, and keep users informed by publishing a short note in the Journal and making an announcement in the Web Page.

The N-terminal Set-β Protein Isoform Induces Neuronal Death*

PubMed Central

Trakhtenberg, Ephraim F.; Morkin, Melina I.; Patel, Karan H.; Fernandez, Stephanie G.; Sang, Alan; Shaw, Peter; Liu, Xiongfei; Wang, Yan; Mlacker, Gregory M.; Gao, Han; Velmeshev, Dmitry; Dombrowski, Susan M.; Vitek, Michael P.; Goldberg, Jeffrey L.

2015-01-01

Set-β protein plays different roles in neurons, but the diversity of Set-β neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-β are altered in Alzheimer disease, cleavage of Set-β leads to neuronal death after stroke, and the full-length Set-β regulates retinal ganglion cell (RGC) and hippocampal neuron axon growth and regeneration in a subcellular localization-dependent manner. Here we used various biochemical approaches to investigate Set-β isoforms and their role in the CNS, using the same type of neurons, RGCs, across studies. We found multiple alternatively spliced isoforms expressed from the Set locus in purified RGCs. Set transcripts containing the Set-β-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-β transcript lacking the nuclear localization signal and demonstrated that the full-length (∼39-kDa) Set-β is localized predominantly in the nucleus, whereas a shorter (∼25-kDa) Set-β isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-β cleavage product can induce neuronal death. PMID:25833944

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.

Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane.

PubMed

Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H; Sosinsky, Gina E

2014-01-01

Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.

Subcellular mechanism of Escherichia coli inactivation during electrochemical disinfection with boron-doped diamond anode: A comparative study of three electrolytes.

PubMed

Long, Yujiao; Ni, Jinren; Wang, Zuhui

2015-11-01

Although the identification of effective oxidant species has been extensively studied, yet the subcellular mechanism of bacterial inactivation has never been clearly elucidated in electrochemical disinfection processes. In this study, subcellular mechanism of Escherichia coli inactivation during electrochemical disinfection was revealed in terms of comprehensive factors such as cell morphology, total organic components, K(+) leakage, membrane permeability, lipid peroxidation, membrane potential, membrane proteins, intracellular enzyme, cellular ATP level and DNA. The electrolysis was conducted with boron-doped diamond anode in three electrolytes including chloride, sulfate and phosphate. Results demonstrated that cell inactivation was mainly attributed to damage to the intracellular enzymatic systems in chloride solution. In sulfate solution, certain essential membrane proteins like the K(+) ion transport systems were eliminated. Thus, the pronounced K(+) leakage from cytosol resulted in gradual collapse of the membrane potential, which would hinder the subcellular localization of cell division-related proteins as well as ATP synthesis and thereby lead to the bacterial inactivation. Remarkable lipid peroxidation was observed, while the intracellular damage was negligible. In phosphate solution, the cells sequentially underwent overall destruction as a whole cell with no captured intermediate state, during which the organic components of the cells were mostly subjected to mineralization. This study provided a thorough insight into the bacterial inactivation mechanism on the subcellular level. Copyright © 2015 Elsevier Ltd. All rights reserved.

Organelle-targeting surface-enhanced Raman scattering (SERS) nanosensors for subcellular pH sensing.

PubMed

Shen, Yanting; Liang, Lijia; Zhang, Shuqin; Huang, Dianshuai; Zhang, Jing; Xu, Shuping; Liang, Chongyang; Xu, Weiqing

2018-01-25

The pH value of subcellular organelles in living cells is a significant parameter in the physiological activities of cells. Its abnormal fluctuations are commonly believed to be associated with cancers and other diseases. Herein, a series of surface-enhanced Raman scattering (SERS) nanosensors with high sensitivity and targeting function was prepared for the quantification and monitoring of pH values in mitochondria, nucleus, and lysosome. The nanosensors were composed of gold nanorods (AuNRs) functionalized with a pH-responsive molecule (4-mercaptopyridine, MPy) and peptides that could specifically deliver the AuNRs to the targeting subcellular organelles. The localization of our prepared nanoprobes in specific organelles was confirmed by super-high resolution fluorescence imaging and bio-transmission electron microscopy (TEM) methods. By the targeting ability, the pH values of the specific organelles can be determined by monitoring the vibrational spectral changes of MPy with different pH values. Compared to the cases of reported lysosome and cytoplasm SERS pH sensors, more accurate pH values of mitochondria and nucleus, which could be two additional intracellular tracers for subcellular microenvironments, were disclosed by this SERS approach, further improving the accuracy of discrimination of related diseases. Our sensitive SERS strategy can also be employed to explore crucial physiological and biological processes that are related to subcellular pH fluctuations.

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.

Advanced Glycation End-Products affect transcription factors regulating insulin gene expression

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

Puddu, A., E-mail: alep100@hotmail.com; Storace, D.; Odetti, P.

2010-04-23

Advanced Glycation End-Products (AGEs) are generated by the covalent interaction of reducing sugars with proteins, lipids or nucleic acids. AGEs are implicated in diabetic complications and pancreatic {beta}-cell dysfunction. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs leads to a significant decrease of insulin secretion and content. Insulin gene transcription is positively regulated by the beta cell specific transcription factor PDX-1 (Pancreatic and Duodenal Homeobox-1). On the contrary, the forkhead transcription factor FoxO1 inhibits PDX-1 gene transcription. Activity of FoxO1 is regulated by post-translational modifications: phosphorylation deactivates FoxO1, and acetylation preventsmore » FoxO1 ubiquitination. In this work we investigated whether AGEs affect expression and subcellular localization of PDX-1 and FoxO1. HIT-T15 cells were cultured for 5 days in presence of AGEs. Cells were then lysed and processed for subcellular fractionation. We determined intracellular insulin content, then we assessed the expression and subcellular localization of PDX-1, FoxO1, phosphoFoxO1 and acetylFoxO1. As expected intracellular insulin content was lower in HIT-T15 cells cultured with AGEs. The results showed that AGEs decreased expression and nuclear localization of PDX-1, reduced phosphorylation of FoxO1, and increased expression and acetylation of FoxO1. These results suggest that AGEs decrease insulin content unbalancing transcription factors regulating insulin gene expression.« less

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.

Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.

PubMed Central

D'Angelo, Maximiliano A; Sanguineti, Santiago; Reece, Jeffrey M; Birnbaumer, Lutz; Torres, Héctor N; Flawiá, Mirtha M

2004-01-01

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites. PMID:14556647

Subcellular localization of leptin and leptin receptor in breast cancer detected in an electron microscopic study.

PubMed

Al-Shibli, Saad M; Amjad, Nasser M; Al-Kubaisi, Muna K; Mizan, Shaikh

2017-01-22

Leptin (LEP) and leptin receptor (LEPR) have long been found associated with breast cancer. So far no high-resolution method such as electron microscopy has been used to investigate the subcellular localization of leptin and leptin receptor in breast cancer. We collected cancer and non-cancer breast tissues from 51 women with invasive ductal breast cancer. Leptin and leptin receptor in the tissues were estimated using immunohistochemistry (IHC). LEP and LEPR were localized at subcellular level by immunocytochemistry (ICC) using ultra-fine gold particle conjugated antibody, and visualized with transmission electron microscopy (TEM). IHC showed high presence of LEP and LEPR in 65% and 67% respectively of the breast cancer samples, 100% and 0% respectively of the adipose tissue samples, and no high presence in the non-cancer breast tissue samples. On TEM views both LEP and LEPR were found highly concentrated within the nucleus of the cancer cells, indicating that nucleus is the principal seat of action. However, presence of high concentration of LEP does not necessarily prove its over-expression, as often concluded, because LEP could be internalized from outside by LEPR in the cells. In contrast, LEPR is definitely over-expressed in the ductal breast cancer cells. Therefore, we hypothesize that over-expression of LEPR, rather than that of LEP has a fundamental role in breast carcinogenesis in particular, and probably for LEP-LEPR associated tumors in general. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

mPLR-Loc: an adaptive decision multi-label classifier based on penalized logistic regression for protein subcellular localization prediction.

PubMed

Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

2015-03-15

Proteins located in appropriate cellular compartments are of paramount importance to exert their biological functions. Prediction of protein subcellular localization by computational methods is required in the post-genomic era. Recent studies have been focusing on predicting not only single-location proteins but also multi-location proteins. However, most of the existing predictors are far from effective for tackling the challenges of multi-label proteins. This article proposes an efficient multi-label predictor, namely mPLR-Loc, based on penalized logistic regression and adaptive decisions for predicting both single- and multi-location proteins. Specifically, for each query protein, mPLR-Loc exploits the information from the Gene Ontology (GO) database by using its accession number (AC) or the ACs of its homologs obtained via BLAST. The frequencies of GO occurrences are used to construct feature vectors, which are then classified by an adaptive decision-based multi-label penalized logistic regression classifier. Experimental results based on two recent stringent benchmark datasets (virus and plant) show that mPLR-Loc remarkably outperforms existing state-of-the-art multi-label predictors. In addition to being able to rapidly and accurately predict subcellular localization of single- and multi-label proteins, mPLR-Loc can also provide probabilistic confidence scores for the prediction decisions. For readers' convenience, the mPLR-Loc server is available online (http://bioinfo.eie.polyu.edu.hk/mPLRLocServer). Copyright © 2014 Elsevier Inc. All rights reserved.

Subcellular characteristics of functional intracellular renin–angiotensin systems☆

PubMed Central

Abadir, Peter M.; Walston, Jeremy D.; Carey, Robert M.

2013-01-01

The renin–angio tensin system (RAS) is now regarded as an integral component in not only the development of hypertension, but also in physiologic and pathophysiologic mechanisms in multiple tissues and chronic disease states. While many of the endocrine (circulating), paracrine (cell-to-different cell) and autacrine (cell-to-same cell) effects of the RAS are believed to be mediated through the canonical extracellular RAS, a complete, independent and differentially regulated intracellular RAS (iRAS) has also been proposed. Angiotensinogen, the enzymes renin and angiotensin-converting enzyme (ACE) and the angiotensin peptides can all be synthesized and retained intracellularly. Angiotensin receptors (types I and 2) are also abundant intracellularly mainly at the nuclear and mitochondrial levels. The aim of this review is to focus on the most recent information concerning the subcellular localization, distribution and functions of the iRAS and to discuss the potential consequences of activation of the subcellular RAS on different organ systems. PMID:23032352

Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus

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

Horie, Ryo; Yoneda, Misako, E-mail: yone@ims.u-tok

Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21–24 and 110–139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60–75 and 72–75 were importantmore » for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm. -- Highlights: •Nipah virus (NiV) infection resulted in high mortality, but effective treatment has not been established. •Several reports revealed that NiV nonstructural C protein (NiV-C) was essential for NiV pathogenicity, however, whole of NiV-C function is still unknown. •Although nonstructural C proteins of other Paramyxoviruses are expressed in similar mechanism and exert similar activity, subcellular localization and cellular targets are different. In this study, we evaluated the subcellular localization of NiV-C. •To our knowledge, this is the first report showing that NiV-C shuttles between the nucleus and cytoplasm. We also clarified that NiV-C has nuclear export signal and nuclear localization signal using NiV-C deleted, alanine substitution mutants and enhanced green fluorescent protein (EGFP) fused proteins. •And we also showed that interferon (IFN) antagonist activity of NiV-C related to its subcellular localization. Our results indicate that NiV-C exert IFN antagonist activity in the cytoplasm.« less

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 localization of transiently expressed fluorescent fusion proteins.

PubMed

Collings, David A

2013-01-01

The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system.

Subcellular localization of the Snf1 kinase is regulated by specific β subunits and a novel glucose signaling mechanism

PubMed Central

Vincent, Olivier; Townley, Robert; Kuchin, Sergei; Carlson, Marian

2001-01-01

The Snf1/AMP-activated protein kinase family has broad roles in transcriptional, metabolic, and developmental regulation in response to stress. In Saccharomyces cerevisiae, Snf1 is required for the response to glucose limitation. Snf1 kinase complexes contain the α (catalytic) subunit Snf1, one of the three related β subunits Gal83, Sip1, or Sip2, and the γ subunit Snf4. We present evidence that the β subunits regulate the subcellular localization of the Snf1 kinase. Green fluorescent protein fusions to Gal83, Sip1, and Sip2 show different patterns of localization to the nucleus, vacuole, and/or cytoplasm. We show that Gal83 directs Snf1 to the nucleus in a glucose-regulated manner. We further identify a novel signaling pathway that controls this nuclear localization in response to glucose phosphorylation. This pathway is distinct from the glucose signaling pathway that inhibits Snf1 kinase activity and responds not only to glucose but also to galactose and sucrose. Such independent regulation of the localization and the activity of the Snf1 kinase, combined with the distinct localization of kinases containing different β subunits, affords versatility in regulating physiological responses. PMID:11331606

Localization of A-type K+ channel subunit Kv4.2 in rat brain.

PubMed

Tsaur, M L; Wu, Y L; Huang, F L; Shih, Y H

2001-09-30

Kv4.2, a voltage-gated K+ (Kv) channel subunit, has been suggested to be the key component of the subthreshold A-type K+ currents (I(SA)s) recorded from the specific subcellular compartments of certain CNS neurons. To correlate Kv4.2 localization with the I(SA)s detected, immunohistochemistry will be useful. Although the Kv4.2 immunostaining pattern in the hippocampus and cerebellum has been reported, the Kv4.2 antibody used was not specific. Furthermore, Kv4.2 localization in other brain regions remains unclear. In this report, we first demonstrated the specificity of a new Kv4.2 antibody, and then used it to examine Kv4.2 localization throughout adult rat brain by immunohistochemistry. At the cellular level, Kv4.2 was found in neurons but not glias. At the subcellular level, Kv4.2 was localized in the somatodendritic compartment of most neurons examined. Nevertheless, our preliminary data indicated that Kv4.2 might be also present in the axon/terminal compartment. At the functional level, our data indicates that Kv4.2 localization and I(SA) correlate quite well in some CNS neurons, supporting that Kv4.2 is the key component of some I(SA)s recorded in vivo.

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

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.

Subcellular Localization and Biochemical Comparison of Cytosolic and Secreted Cytokinin Dehydrogenase Enzymes from Maize

USDA-ARS?s Scientific Manuscript database

Cytokinin dehydrogenase (CKX, EC 1.5.99.12) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in cells of each plant. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a t...

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase in nuclei and rimmed vacuoles of muscle fibers in DMRV (distal myopathy with rimmed vacuoles).

PubMed

Ishihara, Shoichiro; Tomimitsu, Hiroyuki; Fujigasaki, Hiroto; Saito, Fumiaki; Mizusawa, Hidehiro

2008-03-01

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key molecule in the pathogenesis of distal myopathy with rimmed vacuoles (DMRV) and hereditary inclusion body myopathy (HIBM) and almost all such patients have some mutations in GNE. However, subcellular localization of GNE and the mechanism of muscular damage have not been clarified. A rabbit polyclonal antibody for GNE was prepared. Immunohistochemistry was performed using anti-GNE and anti-nuclear protein antibodies. Western blotting with subcellular fractionated proteins was performed to determine subcellular localization of GNE. The sizes of myonuclei were quantified in muscle biopsies from patients with DMRV and amyotrophic lateral sclerosis (ALS). In DMRV muscles, immunohistochemistry identified GNE in sarcoplasm and specifically in myonuclei and rimmed vacuoles (RV). Nuclear proteins were also found in RVs. Immunohistochemistry showed colocalization of GNE and emerin in C2C12 cells. Western blotting revealed the presence of GNE in nuclear fractions of human embryonic kidney (HEK) 293T cells. The mean size of myonuclei of DMRV was significantly larger than that of ALS. GNE is present in myonuclei near nuclear membrane. Our results suggest that myonuclei are involved in RV formation in DMRV, and that mutant GNE in myonuclei seems to play some role in this process.

Motion compensation for in vivo subcellular optical microscopy.

PubMed

Lucotte, B; Balaban, R S

2014-04-01

In this review, we focus on the impact of tissue motion on attempting to conduct subcellular resolution optical microscopy, in vivo. Our position is that tissue motion is one of the major barriers in conducting these studies along with light induced damage, optical probe loading as well as absorbing and scattering effects on the excitation point spread function and collection of emitted light. Recent developments in the speed of image acquisition have reached the limit, in most cases, where the signal from a subcellular voxel limits the speed and not the scanning rate of the microscope. Different schemes for compensating for tissue displacements due to rigid body and deformation are presented from tissue restriction, gating, adaptive gating and active tissue tracking. We argue that methods that minimally impact the natural physiological motion of the tissue are desirable because the major reason to perform in vivo studies is to evaluate normal physiological functions. Towards this goal, active tracking using the optical imaging data itself to monitor tissue displacement and either prospectively or retrospectively correct for the motion without affecting physiological processes is desirable. Critical for this development was the implementation of near real time image processing in conjunction with the control of the microscope imaging parameters. Clearly, the continuing development of methods of motion compensation as well as significant technological solutions to the other barriers to tissue subcellular optical imaging in vivo, including optical aberrations and overall signal-to-noise ratio, will make major contributions to the understanding of cell biology within the body.

Proteome-wide identification of predominant subcellular protein localizations in a bacterial model organism.

PubMed

Stekhoven, Daniel J; Omasits, Ulrich; Quebatte, Maxime; Dehio, Christoph; Ahrens, Christian H

2014-03-17

Proteomics data provide unique insights into biological systems, including the predominant subcellular localization (SCL) of proteins, which can reveal important clues about their functions. Here we analyzed data of a complete prokaryotic proteome expressed under two conditions mimicking interaction of the emerging pathogen Bartonella henselae with its mammalian host. Normalized spectral count data from cytoplasmic, total membrane, inner and outer membrane fractions allowed us to identify the predominant SCL for 82% of the identified proteins. The spectral count proportion of total membrane versus cytoplasmic fractions indicated the propensity of cytoplasmic proteins to co-fractionate with the inner membrane, and enabled us to distinguish cytoplasmic, peripheral inner membrane and bona fide inner membrane proteins. Principal component analysis and k-nearest neighbor classification training on selected marker proteins or predominantly localized proteins, allowed us to determine an extensive catalog of at least 74 expressed outer membrane proteins, and to extend the SCL assignment to 94% of the identified proteins, including 18% where in silico methods gave no prediction. Suitable experimental proteomics data combined with straightforward computational approaches can thus identify the predominant SCL on a proteome-wide scale. Finally, we present a conceptual approach to identify proteins potentially changing their SCL in a condition-dependent fashion. The work presented here describes the first prokaryotic proteome-wide subcellular localization (SCL) dataset for the emerging pathogen B. henselae (Bhen). The study indicates that suitable subcellular fractionation experiments combined with straight-forward computational analysis approaches assessing the proportion of spectral counts observed in different subcellular fractions are powerful for determining the predominant SCL of a large percentage of the experimentally observed proteins. This includes numerous cases where in silico prediction methods do not provide any prediction. Avoiding a treatment with harsh conditions, cytoplasmic proteins tend to co-fractionate with proteins of the inner membrane fraction, indicative of close functional interactions. The spectral count proportion (SCP) of total membrane versus cytoplasmic fractions allowed us to obtain a good indication about the relative proximity of individual protein complex members to the inner membrane. Using principal component analysis and k-nearest neighbor approaches, we were able to extend the percentage of proteins with a predominant experimental localization to over 90% of all expressed proteins and identified a set of at least 74 outer membrane (OM) proteins. In general, OM proteins represent a rich source of candidates for the development of urgently needed new therapeutics in combat of resurgence of infectious disease and multi-drug resistant bacteria. Finally, by comparing the data from two infection biology relevant conditions, we conceptually explore methods to identify and visualize potential candidates that may partially change their SCL in these different conditions. The data are made available to researchers as a SCL compendium for Bhen and as an assistance in further improving in silico SCL prediction algorithms. Copyright © 2014 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.

Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif.

PubMed

Hernández-Sánchez, Itzell E; Maruri-López, Israel; Ferrando, Alejandro; Carbonell, Juan; Graether, Steffen P; Jiménez-Bremont, Juan F

2015-01-01

The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization.

Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif

PubMed Central

Hernández-Sánchez, Itzell E.; Maruri-López, Israel; Ferrando, Alejandro; Carbonell, Juan; Graether, Steffen P.; Jiménez-Bremont, Juan F.

2015-01-01

The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization. PMID:26442018

Geometric approach to segmentation and protein localization in cell culture assays.

PubMed

Raman, S; Maxwell, C A; Barcellos-Hoff, M H; Parvin, B

2007-01-01

Cell-based fluorescence imaging assays are heterogeneous and require the collection of a large number of images for detailed quantitative analysis. Complexities arise as a result of variation in spatial nonuniformity, shape, overlapping compartments and scale (size). A new technique and methodology has been developed and tested for delineating subcellular morphology and partitioning overlapping compartments at multiple scales. This system is packaged as an integrated software platform for quantifying images that are obtained through fluorescence microscopy. Proposed methods are model based, leveraging geometric shape properties of subcellular compartments and corresponding protein localization. From the morphological perspective, convexity constraint is imposed to delineate and partition nuclear compartments. From the protein localization perspective, radial symmetry is imposed to localize punctate protein events at submicron resolution. Convexity constraint is imposed against boundary information, which are extracted through a combination of zero-crossing and gradient operator. If the convexity constraint fails for the boundary then positive curvature maxima are localized along the contour and the entire blob is partitioned into disjointed convex objects representing individual nuclear compartment, by enforcing geometric constraints. Nuclear compartments provide the context for protein localization, which may be diffuse or punctate. Punctate signal are localized through iterative voting and radial symmetries for improved reliability and robustness. The technique has been tested against 196 images that were generated to study centrosome abnormalities. Corresponding computed representations are compared against manual counts for validation.

Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins

PubMed Central

Mosca, Timothy J; Luo, Liqun

2014-01-01

Understanding information flow through neuronal circuits requires knowledge of their synaptic organization. In this study, we utilized fluorescent pre- and postsynaptic markers to map synaptic organization in the Drosophila antennal lobe, the first olfactory processing center. Olfactory receptor neurons (ORNs) produce a constant synaptic density across different glomeruli. Each ORN within a class contributes nearly identical active zone number. Active zones from ORNs, projection neurons (PNs), and local interneurons have distinct subglomerular and subcellular distributions. The correct number of ORN active zones and PN acetylcholine receptor clusters requires the Teneurins, conserved transmembrane proteins involved in neuromuscular synapse organization and synaptic partner matching. Ten-a acts in ORNs to organize presynaptic active zones via the spectrin cytoskeleton. Ten-m acts in PNs autonomously to regulate acetylcholine receptor cluster number and transsynaptically to regulate ORN active zone number. These studies advanced our ability to assess synaptic architecture in complex CNS circuits and their underlying molecular mechanisms. DOI: http://dx.doi.org/10.7554/eLife.03726.001 PMID:25310239

A moving view: subcellular trafficking processes in pattern recognition receptor-triggered plant immunity.

PubMed

Ben Khaled, Sara; Postma, Jelle; Robatzek, Silke

2015-01-01

A significant challenge for plants is to induce localized defense responses at sites of pathogen attack. Therefore, host subcellular trafficking processes enable accumulation and exchange of defense compounds, which contributes to the plant on-site defenses in response to pathogen perception. This review summarizes our current understanding of the transport processes that facilitate immunity, the significance of which is highlighted by pathogens reprogramming membrane trafficking through host cell translocated effectors. Prominent immune-related cargos of plant trafficking pathways are the pattern recognition receptors (PRRs), which must be present at the plasma membrane to sense microbes in the apoplast. We focus on the dynamic localization of the FLS2 receptor and discuss the pathways that regulate receptor transport within the cell and their link to FLS2-mediated immunity. One emerging theme is that ligand-induced late endocytic trafficking is conserved across different PRR protein families as well as across different plant species.

Development of redox-sensitive red fluorescent proteins for imaging redox dynamics in cellular compartments.

PubMed

Fan, Yichong; Ai, Hui-wang

2016-04-01

We recently reported a redox-sensitive red fluorescent protein, rxRFP1, which is one of the first genetically encoded red-fluorescent probes for general redox states in living cells. As individual cellular compartments have different basal redox potentials, we hereby describe a group of rxRFP1 mutants, showing different midpoint redox potentials for detection of redox dynamics in various subcellular domains, such as mitochondria, the cell nucleus, and endoplasmic reticulum (ER). When these redox probes were expressed and subcellularly localized in human embryonic kidney (HEK) 293 T cells, they responded to membrane-permeable oxidants and reductants. In addition, a mitochondrially localized rxRFP1 mutant, Mito-rxRFP1.1, was used to detect mitochondrial oxidative stress induced by doxorubicin-a widely used cancer chemotherapy drug. Our work has expanded the fluorescent protein toolkit with new research tools for studying compartmentalized redox dynamics and oxidative stress under various pathophysiological conditions.

Determining the Localization of Carbohydrate Active Enzymes Within Gram-Negative Bacteria.

PubMed

McLean, Richard; Inglis, G Douglas; Mosimann, Steven C; Uwiera, Richard R E; Abbott, D Wade

2017-01-01

Investigating the subcellular location of secreted proteins is valuable for illuminating their biological function. Although several bioinformatics programs currently exist to predict the destination of a trafficked protein using its signal peptide sequence, these programs have limited accuracy and often require experimental validation. Here, we present a systematic method to fractionate gram-negative cells and characterize the subcellular localization of secreted carbohydrate active enzymes (CAZymes). This method involves four parallel approaches that reveal the relative abundance of protein within the cytoplasm, periplasm, outer membrane, and extracellular environment. Cytoplasmic and periplasmic proteins are fractionated by lysis and osmotic shock, respectively. Outer membrane bound proteins are determined by comparing cells before and after exoproteolytic digestion. Extracellularly secreted proteins are collected from the media and concentrated. These four different fractionations can then be probed for the presence and quantity of target proteins using immunochemical methods such as Western blots and ELISAs, or enzyme activity assays.

Subcellular localization and expression pattern of the neurofibromatosis type 2 protein merlin/schwannomin.

PubMed

Schmucker, B; Ballhausen, W G; Kressel, M

1997-01-01

To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the expression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. In addition, we detected higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa. Although the merlin bands of 100 kDa and 110 kDa may represent homo- or heterodimers, oligomerization due to formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa were quantitatively extractable in Lubrol WX, indicating a localization in or close to the plasma membrane. The 45 kDa band, however, was not soluble in Lubrol WX compatible with a localization of this NF2 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (i) perinuclear in a compartment resembling endoplasmic reticulum, (ii) in ruffling membranes and at the leading edges, (iii) in filopodia, and (iv) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the regulation of cell growth by interaction with cytoskeleton-associated proteins.

SMYD3 interacts with HTLV-1 Tax and regulates subcellular localization of Tax.

PubMed

Yamamoto, Keiyu; Ishida, Takaomi; Nakano, Kazumi; Yamagishi, Makoto; Yamochi, Tadanori; Tanaka, Yuetsu; Furukawa, Yoichi; Nakamura, Yusuke; Watanabe, Toshiki

2011-01-01

HTLV-1 Tax deregulates signal transduction pathways, transcription of genes, and cell cycle regulation of host cells, which is mainly mediated by its protein-protein interactions with host cellular factors. We previously reported an interaction of Tax with a histone methyltransferase (HMTase), SUV39H1. As the interaction was mediated by the SUV39H1 SET domain that is shared among HMTases, we examined the possibility of Tax interaction with another HMTase, SMYD3, which methylates histone H3 lysine 4 and activates transcription of genes, and studied the functional effects. Expression of endogenous SMYD3 in T cell lines and primary T cells was confirmed by immunoblotting analysis. Co-immuno-precipitaion assays and in vitro pull-down assay indicated interaction between Tax and SMYD3. The interaction was largely dependent on the C-terminal 180 amino acids of SMYD3, whereas the interacting domain of Tax was not clearly defined, although the N-terminal 108 amino acids were dispensable for the interaction. In the cotransfected cells, colocalization of Tax and SMYD3 was indicated in the cytoplasm or nuclei. Studies using mutants of Tax and SMYD3 suggested that SMYD3 dominates the subcellular localization of Tax. Reporter gene assays showed that nuclear factor-κB activation promoted by cytoplasmic Tax was enhanced by the presence of SMYD3, and attenuated by shRNA-mediated knockdown of SMYD3, suggesting an increased level of Tax localization in the cytoplasm by SMYD3. Our study revealed for the first time Tax-SMYD3 direct interaction, as well as apparent tethering of Tax by SMYD3, influencing the subcellular localization of Tax. Results suggested that SMYD3-mediated nucleocytoplasmic shuttling of Tax provides one base for the pleiotropic effects of Tax, which are mediated by the interaction of cellular proteins localized in the cytoplasm or nucleus. © 2010 Japanese Cancer Association.

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

Astrocyte-neuron crosstalk regulates the expression and subcellular localization of carbohydrate metabolism enzymes.

PubMed

Mamczur, Piotr; Borsuk, Borys; Paszko, Jadwiga; Sas, Zuzanna; Mozrzymas, Jerzy; Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

2015-02-01

Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes. © 2014 Wiley Periodicals, Inc.

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

PubMed

Fabrick, Jeffrey A; Hull, J Joe

2017-04-20

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Tau regulates the subcellular localization of calmodulin

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

Barreda, Elena Gomez de; Avila, Jesus, E-mail: javila@cbm.uam.es; CIBER de Enfermedades Neurodegenerativas, 28031 Madrid

Highlights: {yields} In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in amore » change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.« 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.

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.

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.

Subcellular targeting of p33ING1b by phosphorylation-dependent 14-3-3 binding regulates p21WAF1 expression.

PubMed

Gong, Wei; Russell, Michael; Suzuki, Keiko; Riabowol, Karl

2006-04-01

ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33(ING1b) splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33(ING1b) protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33(ING1b) increased levels of the p21(Waf1) cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21(Waf1) by p33(ING1b), consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33(ING1b) by directing its subcellular localization.

Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

PubMed

Foureau, E; Carqueijeiro, I; Dugé de Bernonville, T; Melin, C; Lafontaine, F; Besseau, S; Lanoue, A; Papon, N; Oudin, A; Glévarec, G; Clastre, M; St-Pierre, B; Giglioli-Guivarc'h, N; Courdavault, V

2016-01-01

Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform. © 2016 Elsevier Inc. All rights reserved.

Kif4 Is Essential for Mouse Oocyte Meiosis.

PubMed

Camlin, Nicole J; McLaughlin, Eileen A; Holt, Janet E

2017-01-01

Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age.

Nuclear localization signal regulates porcine circovirus type 2 capsid protein nuclear export through phosphorylation.

PubMed

Hou, Qiang; Hou, Shaohua; Chen, Qing; Jia, Hong; Xin, Ting; Jiang, Yitong; Guo, Xiaoyu; Zhu, Hongfei

2018-02-15

The open reading frame 2 (ORF2) of Porcine circovirus type 2 (PCV2) encodes the major Capsid (Cap) protein, which self-assembles into virus-like particle (VLP) of similar morphology to the PCV2 virion and accumulates in the nucleus through the N-terminal arginine-rich nuclear localization signal (NLS). In this study, PCV2 Cap protein and its derivates were expressed via the baculovirus expression system, and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry. Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import, and a phosphorylation site (S17) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the NLS domain to regulate Cap protein nuclear export. Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy. Moreover, the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells. Phosphorylation of NLS regulating Cap protein nuclear export provides more detailed knowledge of the PCV2 viral life cycle. Copyright © 2018 Elsevier B.V. All rights reserved.

Subcellular Fractionation and Localization Studies Reveal a Direct Interaction of the Fragile X Mental Retardation Protein (FMRP) with Nucleolin

PubMed Central

Taha, Mohamed S.; Nouri, Kazem; Milroy, Lech G.; Moll, Jens M.; Herrmann, Christian; Brunsveld, Luc; Piekorz, Roland P.; Ahmadian, Mohammad R.

2014-01-01

Fragile X mental Retardation Protein (FMRP) is a well-known regulator of local translation of its mRNA targets in neurons. However, despite its ubiquitous expression, the role of FMRP remains ill-defined in other cell types. In this study we investigated the subcellular distribution of FMRP and its protein complexes in HeLa cells using confocal imaging as well as detergent-free fractionation and size exclusion protocols. We found FMRP localized exclusively to solid compartments, including cytosolic heavy and light membranes, mitochondria, nuclear membrane and nucleoli. Interestingly, FMRP was associated with nucleolin in both a high molecular weight ribosomal and translation-associated complex (≥6 MDa) in the cytosol, and a low molecular weight complex (∼200 kDa) in the nucleoli. Consistently, we identified two functional nucleolar localization signals (NoLSs) in FMRP that are responsible for a strong nucleolar colocalization of the C-terminus of FMRP with nucleolin, and a direct interaction of the N-terminus of FMRP with the arginine-glycine-glycine (RGG) domain of nucleolin. Taken together, we propose a novel mechanism by which a transient nucleolar localization of FMRP underlies a strong nucleocytoplasmic translocation, most likely in a complex with nucleolin and possibly ribosomes, in order to regulate translation of its target mRNAs. PMID:24658146

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

PubMed

Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

1999-03-05

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

Direct measurement of local material properties within living embryonic tissues

NASA Astrophysics Data System (ADS)

Serwane, Friedhelm; Mongera, Alessandro; Rowghanian, Payam; Kealhofer, David; Lucio, Adam; Hockenbery, Zachary; Campàs, Otger

The shaping of biological matter requires the control of its mechanical properties across multiple scales, ranging from single molecules to cells and tissues. Despite their relevance, measurements of the mechanical properties of sub-cellular, cellular and supra-cellular structures within living embryos pose severe challenges to existing techniques. We have developed a technique that uses magnetic droplets to measure the mechanical properties of complex fluids, including in situ and in vivo measurements within living embryos ,across multiple length and time scales. By actuating the droplets with magnetic fields and recording their deformation we probe the local mechanical properties, at any length scale we choose by varying the droplets' diameter. We use the technique to determine the subcellular mechanics of individual blastomeres of zebrafish embryos, and bridge the gap to the tissue scale by measuring the local viscosity and elasticity of zebrafish embryonic tissues. Using this technique, we show that embryonic zebrafish tissues are viscoelastic with a fluid-like behavior at long time scales. This technique will enable mechanobiology and mechano-transduction studies in vivo, including the study of diseases correlated with tissue stiffness, such as cancer.

Guidelines for the Use of Protein Domains in Acidic Phospholipid Imaging.

PubMed

Platre, Matthieu Pierre; Jaillais, Yvon

2016-01-01

Acidic phospholipids are minor membrane lipids but critically important for signaling events. The main acidic phospholipids are phosphatidylinositol phosphates (PIPs also known as phosphoinositides), phosphatidylserine (PS), and phosphatidic acid (PA). Acidic phospholipids are precursors of second messengers of key signaling cascades or are second messengers themselves. They regulate the localization and activation of many proteins, and are involved in virtually all membrane trafficking events. As such, it is crucial to understand the subcellular localization and dynamics of each of these lipids within the cell. Over the years, several techniques have emerged in either fixed or live cells to analyze the subcellular localization and dynamics of acidic phospholipids. In this chapter, we review one of them: the use of genetically encoded biosensors that are based on the expression of specific lipid binding domains (LBDs) fused to fluorescent proteins. We discuss how to design such sensors, including the criteria for selecting the lipid binding domains of interest and to validate them. We also emphasize the care that must be taken during data analysis as well as the main limitations and advantages of this approach.

In silico cloning, expression of Rieske-like apoprotein gene and protein subcellular localization in the Pacific oyster, Crassostrea gigas.

PubMed

He, Xiaocui; Zhang, Yang; Yu, Ziniu

2010-10-01

Rieske protein gene in the Pacific oyster Crassostrea gigas was obtained by in silico cloning for the first time, and its expression profiles and subcellular localization were determined, respectively. The full-length cDNA of Cgisp is 985 bp in length and contains a 5'- and 3'-untranslated regions of 35 and 161 bp, respectively, with an open reading frame of 786 bp encoding a protein of 262 amino acids. The predicted molecular weight of 30 kDa of Cgisp protein was verified by prokaryotic expression. Conserved Rieske [2Fe-2S] cluster binding sites and highly matched-pair tertiary structure with 3CWB_E (Gallus gallus) were revealed by homologous analysis and molecular modeling. Eleven putative SNP sites and two conserved hexapeptide sequences, box I (THLGC) and II (PCHGS), were detected by multiple alignments. Real-time PCR analysis showed that Cgisp is expressed in a wide range of tissues, with adductor muscle exhibiting the top expression level, suggesting its biological function of energy transduction. The GFP tagging Cgisp indicated a mitochondrial localization, further confirming its physiological function.

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.

[Optimization of labeling and localizing bacterial membrane and nucleus with FM4-64 and Hoechst dyes].

PubMed

Wang, Jing; Han, Yanping; Yang, Ruifu; Zhao, Xingxu

2015-08-04

To observe cell membrane and nucleus in bacteria for subcellular localization. FM4-64 and Hoechst were dyed that can label cell membrane and nucleus, respectively. Both dyes were used to co-stain the membranes and nucleus of eight bacterial strains ( Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Yersinia pestis, Legionella pneumonia, Vibrio cholerae and Bacillus anthracis). E. coli was dyed with different dye concentrations and times and then observed by confocal fluorescence microscopic imaging. Fluorescence intensity of cell membrane and nucleus is affected by dye concentrations and times. The optimal conditions were determined as follows: staining cell membrane with 20 μg/mL FM4-64 for 1 min and cell nucleus with 20 μg/mL Hoechst for 20 min. Gram-negative bacteria were dyed better than gram-positive bacteria with FM4-64dye. FM4-64 and Hoechst can be used to stain membrane and nucleus in different types of bacteria. Co-staining bacterial membrane and nucleus provides the reference to observe cell structure in prokaryotes for studying subcellular localization.

Xuhuai goat H-FABP gene clone, subcellular localization of expression products and the preparation of transgenic mice.

PubMed

Yin, Yan-hui; Li, Bi-chun; Wei, Guang-hui; Zhu, Cai-ye; Li, Wei; Zhang, Ya-ni; Du, Li-xin; Cao, Wen-guang

2012-05-01

The aim of this study was to clone the heart-type fatty acid binding protein (H-FABP) gene of Xuhuai goat, to explore it bioinformatically, and analyze the subcellular localization using enhanced green fluorescent protein (EGFP). The results showed that the coding sequence (CDS) length of Xuhuai goat H-FABP gene was 402 bp, encoding 133 amino acids (GenBank accession number AY466498.1). The H-FABP cDNA coding sequence was compared with the corresponding region of human, chicken, brown rat, cow, wild boar, donkey, and zebrafish. The similarity were 89%, 76%, 85%, 84%, 93%, 91%, 70%, respectively. For the corresponding amino acid sequences, the similarity were 90%, 79%, 88%, 97%, 95%, 94%, 72%, respectively. This study did not find the signal peptide region in the H-FABP protein; it revealed that H-FABP protein might be a nonsecreted protein. H-FABP expression was detected in vitro by reverse transcription-polymerase chain reaction (RT-PCR), and the EGFP-H-FABP fusion protein was localized to the cytoplasm. The gene could also be transiently and permanently expressed in mice.

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.

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

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.

Methods for Detection of Mitochondrial and Cellular Reactive Oxygen Species

PubMed Central

Harrison, David G.

2014-01-01

Abstract Significance: Mitochondrial and cellular reactive oxygen species (ROS) play important roles in both physiological and pathological processes. Different ROS, such as superoxide (O2•−), hydrogen peroxide, and peroxynitrite (ONOO•−), stimulate distinct cell-signaling pathways and lead to diverse outcomes depending on their amount and subcellular localization. A variety of methods have been developed for ROS detection; however, many of these methods are not specific, do not allow subcellular localization, and can produce artifacts. In this review, we will critically analyze ROS detection and present advantages and the shortcomings of several available methods. Recent Advances: In the past decade, a number of new fluorescent probes, electron-spin resonance approaches, and immunoassays have been developed. These new state-of-the-art methods provide improved selectivity and subcellular resolution for ROS detection. Critical Issues: Although new methods for HPLC superoxide detection, application of fluorescent boronate-containing probes, use of cell-targeted hydroxylamine spin probes, and immunospin trapping have been available for several years, there has been lack of translation of these into biomedical research, limiting their widespread use. Future Directions: Additional studies to translate these new technologies from the test tube to physiological applications are needed and could lead to a wider application of these approaches to study mitochondrial and cellular ROS. Antioxid. Redox Signal. 20, 372–382. PMID:22978713

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.

Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*

PubMed Central

Traenkle, Bjoern; Emele, Felix; Anton, Roman; Poetz, Oliver; Haeussler, Ragna S.; Maier, Julia; Kaiser, Philipp D.; Scholz, Armin M.; Nueske, Stefan; Buchfellner, Andrea; Romer, Tina; Rothbauer, Ulrich

2015-01-01

β-catenin is the key component of the canonical Wnt pathway and plays a crucial role in a multitude of developmental and homeostatic processes. The different tasks of β-catenin are orchestrated by its subcellular localization and participation in multiprotein complexes. To gain a better understanding of β-catenin's role in living cells we have generated a new set of single domain antibodies, referred to as nanobodies, derived from heavy chain antibodies of camelids. We selected nanobodies recognizing the N-terminal, core or C-terminal domain of β-catenin and applied these new high-affinity binders as capture molecules in sandwich immunoassays and co-immunoprecipitations of endogenous β-catenin complexes. In addition, we engineered intracellularly functional anti-β-catenin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. For the first time, we were able to visualize the subcellular localization and nuclear translocation of endogenous β-catenin in living cells using these chromobodies. Moreover, the chromobody signal allowed us to trace the accumulation of diffusible, hypo-phosphorylated β-catenin in response to compound treatment in real time using High Content Imaging. The anti-β-catenin nanobodies and chromobodies characterized in this study are versatile tools that enable a novel and unique approach to monitor the dynamics of subcellular β-catenin in biochemical and cell biological assays. PMID:25595278

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.

Quality Control Pathways for Nucleus-Encoded Eukaryotic tRNA Biosynthesis and Subcellular Trafficking

PubMed Central

Huang, Hsiao-Yun

2015-01-01

tRNAs perform an essential role in translating the genetic code. They are long-lived RNAs that are generated via numerous posttranscriptional steps. Eukaryotic cells have evolved numerous layers of quality control mechanisms to ensure that the tRNAs are appropriately structured, processed, and modified. We describe the known tRNA quality control processes that check tRNAs and correct or destroy aberrant tRNAs. These mechanisms employ two types of exonucleases, CCA end addition, tRNA nuclear aminoacylation, and tRNA subcellular traffic. We arrange these processes in order of the steps that occur from generation of precursor tRNAs by RNA polymerase (Pol) III transcription to end maturation and modification in the nucleus to splicing and additional modifications in the cytoplasm. Finally, we discuss the tRNA retrograde pathway, which allows tRNA reimport into the nucleus for degradation or repair. PMID:25848089

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.

A dual transcriptional reporter and CDK-activity sensor marks cell cycle entry and progression in C. elegans

PubMed Central

van Rijnberk, Lotte M.; van der Horst, Suzanne E. M.; van den Heuvel, Sander; Ruijtenberg, Suzan

2017-01-01

Development, tissue homeostasis and tumor suppression depend critically on the correct regulation of cell division. Central in the cell division process is the decision whether to enter the next cell cycle and commit to going through the S and M phases, or to remain temporarily or permanently arrested. Cell cycle studies in genetic model systems could greatly benefit from visualizing cell cycle commitment in individual cells without the need of fixation. Here, we report the development and characterization of a reporter to monitor cell cycle entry in the nematode C. elegans. This reporter combines the mcm-4 promoter, to reveal Rb/E2F-mediated transcriptional control, and a live-cell sensor for CDK-activity. The CDK sensor was recently developed for use in human cells and consists of a DNA Helicase fragment fused to eGFP. Upon phosphorylation by CDKs, this fusion protein changes in localization from the nucleus to the cytoplasm. The combined regulation of transcription and subcellular localization enabled us to visualize the moment of cell cycle entry in dividing seam cells during C. elegans larval development. This reporter is the first to reflect cell cycle commitment in C. elegans and will help further genetic studies of the mechanisms that underlie cell cycle entry and exit. PMID:28158315

L-citrulline immunostaining identifies nitric oxide production sites within neurons

NASA Technical Reports Server (NTRS)

Martinelli, G. P. T.; Friedrich, V. L. Jr; Holstein, G. R.

2002-01-01

The cellular and subcellular localization of L-citrulline was analyzed in the adult rat brain and compared with that of traditional markers for the presence of nitric oxide synthase. Light, transmission electron, and confocal laser scanning microscopy were used to study tissue sections processed for immunocytochemistry employing a monoclonal antibody against L-citrulline or polyclonal anti-neuronal nitric oxide synthase sera, and double immunofluorescence to detect neuronal nitric oxide synthase and L-citrulline co-localization. The results demonstrate that the same CNS regions and cell types are labeled by neuronal nitric oxide synthase polyclonal antisera and L-citrulline monoclonal antibodies, using both immunocytochemistry and immunofluorescence. Short-term pretreatment with a nitric oxide synthase inhibitor reduces L-citrulline immunostaining, but does not affect neuronal nitric oxide synthase immunoreactivity. In the vestibular brainstem, double immunofluorescence studies show that many, but not all, neuronal nitric oxide synthase-positive cells co-express L-citrulline, and that local intracellular patches of intense L-citrulline accumulation are present in some neurons. Conversely, all L-citrulline-labeled neurons co-express neuronal nitric oxide synthase. Cells expressing neuronal nitric oxide synthase alone are interpreted as neurons with the potential to produce nitric oxide under other stimulus conditions, and the subcellular foci of enhanced L-citrulline staining are viewed as intracellular sites of nitric oxide production. This interpretation is supported by ultrastructural observations of subcellular foci with enhanced L-citrulline and/or neuronal nitric oxide synthase staining that are located primarily at postsynaptic densities and portions of the endoplasmic reticulum. We conclude that nitric oxide is produced and released at focal sites within neurons that are identifiable using L-citrulline as a marker. Copyright 2002 IBRO.

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

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.

Identification and Molecular Characterization of the Chloroplast Targeting Domain of Turnip yellow mosaic virus Replication Proteins

PubMed Central

Moriceau, Lucille; Jomat, Lucile; Bressanelli, Stéphane; Alcaide-Loridan, Catherine; Jupin, Isabelle

2017-01-01

Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus infecting plants. The TYMV 140K replication protein is a key organizer of viral replication complex (VRC) assembly, being responsible for recruitment of the viral polymerase and for targeting the VRCs to the chloroplast envelope where viral replication takes place. However, the structural requirements determining the subcellular localization and membrane association of this essential viral protein have not yet been defined. In this study, we investigated determinants for the in vivo chloroplast targeting of the TYMV 140K replication protein. Subcellular localization studies of deletion mutants identified a 41-residue internal sequence as the chloroplast targeting domain (CTD) of TYMV 140K; this sequence is sufficient to target GFP to the chloroplast envelope. The CTD appears to be located in the C-terminal extension of the methyltransferase domain—a region shared by 140K and its mature cleavage product 98K, which behaves as an integral membrane protein during infection. We predicted the CTD to fold into two amphipathic α-helices—a folding that was confirmed in vitro by circular dichroism spectroscopy analyses of a synthetic peptide. The importance for subcellular localization of the integrity of these amphipathic helices, and the function of 140K/98K, was demonstrated by performing amino acid substitutions that affected chloroplast targeting, membrane association and viral replication. These results establish a short internal α-helical peptide as an unusual signal for targeting proteins to the chloroplast envelope membrane, and provide new insights into membrane targeting of viral replication proteins—a universal feature of positive-strand RNA viruses. PMID:29312393

Subcellular localization patterns and their relationship to photodynamic activity of pyropheophorbide-a derivatives.

PubMed

MacDonald, I J; Morgan, J; Bellnier, D A; Paszkiewicz, G M; Whitaker, J E; Litchfield, D J; Dougherty, T J

1999-11-01

To determine if subcellular localization is important to photodynamic therapy (PDT) efficacy, an in vitro fluorescence microscopy study was conducted with a congeneric series of pyropheophorbide-a derivatives in human pharyngeal squamous cell carcinoma (FaDu) cells and murine radiation-induced fibrosarcoma (RIF) mutant cells. In the FaDu cells the octyl, decyl and dodecyl ether derivatives localized to the lysosomes at extracellular concentrations less than needed to produce a 50% cell kill (LD50). At extracellular concentrations equal or greater than the LD50 the compounds localized mainly to mitochondria. The propyl, pentyl, hexyl and heptyl ether derivatives localized mainly to the mitochondria at all concentrations studied. This suggested that mitochondria are a sensitive PDT target for these derivatives. Similar experiments were performed with two Photofrin-PDT resistant RIF cell lines, one of which was found to be resistant to hexyl ether derivative (C6) mediated-PDT and the other sensitive to C6-PDT relative to the parent line. At extracellular concentrations of C6 below the LD50 of each cell line, the mutants exhibited lysosomal localization. At concentrations above these values the patterns shifted to a mainly mitochondrial pattern. In these cell lines mitochondrial localization also correlated with PDT sensitivity. Localization to mitochondria or lysosomes appeared to be affected by the aggregation state of the congeners, all of which are highly aggregated in aqueous medium. Monomers apparently were the active fraction of these compounds because equalizing the extracellular monomer concentrations produced equivalent intracellular concentrations, photoxicity and localization patterns. Compounds that were mainly aggregates localized to the lysosomes where they were rendered less active. Mitochondria appear to be a sensitive target for pyropheophorbide-a-mediated photodamage, and the degree of aggregation seems to be a determinant of the localization site.

Intracellular transport and compartmentation of phosphate in plants.

PubMed

Versaw, Wayne K; Garcia, L Rene

2017-10-01

Phosphate (Pi) is an essential macronutrient with structural and metabolic roles within every compartment of the plant cell. Intracellular Pi transporters direct Pi to each organelle and also control its exchange between subcellular compartments thereby providing the means to coordinate compartmented metabolic processes, including glycolysis, photosynthesis, and respiration. In this review we summarize recent advances in the identification and functional analysis of Pi transporters that localize to vacuoles, chloroplasts, non-photosynthetic plastids, mitochondria, and the Golgi apparatus. Electrical potentials across intracellular membranes and the pH of subcellular environments will also be highlighted as key factors influencing the energetics of Pi transport, and therefore pose limits for Pi compartmentation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Parkin Binds to α/β Tubulin and Increases their Ubiquitination and Degradation

PubMed Central

Ren, Yong; Zhao, Jinghui; Feng, Jian

2007-01-01

In addition to inhibiting the mitochondrial respiratory chain, toxins known to cause Parkinson’s disease (PD), such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone, also strongly depolymerize microtubules and increase tubulin degradation. Microtubules are polymers of tubulin α/β heterodimers, whose correct folding requires coordinated actions of cellular chaperonins and co-factors. Misfolded tubulin monomers are highly toxic and quickly degraded through a hitherto unknown mechanism. Here we report that parkin, a protein-ubiquitin E3 ligase linked to PD, was tightly bound to microtubules in taxol-mediated microtubule co-assembly assays. In lysates from the rat brain or transfected HEK293 cells, α-tubulin and β-tubulin were strongly co-immunoprecipitated with parkin at 4°C in the presence of colchicine, a condition where tubulin exits as α/β heterodimers. At the subcellular level, parkin exhibited punctate immunostaining along microtubules in rat brain sections, cultured primary neurons, glial cells and cell lines. This pattern of subcellular localization was abolished in cells treated with the microtubule-depolymerizing drug colchicine. The binding between parkin and tubulin apparently led to increased ubiquitination and accelerated degradation of α- and β-tubulins in HEK293 cells. Similarly ubiquitinated tubulins were also observed in rat brain lysates. Furthermore, parkin mutants found in PD patients did not ubiquitinate or degrade either tubulin. Taken together, our results show that parkin is a novel tubulin-binding protein, as well as a microtubule-associated protein (MAP). Its ability to enhance the ubiquitination and degradation of misfolded tubulins may play a significant role in protecting neurons from toxins that cause PD. PMID:12716939

Cellular Transfection to Deliver Alanine-Glyoxylate Aminotransferase to Hepatocytes: A Rational Gene Therapy for Primary Hyperoxaluria-1 (PH-1)

PubMed Central

Koul, Sweaty; Johnson, Thomas; Pramanik, Saroj; Koul, Hari

2005-01-01

Background: Primary hyperoxaluria-type 1 (PH-1) is a rare autosomal recessive disorder of glyoxalate metabolism caused by deficiency in the liver-specific peroxisomal enzyme alanine-glyoxalate transaminase 1 (AGT) resulting in the increased oxidation of glyoxalate to oxalate. Accumulation of oxalate in the kidney and other soft tissues results in loss of renal function and significant morbidity. The present treatment options offer some relief in the short term, but they are not completely successful. In the present study, we tested the feasibility of corrective gene therapy for this metabolic disorder. Methods: A cDNA library was made from HepG2 cells. PCR primers were designed for the AGT sequence with modifications to preclude mistargeting during gene delivery. Amplified AGT cDNA was cloned as a fusion protein with green fluorescent protein (GFP) using the vector EGFP-C1 (Clontech) for monitoring subcellular distribution. Sequence and expression of the fusion protein was verified. Fusion protein vectors were transfected into hepatocytes by liposomal transfection. AGT expression and subcellular distribution was monitored by GFP fluorescence. Results: HepG2 cells express full-length mRNA coding for AGT as confirmed by insert size as well as sequence determination. Selective primers allowed us to generate a modified recombinant GFP-AGT fusion protein. Cellular transfections with Lipofectamine resulted in transfection efficiencies of 60–90%. The recombinant AGT did localize to peroxisomes as monitored by GFP fluorescence. Conclusions: The results demonstrate preliminary in vitro feasibility data for AGT transfection into the hepatocytes. To the best of our knowledge, this is the first study to attempt recombinant AGT gene therapy for treatment of primary hyperoxaluria-1. PMID:15849465

PLEKHM2 mutation leads to abnormal localization of lysosomes, impaired autophagy flux and associates with recessive dilated cardiomyopathy and left ventricular noncompaction

PubMed Central

Muhammad, Emad; Levitas, Aviva; Singh, Sonia R.; Braiman, Alex; Ofir, Rivka; Etzion, Sharon; Sheffield, Val C.; Etzion, Yoram; Carrier, Lucie; Parvari, Ruti

2015-01-01

Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients' primary fibroblasts exhibited abnormal subcellular distribution of endosomes marked by Rab5, Rab7 and Rab9, as well as the Golgi apparatus. In addition, lysosomes appeared to be concentrated in the perinuclear region, and autophagy flux was impaired. Transfection of wild-type PLEKHM2 cDNA into patient's fibroblasts corrected the subcellular distribution of the lysosomes, supporting the causal effect of PLEKHM2 mutation. PLEKHM2 joins LAMP-2 and BAG3 as a disease gene altering autophagy resulting in an isolated cardiac phenotype. The association of PLEKHM2 mutation with DCM and LVNC supports the importance of autophagy for normal cardiac function. PMID:26464484

PLEKHM2 mutation leads to abnormal localization of lysosomes, impaired autophagy flux and associates with recessive dilated cardiomyopathy and left ventricular noncompaction.

PubMed

Muhammad, Emad; Levitas, Aviva; Singh, Sonia R; Braiman, Alex; Ofir, Rivka; Etzion, Sharon; Sheffield, Val C; Etzion, Yoram; Carrier, Lucie; Parvari, Ruti

2015-12-20

Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients' primary fibroblasts exhibited abnormal subcellular distribution of endosomes marked by Rab5, Rab7 and Rab9, as well as the Golgi apparatus. In addition, lysosomes appeared to be concentrated in the perinuclear region, and autophagy flux was impaired. Transfection of wild-type PLEKHM2 cDNA into patient's fibroblasts corrected the subcellular distribution of the lysosomes, supporting the causal effect of PLEKHM2 mutation. PLEKHM2 joins LAMP-2 and BAG3 as a disease gene altering autophagy resulting in an isolated cardiac phenotype. The association of PLEKHM2 mutation with DCM and LVNC supports the importance of autophagy for normal cardiac function. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

An intrinsic DFF40/CAD endonuclease deficiency impairs oligonucleosomal DNA hydrolysis during caspase-dependent cell death: a common trait in human glioblastoma cells.

PubMed

Sánchez-Osuna, María; Martínez-Escardó, Laura; Granados-Colomina, Carla; Martínez-Soler, Fina; Pascual-Guiral, Sònia; Iglesias-Guimarais, Victoria; Velasco, Roser; Plans, Gerard; Vidal, Noemi; Tortosa, Avelina; Barcia, Carlos; Bruna, Jordi; Yuste, Victor J

2016-07-01

Glioblastoma (GBM) or grade IV astrocytoma is one of the most devastating human cancers. The loss of DFF40/CAD, the key endonuclease that triggers oligonucleosomal DNA fragmentation during apoptosis, has been linked to genomic instability and cell survival after radiation. Despite the near inevitability of GBM tumor recurrence after treatment, the relationship between DFF40/CAD and GBM remains unexplored. We studied the apoptotic behavior of human GBM-derived cells after apoptotic insult. We analyzed caspase activation and the protein levels and subcellular localization of DFF40/CAD apoptotic endonuclease. DFF40/CAD was also evaluated in histological sections from astrocytic tumors and nontumoral human brain. We showed that GBM cells undergo incomplete apoptosis without generating oligonucleosomal DNA degradation despite the correct activation of executioner caspases. The major defect of GBM cells relied on the improper accumulation of DFF40/CAD at the nucleoplasmic subcellular compartment. Supporting this finding, DFF40/CAD overexpression allowed GBM cells to display oligonucleosomal DNA degradation after apoptotic challenge. Moreover, the analysis of histological slices from astrocytic tumors showed that DFF40/CAD immunoreactivity in tumoral GFAP-positive cells was markedly reduced when compared with nontumoral samples. Our data highlight the low expression levels of DFF40/CAD and the absence of DNA laddering as common molecular traits in GBM. These findings could be of major importance for understanding the malignant behavior of remaining tumor cells after radiochemotherapy. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Monoterpene biosynthesis potential of plant subcellular compartments.

PubMed

Dong, Lemeng; Jongedijk, Esmer; Bouwmeester, Harro; Van Der Krol, Alexander

2016-01-01

Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization

PubMed Central

Ursell, Tristan S.; Nguyen, Jeffrey; Monds, Russell D.; Colavin, Alexandre; Billings, Gabriel; Ouzounov, Nikolay; Gitai, Zemer; Shaevitz, Joshua W.; Huang, Kerwyn Casey

2014-01-01

Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization. PMID:24550515

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

Global genetic analyses reveal strong inter-ethnic variability in the loss of activity of the organic cation transporter OCT1.

PubMed

Seitz, Tina; Stalmann, Robert; Dalila, Nawar; Chen, Jiayin; Pojar, Sherin; Dos Santos Pereira, Joao N; Krätzner, Ralph; Brockmöller, Jürgen; Tzvetkov, Mladen V

2015-01-01

The organic cation transporter OCT1 (SLC22A1) mediates the uptake of vitamin B1, cationic drugs, and xenobiotics into hepatocytes. Nine percent of Caucasians lack or have very low OCT1 activity due to loss-of-function polymorphisms in OCT1 gene. Here we analyzed the global genetic variability in OCT1 to estimate the therapeutic relevance of OCT1 polymorphisms in populations beyond Caucasians and to identify evolutionary patterns of the common loss of OCT1 activity in humans. We applied massively parallel sequencing to screen for coding polymorphisms in 1,079 unrelated individuals from 53 populations worldwide. The obtained data was combined with the existing 1000 Genomes data comprising an additional 1,092 individuals from 14 populations. The identified OCT1 variants were characterized in vitro regarding their cellular localization and their ability to transport 10 known OCT1 substrates. Both the population genetics data and transport data were used in tandem to generate a world map of loss of OCT1 activity. We identified 16 amino acid substitutions potentially causing loss of OCT1 function and analyzed them together with five amino acid substitutions that were not expected to affect OCT1 function. The variants constituted 16 major alleles and 14 sub-alleles. Six major alleles showed improper subcellular localization leading to substrate-wide loss in activity. Five major alleles showed correct subcellular localization, but substrate-specific loss of activity. Striking differences were observed in the frequency of loss of OCT1 activity worldwide. While most East Asian and Oceanian individuals had completely functional OCT1, 80 % of native South American Indians lacked functional OCT1 alleles. In East Asia and Oceania the average nucleotide diversity of the loss-of-function variants was much lower than that of the variants that do not affect OCT1 function (ratio of 0.03) and was significantly lower than the theoretically expected heterozygosity (Tajima's D = -1.64, P < 0.01). Comprehensive genetic analyses showed strong global variations in the frequency of loss of OCT1 activity with selection pressure for maintaining OCT1 activity in East Asia and Oceania. These results not only enable pharmacogenetically-based optimization of drug treatment worldwide, but may help elucidate the functional role of human OCT1.

Subcellular localization of acyl-CoA binding protein in Aspergillus oryzae is regulated by autophagy machinery.

PubMed

Kawaguchi, Kouhei; Kikuma, Takashi; Higuchi, Yujiro; Takegawa, Kaoru; Kitamoto, Katsuhiko

2016-11-04

In eukaryotic cells, acyl-CoA binding protein (ACBP) is important for cellular activities, such as in lipid metabolism. In the industrially important fungus Aspergillus oryzae, the ACBP, known as AoACBP, has been biochemically characterized, but its physiological function is not known. In the present study, although we could not find any phenotype of AoACBP disruptants in the normal growth conditions, we examined the subcellular localization of AoACBP to understand its physiological function. Using an enhanced green fluorescent protein (EGFP)-tagged AoACBP construct we showed that AoACBP localized to punctate structures in the cytoplasm, some of which moved inside the cells in a microtubule-dependent manner. Further microscopic analyses showed that AoACBP-EGFP co-localized with the autophagy marker protein AoAtg8 tagged with red fluorescent protein (mDsRed). Expression of AoACBP-EGFP in disruptants of autophagy-related genes revealed aggregation of AoACBP-EGFP fluorescence in the cytoplasm of Aoatg1, Aoatg4 and Aoatg8 disruptant cells. However, in cells harboring disruption of Aoatg15, which encodes a lipase for autophagic body, puncta of AoACBP-EGFP fluorescence accumulated in vacuoles, indicating that AoACBP is transported to vacuoles via the autophagy machinery. Collectively, these results suggest the existence of a regulatory mechanism between AoACBP localization and autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

Determination of localization accuracy based on experimentally acquired image sets: applications to single molecule microscopy

PubMed Central

Tahmasbi, Amir; Ward, E. Sally; Ober, Raimund J.

2015-01-01

Fluorescence microscopy is a photon-limited imaging modality that allows the study of subcellular objects and processes with high specificity. The best possible accuracy (standard deviation) with which an object of interest can be localized when imaged using a fluorescence microscope is typically calculated using the Cramér-Rao lower bound, that is, the inverse of the Fisher information. However, the current approach for the calculation of the best possible localization accuracy relies on an analytical expression for the image of the object. This can pose practical challenges since it is often difficult to find appropriate analytical models for the images of general objects. In this study, we instead develop an approach that directly uses an experimentally collected image set to calculate the best possible localization accuracy for a general subcellular object. In this approach, we fit splines, i.e. smoothly connected piecewise polynomials, to the experimentally collected image set to provide a continuous model of the object, which can then be used for the calculation of the best possible localization accuracy. Due to its practical importance, we investigate in detail the application of the proposed approach in single molecule fluorescence microscopy. In this case, the object of interest is a point source and, therefore, the acquired image set pertains to an experimental point spread function. PMID:25837101

Hum-mPLoc: an ensemble classifier for large-scale human protein subcellular location prediction by incorporating samples with multiple sites.

PubMed

Shen, Hong-Bin; Chou, Kuo-Chen

2007-04-20

Proteins may simultaneously exist at, or move between, two or more different subcellular locations. Proteins with multiple locations or dynamic feature of this kind are particularly interesting because they may have some very special biological functions intriguing to investigators in both basic research and drug discovery. For instance, among the 6408 human protein entries that have experimentally observed subcellular location annotations in the Swiss-Prot database (version 50.7, released 19-Sept-2006), 973 ( approximately 15%) have multiple location sites. The number of total human protein entries (except those annotated with "fragment" or those with less than 50 amino acids) in the same database is 14,370, meaning a gap of (14,370-6408)=7962 entries for which no knowledge is available about their subcellular locations. Although one can use the computational approach to predict the desired information for the gap, so far all the existing methods for predicting human protein subcellular localization are limited in the case of single location site only. To overcome such a barrier, a new ensemble classifier, named Hum-mPLoc, was developed that can be used to deal with the case of multiple location sites as well. Hum-mPLoc is freely accessible to the public as a web server at http://202.120.37.186/bioinf/hum-multi. Meanwhile, for the convenience of people working in the relevant areas, Hum-mPLoc has been used to identify all human protein entries in the Swiss-Prot database that do not have subcellular location annotations or are annotated as being uncertain. The large-scale results thus obtained have been deposited in a downloadable file prepared with Microsoft Excel and named "Tab_Hum-mPLoc.xls". This file is available at the same website and will be updated twice a year to include new entries of human proteins and reflect the continuous development of Hum-mPLoc.

Proteomic analysis of synaptoneurosomes highlights the relevant role of local translation in the hippocampus.

PubMed

Benito, Itziar; Casañas, Juan José; Montesinos, María Luz

2018-06-19

Several proteomic analyses have been performed on synaptic fractions isolated from cortex or even total brain, resulting in preparations with a high synaptic heterogeneity and complexity. Synaptoneurosomes (SNs) are subcellular membranous elements that contain sealed pre- and post-synaptic components. They are obtained by subcellular fractionation of brain homogenates and serve as a suitable model to study many aspects of the synapse physiology. Here we report the proteomic content of SNs isolated from hippocampus of adult mice, a brain region involved in memory that presents lower synaptic heterogeneity than cortex. Interestingly, in addition to pre- and post-synaptic proteins, we found that proteins involved in RNA binding and translation were overrepresented in our preparation. These results validate the protocol we previously reported for SNs isolation, and, as reported by other authors, highlight the relevance of local synaptic translation for hippocampal physiology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Uses of Ku70

DOEpatents

Li, Gloria C.; Cordon-Cardo, Carlos; Ouyang, Honghai

2004-10-26

This invention provides a method of diagnosing a predisposition to cancer in a subject comprising: (a) obtaining a nucleic acid sample from the subject; and; (b) determining whether one or more of the subject's Ku70 alleles or regulatory regions to those alleles are deleted or different from the wild type so as to reduce or eliminate the subject's expression of polypeptide having tumor suppressor activity. This invention also provides a method of assessing the severity of cancer in a subject comprising: (a) obtaining a nucleic acid sample from the subject; and (b) determining whether one or more of the subject's Ku70 alleles or regulatory regions to those alleles are deleted or different from the wild type so as to reduce or eliminate the subject's expression of polypeptide having tumor suppressor activity. This invention also provides a method of assessing the severity of cancer in a subject comprising: determining the subcellular localization of Ku70 in the subject, wherein an abnormal subcellular localization of Ku70 indicates a predisposition to cancer.

Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle

PubMed Central

Peterson, Soren J.; Krasnow, Mark A.

2015-01-01

SUMMARY To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains including inside the cell. PMID:25557078

Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle.

PubMed

Peterson, Soren J; Krasnow, Mark A

2015-01-15

To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here, we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains, including inside the cell. Copyright © 2015 Elsevier Inc. All rights reserved.

Subcellular Localization of a Plant Catalase-Phenol Oxidase, AcCATPO, from Amaranthus and Identification of a Non-canonical Peroxisome Targeting Signal

PubMed Central

Chen, Ning; Teng, Xiao-Lu; Xiao, Xing-Guo

2017-01-01

AcCATPO is a plant catalase-phenol oxidase recently identified from red amaranth. Its physiological function remains unexplored. As the starting step of functional analysis, here we report its subcellular localization and a non-canonical targeting signal. Commonly used bioinformatics programs predicted a peroxisomal localization for AcCATPO, but failed in identification of canonical peroxisomal targeting signals (PTS). The C-terminal GFP tagging led the fusion protein AcCATPO-GFP to the cytosol and the nucleus, but N-terminal tagging directed the GFP-AcCATPO to peroxisomes and nuclei, in transgenic tobacco. Deleting the tripeptide (PTM) at the extreme C-terminus almost ruled out the peroxisomal localization of GFP-AcCATPOΔ3, and removing the C-terminal decapeptide completely excluded peroxisomes as the residence of GFP-AcCATPOΔ10. Furthermore, this decapeptide as a targeting signal could import GFP-10aa to the peroxisome exclusively. Taken together, these results demonstrate that AcCATPO is localized to the peroxisome and the nucleus, and its peroxisomal localization is attributed to a non-canonical PTS1, the C-terminal decapeptide which contains an internal SRL motif and a conserved tripeptide P-S/T-I/M at the extreme of C-terminus. This work may further the study as to the physiological function of AcCATPO, especially clarify its involvement in betalain biosynthesis, and provide a clue to elucidate more non-canonic PTS. PMID:28824680

Subcellular Localization of a Plant Catalase-Phenol Oxidase, AcCATPO, from Amaranthus and Identification of a Non-canonical Peroxisome Targeting Signal.

PubMed

Chen, Ning; Teng, Xiao-Lu; Xiao, Xing-Guo

2017-01-01

AcCATPO is a plant catalase-phenol oxidase recently identified from red amaranth. Its physiological function remains unexplored. As the starting step of functional analysis, here we report its subcellular localization and a non-canonical targeting signal. Commonly used bioinformatics programs predicted a peroxisomal localization for AcCATPO, but failed in identification of canonical peroxisomal targeting signals (PTS). The C-terminal GFP tagging led the fusion protein AcCATPO-GFP to the cytosol and the nucleus, but N-terminal tagging directed the GFP-AcCATPO to peroxisomes and nuclei, in transgenic tobacco. Deleting the tripeptide (PTM) at the extreme C-terminus almost ruled out the peroxisomal localization of GFP-AcCATPOΔ3, and removing the C-terminal decapeptide completely excluded peroxisomes as the residence of GFP-AcCATPOΔ10. Furthermore, this decapeptide as a targeting signal could import GFP-10aa to the peroxisome exclusively. Taken together, these results demonstrate that AcCATPO is localized to the peroxisome and the nucleus, and its peroxisomal localization is attributed to a non-canonical PTS1, the C-terminal decapeptide which contains an internal SRL motif and a conserved tripeptide P-S/T-I/M at the extreme of C-terminus. This work may further the study as to the physiological function of AcCATPO, especially clarify its involvement in betalain biosynthesis, and provide a clue to elucidate more non-canonic PTS.

Nucleobindin Co-Localizes and Associates with Cyclooxygenase (COX)-2 in Human Neutrophils

PubMed Central

Leclerc, Patrick; Biarc, Jordane; St-Onge, Mireille; Gilbert, Caroline; Dussault, Andrée-Anne; Laflamme, Cynthia; Pouliot, Marc

2008-01-01

The inducible cyclooxygenase isoform (COX-2) is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc), a ubiquitous Ca2+-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE2 biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr) Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE2 biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE2. Moreover, neutrophil transfection with hrNuc specifically enhanced PGE2 biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis. PMID:18493301

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

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.

WegoLoc: accurate prediction of protein subcellular localization using weighted Gene Ontology terms.

PubMed

Chi, Sang-Mun; Nam, Dougu

2012-04-01

We present an accurate and fast web server, WegoLoc for predicting subcellular localization of proteins based on sequence similarity and weighted Gene Ontology (GO) information. A term weighting method in the text categorization process is applied to GO terms for a support vector machine classifier. As a result, WegoLoc surpasses the state-of-the-art methods for previously used test datasets. WegoLoc supports three eukaryotic kingdoms (animals, fungi and plants) and provides human-specific analysis, and covers several sets of cellular locations. In addition, WegoLoc provides (i) multiple possible localizations of input protein(s) as well as their corresponding probability scores, (ii) weights of GO terms representing the contribution of each GO term in the prediction, and (iii) a BLAST E-value for the best hit with GO terms. If the similarity score does not meet a given threshold, an amino acid composition-based prediction is applied as a backup method. WegoLoc and User's guide are freely available at the website http://www.btool.org/WegoLoc smchiks@ks.ac.kr; dougnam@unist.ac.kr Supplementary data is available at http://www.btool.org/WegoLoc.

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.

Periplasmic localization of a GroES homologue in Escherichia coli transformed with groESx cloned from Legionella-like endosymbionts in Amoeba proteus.

PubMed

Lee, J E; Ahn, T I

2000-10-01

Escherichia coli MC4100 transformed with a groE homologous operon cloned from X-bacteria accumulated large amounts of the gene product when cultured at 30 or 37 degrees C. Heat shock for 10-30 min at 42 degrees C or ethanol (5%) shock for 2 h increased GroESx levels to about twice that in E. coli grown at 30 degrees C. The subcellular localization of GroESx in transformed E. coli was determined by several subcellular fractionation methods, by the analysis of extracted proteins in SDS polyacrylamide gels and by assays of marker enzymes. The GroESx protein was detected in both the periplasmic and cytoplasmic extracts and a large amount of the protein was accumulated in the periplasm. The GroEL protein and recombinant beta-galactosidase were exclusively localized in the cytoplasmic fraction, eliminating the possibility that periplasmic GroESx might be due to simple overproduction. N-terminal amino acid sequencing confirmed that the protein resolved on a 2-D gel was GroESx. This work represents the first report of the periplasmic location of GroES homologues in E. coli.

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

Identification and functional characterization of effectors in expressed sequence tags from various life cycle stages of the potato cyst nematode Globodera pallida.

PubMed

Jones, John T; Kumar, Amar; Pylypenko, Liliya A; Thirugnanasambandam, Amarnath; Castelli, Lydia; Chapman, Sean; Cock, Peter J A; Grenier, Eric; Lilley, Catherine J; Phillips, Mark S; Blok, Vivian C

2009-11-01

In this article, we describe the analysis of over 9000 expressed sequence tags (ESTs) from cDNA libraries obtained from various life cycle stages of Globodera pallida. We have identified over 50 G. pallida effectors from this dataset using bioinformatics analysis, by screening clones in order to identify secreted proteins up-regulated after the onset of parasitism and using in situ hybridization to confirm the expression in pharyngeal gland cells. A substantial gene family encoding G. pallida SPRYSEC proteins has been identified. The expression of these genes is restricted to the dorsal pharyngeal gland cell. Different members of the SPRYSEC family of proteins from G. pallida show different subcellular localization patterns in plants, with some localized to the cytoplasm and others to the nucleus and nucleolus. Differences in subcellular localization may reflect diverse functional roles for each individual protein or, more likely, variety in the compartmentalization of plant proteins targeted by the nematode. Our data are therefore consistent with the suggestion that the SPRYSEC proteins suppress host defences, as suggested previously, and that they achieve this through interaction with a range of host targets.

Ultrastructural study of liver and lead tissue concentrations in young mallard ducks (Anas platyrhynchos) after ingestion of single lead shot.

PubMed

Pineau, Alain; Fauconneau, Bernard; Plouzeau, Eric; Fernandez, Béatrice; Quellard, Nathalie; Levillain, Pierre; Guillard, Olivier

2017-01-01

Lead (Pb) represents a serious threat to wildlife and ecosystems. The aim of this study was to examine the subcellular effects of dietary Pb pellet ingestion on mallard (Anas platyrhynchos) livers. After ingestion of a single Pb shot (LS4 size class: 0.177 ± 0.03 g) in 41 mallard ducks (22 males and 19 females) versus 10 controls (5 males and 5 females), all 7-week old, a morphologic study was conducted by TEM (transmission electron microscopy) of liver at the subcellular level. The results in treated mallards showed at a magnification of 2500 X that hepatic parenchyma was altered as evidenced by intralysosomal electron-dense deposits, which are compatible with Pb deposits. Further, at a higher magnification (15,000 X) in both genders, deterioration of mitochondria was observed in which the crests and, to a lesser extent, outer membrane were lysed. While the rough endoplasmic reticulum was fragmented, intracytoplasmic electron-dense material compatible with Pb deposits was maximally visible, thereby underscoring the deeply destructive effect of this metal on the subcellular architecture of the liver. In addition, applying an optimized and validated method in a clean room using electrothermal atomic absorption spectrophotometer (ETAAS) with Zeeman background correction, the objective was to improve and refine certain indispensable measurements pertaining to Pb impregnation in tissues other than liver such as kidneys, bones, and feathers of mallards. Data demonstrated show that compared with controls, Pb accumulation increases significantly, not only in the liver (3-fold), but also in the bones and the feathers (14-fold). No significant difference was noted between males and females. Bearing in mind the marked subcellular toxicity attributed to Pb, this study reinforces present-day arguments advocating limitation of game consumption.

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

Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

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

Kwon, ChangHyuk, E-mail: netbuyer@hanmail.net; Tak, Hyosun, E-mail: chuberry@naver.com; Rho, Mina, E-mail: minarho@hanyang.ac.kr

2014-03-28

Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWImore » and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies.« less

Prediction of protein subcellular locations by GO-FunD-PseAA predictor.

PubMed

Chou, Kuo-Chen; Cai, Yu-Dong

2004-08-06

The localization of a protein in a cell is closely correlated with its biological function. With the explosion of protein sequences entering into DataBanks, it is highly desired to develop an automated method that can fast identify their subcellular location. This will expedite the annotation process, providing timely useful information for both basic research and industrial application. In view of this, a powerful predictor has been developed by hybridizing the gene ontology approach [Nat. Genet. 25 (2000) 25], functional domain composition approach [J. Biol. Chem. 277 (2002) 45765], and the pseudo-amino acid composition approach [Proteins Struct. Funct. Genet. 43 (2001) 246; Erratum: ibid. 44 (2001) 60]. As a showcase, the recently constructed dataset [Bioinformatics 19 (2003) 1656] was used for demonstration. The dataset contains 7589 proteins classified into 12 subcellular locations: chloroplast, cytoplasmic, cytoskeleton, endoplasmic reticulum, extracellular, Golgi apparatus, lysosomal, mitochondrial, nuclear, peroxisomal, plasma membrane, and vacuolar. The overall success rate of prediction obtained by the jackknife cross-validation was 92%. This is so far the highest success rate performed on this dataset by following an objective and rigorous cross-validation procedure.

HPSLPred: An Ensemble Multi-Label Classifier for Human Protein Subcellular Location Prediction with Imbalanced Source.

PubMed

Wan, Shixiang; Duan, Yucong; Zou, Quan

2017-09-01

Predicting the subcellular localization of proteins is an important and challenging problem. Traditional experimental approaches are often expensive and time-consuming. Consequently, a growing number of research efforts employ a series of machine learning approaches to predict the subcellular location of proteins. There are two main challenges among the state-of-the-art prediction methods. First, most of the existing techniques are designed to deal with multi-class rather than multi-label classification, which ignores connections between multiple labels. In reality, multiple locations of particular proteins imply that there are vital and unique biological significances that deserve special focus and cannot be ignored. Second, techniques for handling imbalanced data in multi-label classification problems are necessary, but never employed. For solving these two issues, we have developed an ensemble multi-label classifier called HPSLPred, which can be applied for multi-label classification with an imbalanced protein source. For convenience, a user-friendly webserver has been established at http://server.malab.cn/HPSLPred. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimization of immunostaining on flat-mounted human corneas.

PubMed

Forest, Fabien; Thuret, Gilles; Gain, Philippe; Dumollard, Jean-Marc; Peoc'h, Michel; Perrache, Chantal; He, Zhiguo

2015-01-01

In the literature, immunohistochemistry on cross sections is the main technique used to study protein expression in corneal endothelial cells (ECs), even though this method allows visualization of few ECs, without clear subcellular localization, and is subject to the staining artifacts frequently encountered at tissue borders. We previously proposed several protocols, using fixation in 0.5% paraformaldehyde (PFA) or in methanol, allowing immunostaining on flatmounted corneas for proteins of different cell compartments. In the present study, we further refined the technique by systematically assessing the effect of fixative temperature. Last, we used optimized protocols to further demonstrate the considerable advantages of immunostaining on flatmounted intact corneas: detection of rare cells in large fields of thousands of ECs and epithelial cells, and accurate subcellular localization of given proteins. The staining of four ubiquitous proteins, ZO-1, hnRNP L, actin, and histone H3, with clearly different subcellular localizations, was analyzed in ECs of organ-cultured corneas. Whole intact human corneas were fixed for 30 min in 0.5% paraformaldehyde or pure methanol at four temperatures (4 °C for PFA, -20 °C for methanol, and 23, 37, and 50 °C for both). Experiments were performed in duplicate and repeated on three corneas. Standardized pictures were analyzed independently by two experts. Second, optimized immunostaining protocols were applied to fresh corneas for three applications: identification of rare cells that express KI67 in the endothelium of specimens with Fuch's endothelial corneal dystrophy (FECD), the precise localization of neural cell adhesion molecules (NCAMs) in normal ECs and of the cytokeratin pair K3/12 and CD44 in normal epithelial cells, and the identification of cells that express S100b in the normal epithelium. Temperature strongly influenced immunostaining quality. There was no ubiquitous protocol, but nevertheless, room temperature may be recommended as first-line temperature during fixation, instead of the conventional -20 °C for methanol and 4 °C for PFA. Further optimization may be required for certain target proteins. Optimized protocols allowed description of two previously unknown findings: the presence of a few proliferating ECs in FECD specimens, suggesting ineffective compensatory mechanisms against premature EC death, and the localization of NCAMs exclusively in the lateral membranes of ECs, showing hexagonal organization at the apical pole and an irregular shape with increasing complexity toward the basal pole. Optimized protocols were also effective for the epithelium, allowing clear localization of cytokeratin 3/12 and CD44 in superficial and basal epithelial cells, respectively. Finally, S100b allowed identification of clusters of epithelial Langerhans cells near the limbus and more centrally. Fixative temperature is a crucial parameter in optimizing immunostaining on flatmounted intact corneas. Whole-tissue overview and precise subcellular staining are significant advantages over conventional immunohistochemistry (IHC) on cross sections. This technique, initially developed for the corneal endothelium, proved equally suitable for the corneal epithelium and could be used for other superficial mono- and multilayered epithelia.

Biological effects of simple changes in functionality on rhodium metalloinsertors

PubMed Central

Weidmann, Alyson G.; Komor, Alexis C.; Barton, Jacqueline K.

2013-01-01

DNA mismatch repair (MMR) is crucial to ensuring the fidelity of the genome. The inability to correct single base mismatches leads to elevated mutation rates and carcinogenesis. Using metalloinsertors–bulky metal complexes that bind with high specificity to mismatched sites in the DNA duplex–our laboratory has adopted a new chemotherapeutic strategy through the selective targeting of MMR-deficient cells, that is, those that have a propensity for cancerous transformation. Rhodium metalloinsertors display inhibitory effects selectively in cells that are deficient in the MMR machinery, consistent with this strategy. However, a highly sensitive structure–function relationship is emerging with the development of new complexes that highlights the importance of subcellular localization. We have found that small structural modifications, for example a hydroxyl versus a methyl functional group, can yield profound differences in biological function. Despite similar binding affinities and selectivities for DNA mismatches, only one metalloinsertor shows selective inhibition of cellular proliferation in MMR-deficient versus -proficient cells. Studies of whole-cell, nuclear and mitochondrial uptake reveal that this selectivity depends upon targeting DNA mismatches in the cell nucleus. PMID:23776288

Emory University: "LC-MS analysis of PRAS40 protein-protein interactions" | Office of Cancer Genomics

Cancer.gov

This study focuses on subcellular localization and interactome of nuclear PRAS40 in HeLa cells.  Read the abstract.  Experimental Approaches Read the detailed Experimental Approaches. If you cannot access the manuscript, or if you have additional questions, please email Andrei Ivanov.

[Regulation of terpene metabolism]. [Mentha piperita, Mentha spicata

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

Croteau, R.

1989-01-01

Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

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.

Divisome-dependent subcellular localization of cell-cell joining protein SepJ in the filamentous cyanobacterium Anabaena.

PubMed

Ramos-León, Félix; Mariscal, Vicente; Frías, José E; Flores, Enrique; Herrero, Antonia

2015-05-01

Heterocyst-forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA-dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two-hybrid system. We found SepJ self-interaction and a specific interaction with FtsQ, confirmed by co-purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity. © 2015 John Wiley & Sons Ltd.

The accumulation and localization of chalcone synthase in grapevine (Vitis vinifera L.).

PubMed

Wang, Huiling; Wang, Wei; Zhan, JiCheng; Yan, Ailing; Sun, Lei; Zhang, Guojun; Wang, Xiaoyue; Ren, Jiancheng; Huang, Weidong; Xu, Haiying

2016-09-01

Chalcone synthase (CHS, E.C.2.3.1.74) is the first committed enzyme in the flavonoid pathway. Previous studies have primarily focused on the cloning, expression and regulation of the gene at the transcriptional level. Little is yet known about the enzyme accumulation, regulation at protein level, as well as its localization in grapevine. In present study, the accumulation, tissue and subcellular localization of CHS in different grapevine tissues (Vitis vinifera L. Cabernet Sauvignon) were investigated via the techniques of Western blotting, immunohistochemical localization, immunoelectron microscopy and confocal microscopy. The results showed that CHS were mainly accumulated in the grape berry skin, leaves, stem tips and stem phloem, correlated with flavonoids accumulation. The accumulation of CHS is developmental dependent in grape berry skin and flesh. Immunohistochemical analysis revealed that CHS were primarily localized in the exocarp and vascular bundles of the fruits during berry development; in palisade, spongy tissues and vascular bundles of the leaves; in the primary phloem and pith ray in the stems; in the growth point, leaf primordium, and young leaves of leaf buds; and in the endoderm and primary phloem of grapevine roots. Furthermore, at the subcellular level, the cell wall, cytoplasm and nucleus localized patterns of CHS were observed in the grapevine vegetative tissue cells. Results above indicated that distribution of CHS in grapevine was organ-specific and tissue-specific. This work will provide new insight for the biosynthesis and regulation of diverse flavonoid compounds in grapevine. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Tilted light sheet microscopy with 3D point spread functions for single-molecule super-resolution imaging in mammalian cells

NASA Astrophysics Data System (ADS)

Gustavsson, Anna-Karin; Petrov, Petar N.; Lee, Maurice Y.; Shechtman, Yoav; Moerner, W. E.

2018-02-01

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D superresolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Emory University: "LC-MS analysis of PRAS40 protein-protein interactions" | Office of Cancer Genomics

Cancer.gov

This study focuses on subcellular localization and interactome of nuclear PRAS40 in HeLa cells.  Read the abstract.  Experimental Approaches Read the detailed Experimental Approaches. If you cannot access the manuscript, or if you have additional questions, please email Andrei Ivanov. Data

Nuclear glutaredoxin 3 is critical for protection against oxidative stress-induced cell death

USDA-ARS?s Scientific Manuscript database

Mammalian glutaredoxin 3 (Grx3) has been shown to be critical in maintaining redox homeostasis and regulating cell survival pathways in cancer cells. However, the regulation of Grx3 is not fully understood. In the present study, we investigate the subcellular localization of Grx3 under normal growth...

[Regulation of terpene metabolism]. Annual progress report, March 15, 1988--March 14, 1989

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

Croteau, R.

1989-12-31

Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

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

Local structure of subcellular input retinotopy in an identified visual interneuron

NASA Astrophysics Data System (ADS)

Zhu, Ying; Gabbiani, Fabrizio; Fabrizio Gabbiani's lab Team

2015-03-01

How does the spatial layout of the projections that a neuron receives impact its synaptic integration and computation? What is the mapping topography of subcellular wiring at the single neuron level? The LGMD (lobula giant movement detector) neuron in the locust is an identified neuron that responds preferentially to objects approaching on a collision course. It receives excitatory inputs from the entire visual hemifield through calcium-permeable nicotinic acetylcholine receptors. Previous work showed that the projection from the locust compound eye to the LGMD preserved retinotopy down to the level of a single ommatidium (facet) by employing in vivo widefield calcium imaging. Because widefield imaging relies on global excitation of the preparation and has a relatively low resolution, previous work could not investigate this retinotopic mapping at the level of individual thin dendritic branches. Our current work employs a custom-built two-photon microscope with sub-micron resolution in conjunction with a single-facet stimulation setup that provides visual stimuli to the single ommatidium of locust adequate to explore the local structure of this retinotopy at a finer level. We would thank NIMH for funding this research.

Expression and subcellular localization of ORC1 in Leishmania major

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

Kumar, Diwakar; Mukherji, Agnideep; Saha, Swati

2008-10-10

The mechanism of DNA replication is highly conserved in eukaryotes, with the process being preceded by the ordered assembly of pre-replication complexes (pre-RCs). Pre-RC formation is triggered by the association of the origin replication complex (ORC) with chromatin. Leishmania major appears to have only one ORC ortholog, ORC1. ORC1 in other eukaryotes is the largest of the ORC subunits and is believed to play a significant role in modulating replication initiation. Here we report for the first time, the cloning of ORC1 from L. major, and the analysis of its expression in L. major promastigotes. In human cells ORC1 levelsmore » have been found to be upregulated in G1 and subsequently degraded, thus playing a role in controlling replication initiation. We examine the subcellular localization of L. major ORC1 in relation to the different stages of the cell cycle. Our results show that, unlike what is widely believed to be the case with ORC1 in human cells, ORC1 in L. major is nuclear at all stages of the cell cycle.« less

Bioinformatics approach of three partial polyprenol reductase genes in Kandelia obovata

NASA Astrophysics Data System (ADS)

Basyuni, M.; Wati, R.; Sagami, H.; Oku, H.; Baba, S.

2018-03-01

This present study describesthe bioinformatics approach to analyze three partial polyprenol reductase genes from mangrove plant, Kandeliaobovataas well aspredictedphysical and chemical properties, potential peptide, subcellular localization, and phylogenetic. The diversity was noted in the physical and chemical properties of three partial polyprenol reductase genes. The values of chloroplast were relatively high, showed that chloroplast transit peptide occurred in mangrove polyprenol reductase. The target peptide value of mitochondria varied from 0.088 to 0.198 indicated it was possible to be present. These results suggested the importance of understanding the diversity of physicochemical properties of the different amino acids in polyprenol reductase. The subcellular localization of two partial genes located in the plasma membrane. To confirm the homology among the polyprenol reductase in the database, a dendrogram was drawn. The phylogenetic tree depicts that there are three clusters, the partial genes of K. obovata joined the largest one: C23157 was close to Ricinus communis polyprenol reductase. Whereas, C23901 and C24171 were grouped with Ipomoea nil polyprenol reductase, suggested that these polyprenol reductase genes form distinct separation into tropical habitat plants.

Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins.

PubMed

Chanoca, Alexandra; Burkel, Brian; Kovinich, Nik; Grotewold, Erich; Eliceiri, Kevin W; Otegui, Marisa S

2016-12-01

Anthocyanins are flavonoid pigments that accumulate in most seed plants. They are synthesized in the cytoplasm but accumulate inside the vacuoles. Anthocyanins are pigmented at the lower vacuolar pH, but in the cytoplasm they can be visualized based on their fluorescence properties. Thus, anthocyanins provide an ideal system for the development of new methods to investigate cytoplasmic pools and association with other molecular components. We have analyzed the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), in both in vitro and in vivo conditions, using wild-type and mutant Arabidopsis thaliana seedlings. Within plant cells, the amplitude-weighted mean fluorescence lifetime (τ m ) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter τ m than the cytoplasmic pool. Consistently, lowering the pH of anthocyanins in solution shortened their fluorescence decay. We propose that FLIM is a useful tool for understanding the trafficking of anthocyanins and, potentially, for estimating vacuolar pH inside intact plant cells. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

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.

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

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.

Functional relationship between CABIT, SAM and 14-3-3 binding domains of GAREM1 that play a role in its subcellular localization

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

Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki, E-mail: hkonishi@pu-hiroshima.ac.jp

2015-08-21

GAREM1 (Grb2-associated regulator of Erk/MAPK1) is an adaptor protein that is involved in the epidermal growth factor (EGF) pathway. The nuclear localization of GAREM1 depends on the nuclear localization sequence (NLS), which is located at the N-terminal CABIT (cysteine-containing, all in Themis) domain. Here, we identified 14-3-3ε as a GAREM-binding protein, and its binding site is closely located to the NLS. This 14-3-3 binding site was of the atypical type and independent of GAREM phosphorylation. Moreover, the binding of 14-3-3 had an effect on the nuclear localization of GAREM1. Unexpectedly, we observed that the CABIT domain had intramolecular association withmore » the C-terminal SAM (sterile alpha motif) domain. This association might be inhibited by binding of 14-3-3 at the CABIT domain. Our results demonstrate that the mechanism underlying the nuclear localization of GAREM1 depends on its NLS in the CABIT domain, which is controlled by the binding of 14-3-3 and the C-terminal SAM domain. We suggest that the interplay between 14-3-3, SAM domain and CABIT domain might be responsible for the distribution of GAREM1 in mammalian cells. - Highlights: • 14-3-3ε regulated the nuclear localization of GAREM1 as its binding partner. • The atypical 14-3-3 binding site of GAREM1 is located near the NLS in CABIT domain. • The CABIT domain had intramolecular association with the SAM domain in GAREM1. • Subcellular localization of GAREM1 is affected with its CABIT-SAM interaction.« less

Extrasynaptic N-methyl-D-aspartate (NMDA) receptor stimulation induces cytoplasmic translocation of the CDKL5 kinase and its proteasomal degradation.

PubMed

Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2011-10-21

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-D-aspartate receptors and suggest regulation of CDKL5 by cell death pathways.

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

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

Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization

PubMed Central

Komor, Alexis C.; Schneider, Curtis J.; Weidmann, Alyson G.; Barton, Jacqueline K.

2013-01-01

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biological activities determined. Although DNA photocleavage experiments demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments have uncovered a relationship between the subcellular distribution of these metalloinsertors and their biological activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient versus proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA. PMID:23137296

Proteome-wide identification of predominant subcellular protein localizations in a bacterial model organism

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

Stekhoven, Daniel J.; Omasits, Ulrich; Quebatte, Maxime

2014-03-01

Proteomics data provide unique insights into biological systems, including the predominant subcellular localization (SCL) of proteins, which can reveal important clues about their functions. Here we analyzed data of a complete prokaryotic proteome expressed under two conditions mimicking interaction of the emerging pathogen Bartonella henselae with its mammalian host. Normalized spectral count data from cytoplasmic, total membrane, inner and outer membrane fractions allowed us to identify the predominant SCL for 82% of the identified proteins. The spectral count proportion of total membrane versus cytoplasmic fractions indicated the propensity of cytoplasmic proteins to co-fractionate with the inner membrane, and enabled usmore » to distinguish cytoplasmic, peripheral innermembrane and bona fide inner membrane proteins. Principal component analysis and k-nearest neighbor classification training on selected marker proteins or predominantly localized proteins, allowed us to determine an extensive catalog of at least 74 expressed outer membrane proteins, and to extend the SCL assignment to 94% of the identified proteins, including 18% where in silico methods gave no prediction. Suitable experimental proteomics data combined with straightforward computational approaches can thus identify the predominant SCL on a proteome-wide scale. Finally, we present a conceptual approach to identify proteins potentially changing their SCL in a condition-dependent fashion.« less

Ultraflexible nanoelectronic probes form reliable, glial scar–free neural integration

PubMed Central

Luan, Lan; Wei, Xiaoling; Zhao, Zhengtuo; Siegel, Jennifer J.; Potnis, Ojas; Tuppen, Catherine A; Lin, Shengqing; Kazmi, Shams; Fowler, Robert A.; Holloway, Stewart; Dunn, Andrew K.; Chitwood, Raymond A.; Xie, Chong

2017-01-01

Implanted brain electrodes construct the only means to electrically interface with individual neurons in vivo, but their recording efficacy and biocompatibility pose limitations on scientific and clinical applications. We showed that nanoelectronic thread (NET) electrodes with subcellular dimensions, ultraflexibility, and cellular surgical footprints form reliable, glial scar–free neural integration. We demonstrated that NET electrodes reliably detected and tracked individual units for months; their impedance, noise level, single-unit recording yield, and the signal amplitude remained stable during long-term implantation. In vivo two-photon imaging and postmortem histological analysis revealed seamless, subcellular integration of NET probes with the local cellular and vasculature networks, featuring fully recovered capillaries with an intact blood-brain barrier and complete absence of chronic neuronal degradation and glial scar. PMID:28246640

Tuning the Catalytic Activity of Subcellular Nanoreactors.

PubMed

Jakobson, Christopher M; Chen, Yiqun; Slininger, Marilyn F; Valdivia, Elias; Kim, Edward Y; Tullman-Ercek, Danielle

2016-07-31

Bacterial microcompartments are naturally occurring subcellular organelles of bacteria and serve as a promising scaffold for the organization of heterologous biosynthetic pathways. A critical element in the design of custom biosynthetic organelles is quantitative control over the loading of heterologous enzymes to the interior of the organelles. We demonstrate that the loading of heterologous proteins to the 1,2-propanediol utilization microcompartment of Salmonella enterica can be controlled using two strategies: by modulating the transcriptional activation of the microcompartment container and by coordinating the expression of the microcompartment container and the heterologous cargo. These strategies allow general control over the loading of heterologous proteins localized by two different N-terminal targeting peptides and represent an important step toward tuning the catalytic activity of bacterial microcompartments for increased biosynthetic productivity. Copyright © 2016. Published by Elsevier Ltd.

Cdk2 Phosphorylation on Threonine39 by AKT and Its Implication on Cyclin Binding, Cellular Localization, and Cell Cycle Progression

DTIC Science & Technology

2008-10-01

cell cycle progression in most cell types. Mouse embryos develop normally until mid gestation without all interphase Cdks 28. Pertinent to the...Ciemerych and P. Sicinski, "Cell cycle in mouse development ," 24(17), 2877 (2005). Ref Type: Journal 5 K. Coulonval, et al., "Phosphorylations of...34 Development 135(20), 3389 (2008). Ref Type: Journal 30 J. P. Tassan, et al., "Cell cycle analysis of the activity, subcellular localization, and subunit

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

Kim, Woo Taek; Franceschi, V.R.; Okita, T.W.

The subcellular localization of ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis, was determined in developing potato tuber cells by immunocytochemical localization techniques at the light microscopy level. Specific labeling of ADPglucose pyrophosphorylase by either immunofluorescence or immunogold followed by silver enhancement was detected only in the amyloplasts and indicates that this enzyme is located exclusively in the amyloplasts in developing potato tuber cells. Labeling occurred on the starch grains and, in some instances, specific labeling patterns were evident which may be related to sites active in starch deposition.

Modeling curvature-dependent subcellular localization of a small sporulation protein in Bacillus subtilis

NASA Astrophysics Data System (ADS)

Wasnik, Vaibhav; Wingreen, Ned; Mukhopadhyay, Ranjan

2012-02-01

Recent experiments suggest that in the bacterium, B. subtilis, the cue for the localization of small sporulation protein, SpoVM, that plays a central role in spore coat formation, is curvature of the bacterial plasma membrane. This curvature-dependent localization is puzzling given the orders of magnitude difference in lengthscale of an individual protein and radius of curvature of the membrane. Here we develop a minimal model to study the relationship between curvature-dependent membrane absorption of SpoVM and clustering of membrane-associated SpoVM and compare our results with experiments.

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa.

PubMed

Nag, Ambarish; Karpinets, Tatiana V; Chang, Christopher H; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can be directly accessed at http://cricket.ornl.gov:1555/PTR/new-image?object=SUGAR-NUCLEOTIDES.

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa

PubMed Central

Nag, Ambarish; Karpinets, Tatiana V.; Chang, Christopher H.; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can be directly accessed at http://cricket.ornl.gov:1555/PTR/new-image?object=SUGAR-NUCLEOTIDES. PMID:22465851

Isoform-specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis.

PubMed

Xiao, Shangxi; MacNair, Laura; McGoldrick, Philip; McKeever, Paul M; McLean, Jesse R; Zhang, Ming; Keith, Julia; Zinman, Lorne; Rogaeva, Ekaterina; Robertson, Janice

2015-10-01

A noncoding hexanucleotide repeat expansion in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). It has been reported that the repeat expansion causes a downregulation of C9orf72 transcripts, suggesting that haploinsufficiency may contribute to disease pathogenesis. Two protein isoforms are generated from three alternatively spliced transcripts of C9orf72; a long form (C9-L) and a short form (C9-S), and their function(s) are largely unknown owing to lack of specific antibodies. To investigate C9orf72 protein properties, we developed novel antibodies that recognize either C9-L or C9-S. Multiple techniques, including Western blot, immunohistochemistry, and coimmunoprecipitation, were used to determine the expression levels and subcellular localizations of C9-L and C9-S. Investigation of expression of C9-L and C9-S demonstrated distinct biochemical profiles, region-specific changes, and distinct subcellular localizations in ALS tissues. In particular, C9-L antibody exhibited a diffuse cytoplasmic staining in neurons and labeled large speckles in cerebellar Purkinje cells. In contrast, C9-S antibody gave very specific labeling of the nuclear membrane in healthy neurons, with apparent relocalization to the plasma membrane of diseased motor neurons in ALS. Coimmunoprecipitation experiments revealed an interaction of the C9-isoforms with both Importin β1 and Ran-GTPase, components of the nuclear pore complex. Using these antibodies, we have shown that C9orf72 may be involved in nucleocytoplasmic shuttling and this may have relevance to pathophysiology of ALS/FTLD. Our antibodies have provided improved detection of C9orf72 protein isoforms, which will help elucidate its physiological function and role in ALS/FTLD. © 2015 The Authors Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

Expression, subcellular localization and regulation of sigma receptor in retinal Müller cells

PubMed Central

Jiang, Guoliang; Mysona, Barbara; Dun, Ying; Gnana-Prakasam, Jaya P.; Pabla, Navjotsin; Li, Weiguo; Dong, Zheng; Ganapathy, Vadivel; Smith, Sylvia B.

2013-01-01

Purpose Sigma receptors (σR) are non-opioid, non-phencyclidine binding sites with robust neuroprotective properties. σR1 is expressed in brain oligodendrocytes, but its expression and binding capacity have not been analyzed in retinal glial cells. This study examined the expression, subcellular localization, binding activity and regulation of σR1 in retinal Müller cells. Methods Primary mouse Müller cells (1°MC) were analyzed by RT-PCR, immunoblotting and immunocytochemistry for the expression of σR1 and data were compared to the rat Müller cell line, rMC-1 and rat ganglion cell line, RGC-5. Confocal microscopy was used to determine the subcellular σR1 location in 1°MC. Membranes prepared from these cells were used for binding assays using [3H]-pentazocine (PTZ). The kinetics of binding, the ability of various σR1 ligands to compete with σR1 binding and the effects of nitric oxide (NO) and reactive oxygen species (ROS) donors on binding were examined. Results σR1 is expressed in 1°MC and is localized to the nuclear and endoplasmic reticulum membranes. Binding assays showed that in 1°MCs, rMC-1 and RGC-5 cells, the binding of PTZ was saturable. [3H]-PTZ bound with high affinity in RGC-5 and rMC-1 cells and the binding was similarly robust in 1°MC. Competition studies showed marked inhibition of [3H]-PTZ binding in the presence of σR1-specific ligands. Incubation of cells with NO and ROS donors markedly increased σR1 binding activity. Conclusions Müller cells express σR1 and demonstrate robust σR1 binding activity, which is inhibited by σR1 ligands and is stimulated during oxidative stress. The potential of Müller cells to bind σR1 ligands may prove beneficial in retinal degenerative diseases such as diabetic retinopathy. PMID:17122151

Expression, subcellular localization, and regulation of sigma receptor in retinal muller cells.

PubMed

Jiang, Guoliang; Mysona, Barbara; Dun, Ying; Gnana-Prakasam, Jaya P; Pabla, Navjotsin; Li, Weiguo; Dong, Zheng; Ganapathy, Vadivel; Smith, Sylvia B

2006-12-01

Sigma receptors (sigmaRs) are nonopioid, nonphencyclidine binding sites with robust neuroprotective properties. Type 1 sigmaR1 (sigmaR1) is expressed in brain oligodendrocytes, but its expression and binding capacity have not been analyzed in retinal glial cells. This study examined the expression, subcellular localization, binding activity, and regulation of sigmaR1 in retinal Müller cells. Primary mouse Müller cells (MCs) were analyzed by RT-PCR, immunoblotting, and immunocytochemistry for the expression of sigmaR1, and data were compared with those of the rat Müller cell line (rMC-1) and the rat ganglion cell line (RGC-5). Confocal microscopy was used to determine the subcellular sigmaR1 location in primary mouse MCs. Membranes prepared from these cells were used for binding assays with [3H]-pentazocine (PTZ). The kinetics of binding, the ability of various sigmaR1 ligands to compete with sigmaR1 binding, and the effects of donated nitric oxide (NO) and reactive oxygen species (ROS) on binding were examined. sigmaR1 is expressed in primary mouse MCs and is localized to the nuclear and endoplasmic reticulum membranes. Binding assays showed that in primary mouse MCs, rMC-1, and RGC-5, the binding of PTZ was saturable. [3H]-PTZ bound with high affinity in RGC-5 and rMC-1 cells, and the binding was similarly robust in primary mouse MCs. Competition studies showed marked inhibition of [3H]-PTZ binding in the presence of sigmaR1-specific ligands. Incubation of cells with NO and ROS donors markedly increased sigmaR1 binding activity. MCs express sigmaR1 and demonstrate robust sigmaR1 binding activity, which is inhibited by sigmaR1 ligands and is stimulated during oxidative stress. The potential of Müller cells to bind sigmaR1 ligands may prove beneficial in retinal degenerative diseases such as diabetic retinopathy.

Prediction of subcellular localization of eukaryotic proteins using position-specific profiles and neural network with weighted inputs.

PubMed

Zou, Lingyun; Wang, Zhengzhi; Huang, Jiaomin

2007-12-01

Subcellular location is one of the key biological characteristics of proteins. Position-specific profiles (PSP) have been introduced as important characteristics of proteins in this article. In this study, to obtain position-specific profiles, the Position Specific Iterative-Basic Local Alignment Search Tool (PSI-BLAST) has been used to search for protein sequences in a database. Position-specific scoring matrices are extracted from the profiles as one class of characteristics. Four-part amino acid compositions and 1st-7th order dipeptide compositions have also been calculated as the other two classes of characteristics. Therefore, twelve characteristic vectors are extracted from each of the protein sequences. Next, the characteristic vectors are weighed by a simple weighing function and inputted into a BP neural network predictor named PSP-Weighted Neural Network (PSP-WNN). The Levenberg-Marquardt algorithm is employed to adjust the weight matrices and thresholds during the network training instead of the error back propagation algorithm. With a jackknife test on the RH2427 dataset, PSP-WNN has achieved a higher overall prediction accuracy of 88.4% rather than the prediction results by the general BP neural network, Markov model, and fuzzy k-nearest neighbors algorithm on this dataset. In addition, the prediction performance of PSP-WNN has been evaluated with a five-fold cross validation test on the PK7579 dataset and the prediction results have been consistently better than those of the previous method on the basis of several support vector machines, using compositions of both amino acids and amino acid pairs. These results indicate that PSP-WNN is a powerful tool for subcellular localization prediction. At the end of the article, influences on prediction accuracy using different weighting proportions among three characteristic vector categories have been discussed. An appropriate proportion is considered by increasing the prediction accuracy.

Cell- and virus-mediated regulation of the barrier-to-autointegration factor's phosphorylation state controls its DNA binding, dimerization, subcellular localization, and antipoxviral activity.

PubMed

Jamin, Augusta; Wicklund, April; Wiebe, Matthew S

2014-05-01

Barrier-to-autointegration factor (BAF) is a DNA binding protein with multiple cellular functions, including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAF's ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia virus B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however, the precise mechanisms are poorly understood. Here we analyzed how phosphorylation impacts BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable mutants. Our studies demonstrate that increased phosphorylation enhances BAF's mobilization from the nucleus to the cytosol, while dephosphorylation restricts BAF to the nucleus. Phosphorylation also impairs both BAF's dimerization and its DNA binding activity. Furthermore, our studies of BAF's antiviral activity revealed that hyperphosphorylated BAF is unable to suppress viral DNA replication or virus production. Interestingly, the unphosphorylatable BAF mutant, which is capable of binding DNA but localizes predominantly to the nucleus, was also incapable of suppressing viral replication. Thus, both DNA binding and localization are important determinants of BAF's antiviral function. Finally, our examination of how phosphatases are involved in regulating BAF revealed that PP2A dephosphorylates BAF during vaccinia infection, thus counterbalancing the activity of the B1 kinase. Altogether, these data demonstrate that phosphoregulation of BAF by viral and cellular enzymes modulates this protein at multiple molecular levels, thus determining its effectiveness as an antiviral factor and likely other functions as well. The barrier-to-autointegration factor (BAF) contributes to cellular genomic integrity in multiple ways, the best characterized of which are as a host defense against cytoplasmic DNA and as a regulator of mitotic nuclear reassembly. Although dynamic phosphorylation involving both viral and cellular enzymes is likely a key regulator of multiple BAF functions, the precise mechanisms involved are poorly understood. Here we demonstrate that phosphorylation coordinately regulates BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity. Overall, our findings provide new insights into how phosphoregulation of BAF modulates this protein at multiple levels and governs its effectiveness as an antiviral factor against foreign DNA.

Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

NASA Astrophysics Data System (ADS)

Chandra, Subhash

2008-12-01

Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2+) revealed local secondary ion signal enhancements correlated with the water image signals of 19(H 3O) +. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells provided a rule-of-thumb criterion for the validation of sample preparation. The fractured freeze-dried cells allowed 3-D SIMS imaging and localization of 13C 15N labeled molecules and therapeutic drugs containing an elemental tag. Examples are shown to demonstrate that both diffusible elements and molecules are prone to artifact-induced relocation at subcellular scale if the sample preparation is compromised. The sample preparation is problem dependent and may vary widely between the diverse sample types of biological systems and the type of instrument used for SIMS analysis. The sample preparation, however, must be validated so that SIMS can be applied with confidence in biology and medicine.

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

Novel Therapeutic Targets to Inhibit Tumor Microenvironment Induced Castration-resistant Prostate Cancer

DTIC Science & Technology

2015-10-01

CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a. REPORT Unclassified b. ABSTRACT Unclassified c...kinase 5 activity and subcellular localization by the atypical MAPK ERK4/MAPK4. J Biol Chem 281, 35499-510 (2006). 5. Kant , S. et al. Characterization of

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...

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.

Multiple-Localization and Hub Proteins

PubMed Central

Ota, Motonori; Gonja, Hideki; Koike, Ryotaro; Fukuchi, Satoshi

2016-01-01

Protein-protein interactions are fundamental for all biological phenomena, and protein-protein interaction networks provide a global view of the interactions. The hub proteins, with many interaction partners, play vital roles in the networks. We investigated the subcellular localizations of proteins in the human network, and found that the ones localized in multiple subcellular compartments, especially the nucleus/cytoplasm proteins (NCP), the cytoplasm/cell membrane proteins (CMP), and the nucleus/cytoplasm/cell membrane proteins (NCMP), tend to be hubs. Examinations of keywords suggested that among NCP, those related to post-translational modifications and transcription functions are the major contributors to the large number of interactions. These types of proteins are characterized by a multi-domain architecture and intrinsic disorder. A survey of the typical hub proteins with prominent numbers of interaction partners in the type revealed that most are either transcription factors or co-regulators involved in signaling pathways. They translocate from the cytoplasm to the nucleus, triggered by the phosphorylation and/or ubiquitination of intrinsically disordered regions. Among CMP and NCMP, the contributors to the numerous interactions are related to either kinase or ubiquitin ligase activity. Many of them reside on the cytoplasmic side of the cell membrane, and act as the upstream regulators of signaling pathways. Overall, these hub proteins function to transfer external signals to the nucleus, through the cell membrane and the cytoplasm. Our analysis suggests that multiple-localization is a crucial concept to characterize groups of hub proteins and their biological functions in cellular information processing. PMID:27285823

Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control.

PubMed

Omori, Yoshihiro; Chaya, Taro; Yoshida, Satoyo; Irie, Shoichi; Tsujii, Toshinori; Furukawa, Takahisa

2015-01-01

Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs). We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR), neuropeptide FF receptor 1 (NPFFR1), and neuromedin U receptor 1 (NMUR1), localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S) is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L) is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO) mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control.

The potato-specific apyrase is apoplastically localized and has influence on gene expression, growth, and development.

PubMed

Riewe, David; Grosman, Lukasz; Fernie, Alisdair R; Wucke, Cornelia; Geigenberger, Peter

2008-07-01

Apyrases hydrolyze nucleoside triphosphates and diphosphates and are found in all eukaryotes and a few prokaryotes. Although their enzymatic properties have been well characterized, relatively little is known regarding their subcellular localization and physiological function in plants. In this study, we used reverse genetic and biochemical approaches to investigate the role of potato (Solanum tuberosum)-specific apyrase. Silencing of the apyrase gene family with RNA interference constructs under the control of the constitutive 35S promoter led to a strong decrease in apyrase activity to below 10% of the wild-type level. This decreased activity led to phenotypic changes in the transgenic lines, including a general retardation in growth, an increase in tuber number per plant, and differences in tuber morphology. Silencing of apyrase under the control of a tuber-specific promoter led to similar changes in tuber morphology; however, there were no direct effects of apyrase inhibition on tuber metabolism. DNA microarrays revealed that decreased expression of apyrase leads to increased levels of transcripts coding for cell wall proteins involved in growth and genes involved in energy transfer and starch synthesis. To place these results in context, we determined the subcellular localization of the potato-specific apyrase. Using a combination of approaches, we were able to demonstrate that this enzyme is localized to the apoplast. We describe the evidence that underlies both this fact and that potato-specific apyrase has a crucial role in regulating growth and development.

Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.

PubMed

Robin, Guillaume P; Kleemann, Jochen; Neumann, Ulla; Cabre, Lisa; Dallery, Jean-Félix; Lapalu, Nicolas; O'Connell, Richard J

2018-01-01

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum , encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium -mediated transformation to transiently express them as N -terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana , and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C -terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.

Approaches for Studying the Subcellular Localization, Interactions, and Regulation of Histone Deacetylase 5 (HDAC5)

PubMed Central

Guise, Amanda J.; Cristea, Ileana M.

2017-01-01

As a member of the class IIa family of histone deacetylases, the histone deacetylase 5 (HDAC5) is known to undergo nuclear–cytoplasmic shuttling and to be a critical transcriptional regulator. Its misregulation has been linked to prominent human diseases, including cardiac diseases and tumorigenesis. In this chapter, we describe several experimental methods that have proven effective for studying the functions and regulatory features of HDAC5. We present methods for assessing the subcellular localization, protein interactions, posttranslational modifications (PTMs), and activity of HDAC5 from the standpoint of investigating either the endogenous protein or tagged protein forms in human cells. Specifically, given that at the heart of HDAC5 regulation lie its dynamic localization, interactions, and PTMs, we present methods for assessing HDAC5 localization in fixed and live cells, for isolating HDAC5-containing protein complexes to identify its interactions and modifications, and for determining how these PTMs map to predicted HDAC5 structural motifs. Lastly, we provide examples of approaches for studying HDAC5 functions with a focus on its regulation during cell-cycle progression. These methods can readily be adapted for the study of other HDACs or non-HDAC-proteins of interest. Individually, these techniques capture temporal and spatial snapshots of HDAC5 functions; yet together, these approaches provide powerful tools for investigating both the regulation and regulatory roles of HDAC5 in different cell contexts relevant to health and disease. PMID:27246208

DRP1-Dependent Endocytosis is Essential for Polar Localization and Boron-Induced Degradation of the Borate Transporter BOR1 in Arabidopsis thaliana.

PubMed

Yoshinari, Akira; Fujimoto, Masaru; Ueda, Takashi; Inada, Noriko; Naito, Satoshi; Takano, Junpei

2016-09-01

Boron (B) is essential for plants but toxic in excess. The borate efflux transporter BOR1 is expressed in various root cells and localized to the inner/stele-side domain of the plasma membrane (PM) under low-B conditions. BOR1 is rapidly degraded through endocytosis upon sufficient B supply. The polar localization and degradation of BOR1 are considered important for efficient B translocation and avoidance of B toxicity, respectively. In this study, we first analyzed the subcellular localization of BOR1 in roots, cotyledons and hypocotyls, and revealed a polar localization in various cell types. We also found that the inner polarity of BOR1 is established after completion of cytokinesis in the root meristem. Moreover, variable-angle epifluorescence microscopy visualized BOR1-green fluorescent protein (GFP) as particles in the PM with significant lateral movements but in restricted areas. Importantly, a portion of BOR1-GFP particles co-localized with DYNAMIN-RELATED PROTEIN 1A (DRP1A), which is involved in scission of the clathrin-coated vesicles, and they disappeared together from the PM. To examine the contribution of DRP1A-mediated endocytosis to BOR1 localization and degradation, we developed an inducible expression system of the DRP1A K47A variant. The DRP1A variant prolonged the residence time of clathrin on the PM and inhibited endocytosis of membrane lipids. The dominant-negative DRP1A blocked endocytosis of BOR1 and disturbed its polar localization and B-induced degradation. Our results provided insight into the endocytic mechanisms that modulate the subcellular localization and abundance of a mineral transporter for nutrient homeostasis in plant cells. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A Concerted Action of Hepatitis C Virus P7 and Nonstructural Protein 2 Regulates Core Localization at the Endoplasmic Reticulum and Virus Assembly

PubMed Central

Boson, Bertrand; Granio, Ophélia; Bartenschlager, Ralf; Cosset, François-Loïc

2011-01-01

Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly. PMID:21814513

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

Off to the Organelles - Killing Cancer Cells with Targeted Gold Nanoparticles

PubMed Central

Kodiha, Mohamed; Wang, Yi Meng; Hutter, Eliza; Maysinger, Dusica; Stochaj, Ursula

2015-01-01

Gold nanoparticles (AuNPs) are excellent tools for cancer cell imaging and basic research. However, they have yet to reach their full potential in the clinic. At present, we are only beginning to understand the molecular mechanisms that underlie the biological effects of AuNPs, including the structural and functional changes of cancer cells. This knowledge is critical for two aspects of nanomedicine. First, it will define the AuNP-induced events at the subcellular and molecular level, thereby possibly identifying new targets for cancer treatment. Second, it could provide new strategies to improve AuNP-dependent cancer diagnosis and treatment. Our review summarizes the impact of AuNPs on selected subcellular organelles that are relevant to cancer therapy. We focus on the nucleus, its subcompartments, and mitochondria, because they are intimately linked to cancer cell survival, growth, proliferation and death. While non-targeted AuNPs can damage tumor cells, concentrating AuNPs in particular subcellular locations will likely improve tumor cell killing. Thus, it will increase cancer cell damage by photothermal ablation, mechanical injury or localized drug delivery. This concept is promising, but AuNPs have to overcome multiple hurdles to perform these tasks. AuNP size, morphology and surface modification are critical parameters for their delivery to organelles. Recent strategies explored all of these variables, and surface functionalization has become crucial to concentrate AuNPs in subcellular compartments. Here, we highlight the use of AuNPs to damage cancer cells and their organelles. We discuss current limitations of AuNP-based cancer research and conclude with future directions for AuNP-dependent cancer treatment. PMID:25699096

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

Biosensor reveals multiple sources for mitochondrial NAD⁺.

PubMed

Cambronne, Xiaolu A; Stewart, Melissa L; Kim, DongHo; Jones-Brunette, Amber M; Morgan, Rory K; Farrens, David L; Cohen, Michael S; Goodman, Richard H

2016-06-17

Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations. Copyright © 2016, American Association for the Advancement of Science.

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.

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.

The Native Form and Maturation Process of Hepatitis C Virus Core Protein

PubMed Central

Yasui, Kohichiroh; Wakita, Takaji; Tsukiyama-Kohara, Kyoko; Funahashi, Shin-Ichi; Ichikawa, Masumi; Kajita, Tadahiro; Moradpour, Darius; Wands, Jack R.; Kohara, Michinori

1998-01-01

The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles. PMID:9621068

Spatio-temporal manipulation of small GTPase activity at subcellular level and on timescale of seconds in living cells.

PubMed

DeRose, Robert; Pohlmeyer, Christopher; Umeda, Nobuhiro; Ueno, Tasuku; Nagano, Tetsuo; Kuo, Scot; Inoue, Takanari

2012-03-09

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events(1, 2). Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time(3). In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac(4), Cdc42(4), RhoA(4) and Ras(5), in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells(6). Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level(6).

Substrate specificity and subcellular localization of the aldehyde-alcohol redox-coupling reaction in carp cones.

PubMed

Sato, Shinya; Fukagawa, Takashi; Tachibanaki, Shuji; Yamano, Yumiko; Wada, Akimori; Kawamura, Satoru

2013-12-20

Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.

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

Segment and fit thresholding: a new method for image analysis applied to microarray and immunofluorescence data.

PubMed

Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E; Allen, Peter J; Sempere, Lorenzo F; Haab, Brian B

2015-10-06

Experiments involving the high-throughput quantification of image data require algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multicolor, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu's method for selected images. SFT promises to advance the goal of full automation in image analysis.

Segment and Fit Thresholding: A New Method for Image Analysis Applied to Microarray and Immunofluorescence Data

PubMed Central

Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M.; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E.; Allen, Peter J.; Sempere, Lorenzo F.; Haab, Brian B.

2016-01-01

Certain experiments involve the high-throughput quantification of image data, thus requiring algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multi-color, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu’s method for selected images. SFT promises to advance the goal of full automation in image analysis. PMID:26339978

Unique nuclear localization of Nile tilapia (Oreochromis niloticus) Neu4 sialidase is regulated by nuclear transport receptor importin α/β.

PubMed

Honda, Akinobu; Chigwechokha, Petros Kingstone; Kamada-Futagami, Yuko; Komatsu, Masaharu; Shiozaki, Kazuhiro

2018-06-01

Sialidase catalyzes the removal of sialic acids from glycoconjugates. Different from Neu1 and Neu3 sialidases, Neu4 enzymatic properties such as substrate specificity and subcellular localization are not well-conserved among vertebrates. In fish only zebrafish and medaka neu4 genes have been cloned and their polypeptides have been characterized so far. Thus, characterization of Neu4 from other fish species is necessary to evaluate Neu4 physiological functions. Here, Nile tilapia was chosen for the characterization of Neu4 polypeptide considering that it is one of the major cultured fish all over the world and that its genomic sequences are now available. Coding DNA sequence of tilapia Neu4 was identified as 1,497 bp and its recombinant protein showed broad substrate specificity and optimal sialidase enzyme activity pH at 4.0. Neu4 activity was sustained even in neutral and alkali pH. Interestingly, immunofluorescence analysis revealed that major subcellular localization of tilapia Neu4 was nuclear, quite distinct from zebrafish (ER) and medaka Neu4 (lysosome). Bioinformatic analysis showed the existence of putative nuclear localization signal (NLS) in tilapia Neu4. In general, it is known that importin families bind to several proteins via NLS and transfer them into nucleus. Therefore, to determine the involvement of putative NLS in Neu4 nuclear localization, Neu4 mutant deleting NLS was constructed and expressed in cultured cells. As a result, NLS deletion significantly diminished the nuclear localization. Furthermore, treatment of importazole, interrupter of binding importin β and RanGTP, significantly suppressed Neu4 nuclear localization. In summary, tilapia Neu4 is a unique sialidase localized at nucleus and its transport system into nucleus is regulated by importin. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues.

PubMed

Takeuchi, Miyuki; Takabe, Keiji; Mineyuki, Yoshinobu

2016-01-01

Cryofixation and freeze-substitution techniques provide excellent preservation of plant ultrastructure. The advantage of cryofixation is not only in structural preservation, as seen in the smooth plasma membrane, but also in the speed in arresting cell activity. Immunoelectron microscopy reveals the subcellular localization of molecules within cells. Immunolabeling in combination with cryofixation and freeze-substitution techniques provides more detailed information on the immunoelectron-microscopic localization of molecules in the plant cell than can be obtained from chemically fixed tissues. Here, we introduce methods for immunoelectron microscopy of cryofixed and freeze-substituted plant tissues.

Traffic of chitin synthase 1 (CHS-1) to the Spitzenkörper and developing septa in hyphae of Neurospora crassa: actin dependence and evidence of distinct microvesicle populations.

PubMed

Sánchez-León, Eddy; Verdín, Jorge; Freitag, Michael; Roberson, Robert W; Bartnicki-Garcia, Salomon; Riquelme, Meritxell

2011-05-01

We describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases in Neurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1-green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkörper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1-GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1-GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1-GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex.

Extrasynaptic N-Methyl-d-aspartate (NMDA) Receptor Stimulation Induces Cytoplasmic Translocation of the CDKL5 Kinase and Its Proteasomal Degradation*

PubMed Central

Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2011-01-01

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-d-aspartate receptors and suggest regulation of CDKL5 by cell death pathways. PMID:21832092

[Cloning, subcellular localization, and heterologous expression of ApNAC1 gene from Andrographis paniculata].

PubMed

Wang, Jian; Qi, Meng-Die; Guo, Juan; Shen, Ye; Lin, Hui-Xin; Huang, Lu-Qi

2017-03-01

Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis. Copyright© by the Chinese Pharmaceutical Association.

Subcellular localization and photodynamic activity of Photodithazine (glucosamine salt of chlorin e6) in murine melanoma B16-F10: an in vitro and in vivo study

NASA Astrophysics Data System (ADS)

Ono, Bruno Andrade; Pires, Layla; Nogueira, Marcelo Saito; Kurachi, Cristina; Pratavieira, Sebastião.

2018-02-01

Photodynamic therapy (PDT) is already a good option for the clinical treatment of several lesions, including mainly nonmelanoma skin cancers. However, cutaneous melanoma treatment remains a challenge when using PDT. One of the reasons for its reduced efficacy is the high pigmentation of melanoma cells. The object of our study is to evaluate the feasibility of the Photodithazine as a photosensitizer for melanoma. Photodithazine is already used in some malignant tumors with satisfactory results and has significant absorption band around 660 nm where the absorption of melanin is low. In this study, we measured the subcellular localization and photodynamic activity of Photodithazine (PDZ) in murine melanoma B16-F10 cell culture. Additionally, a PDT procedure was applied in an animal melanoma model. This first result demonstrates that Photodithazine is more localized at mitochondria in B16F10 cell culture and the cell viability is reduced to less than 90% using 1 µg/mL (PDZ) and 2 J/cm2. We also noticed a rapid PDZ (less than one hour) accumulation in a murine melanoma model. The treatment of melanoma resulted in 20 % more animal survival after one session of PDT compared with the control group. More studies are required to evaluate the cytotoxic effects of Photodithazine at human melanoma.

Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

PubMed

Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Vázquez-Chávez, Elena; Lasserre, Rémi; Agüera-González, Sonia; Cuche, Céline; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

2017-04-01

The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production. Copyright © 2017 by The American Association of Immunologists, Inc.

LIMK1 regulates Golgi dynamics, traffic of Golgi-derived vesicles, and process extension in primary cultured neurons.

PubMed

Rosso, Silvana; Bollati, Flavia; Bisbal, Mariano; Peretti, Diego; Sumi, Tomoyuki; Nakamura, Toshikazu; Quiroga, Santiago; Ferreira, Adriana; Cáceres, Alfredo

2004-07-01

In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.

A potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein.

PubMed

Majer, Eszter; Navarro, José-Antonio; Daròs, José-Antonio

2015-09-01

Plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino-terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino-terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus-based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Robust prediction of protein subcellular localization combining PCA and WSVMs.

PubMed

Tian, Jiang; Gu, Hong; Liu, Wenqi; Gao, Chiyang

2011-08-01

Automated prediction of protein subcellular localization is an important tool for genome annotation and drug discovery, and Support Vector Machines (SVMs) can effectively solve this problem in a supervised manner. However, the datasets obtained from real experiments are likely to contain outliers or noises, which can lead to poor generalization ability and classification accuracy. To explore this problem, we adopt strategies to lower the effect of outliers. First we design a method based on Weighted SVMs, different weights are assigned to different data points, so the training algorithm will learn the decision boundary according to the relative importance of the data points. Second we analyse the influence of Principal Component Analysis (PCA) on WSVM classification, propose a hybrid classifier combining merits of both PCA and WSVM. After performing dimension reduction operations on the datasets, kernel-based possibilistic c-means algorithm can generate more suitable weights for the training, as PCA transforms the data into a new coordinate system with largest variances affected greatly by the outliers. Experiments on benchmark datasets show promising results, which confirms the effectiveness of the proposed method in terms of prediction accuracy. Copyright © 2011 Elsevier Ltd. All rights reserved.

Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

PubMed Central

Barlag, Britta; Beutel, Oliver; Janning, Dennis; Czarniak, Frederik; Richter, Christian P.; Kommnick, Carina; Göser, Vera; Kurre, Rainer; Fabiani, Florian; Erhardt, Marc; Piehler, Jacob; Hensel, Michael

2016-01-01

The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells. PMID:27534893

The laforin-malin complex negatively regulates glycogen synthesis by modulating cellular glucose uptake via glucose transporters.

PubMed

Singh, Pankaj Kumar; Singh, Sweta; Ganesh, Subramaniam

2012-02-01

Lafora disease (LD), an inherited and fatal neurodegenerative disorder, is characterized by increased cellular glycogen content and the formation of abnormally branched glycogen inclusions, called Lafora bodies, in the affected tissues, including neurons. Therefore, laforin phosphatase and malin ubiquitin E3 ligase, the two proteins that are defective in LD, are thought to regulate glycogen synthesis through an unknown mechanism, the defects in which are likely to underlie some of the symptoms of LD. We show here that laforin's subcellular localization is dependent on the cellular glycogen content and that the stability of laforin is determined by the cellular ATP level, the activity of 5'-AMP-activated protein kinase, and the affinity of malin toward laforin. By using cell and animal models, we further show that the laforin-malin complex regulates cellular glucose uptake by modulating the subcellular localization of glucose transporters; loss of malin or laforin resulted in an increased abundance of glucose transporters in the plasma membrane and therefore excessive glucose uptake. Loss of laforin or malin, however, did not affect glycogen catabolism. Thus, the excessive cellular glucose level appears to be the primary trigger for the abnormally higher levels of cellular glycogen seen in LD.

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.

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

Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells.

PubMed

Kwon, ChangHyuk; Tak, Hyosun; Rho, Mina; Chang, Hae Ryung; Kim, Yon Hui; Kim, Kyung Tae; Balch, Curt; Lee, Eun Kyung; Nam, Seungyoon

2014-03-28

Piwi-interacting RNAs (piRNAs) are 26-31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Dual-channel (green and red) fluorescence microendoscope with subcellular resolution

NASA Astrophysics Data System (ADS)

de Paula D'Almeida, Camila; Fortunato, Thereza Cury; Teixeira Rosa, Ramon Gabriel; Romano, Renan Arnon; Moriyama, Lilian Tan; Pratavieira, Sebastião.

2018-02-01

Usually, tissue images at cellular level need biopsies to be done. Considering this, diagnostic devices, such as microendoscopes, have been developed with the purpose of do not be invasive. This study goal is the development of a dual-channel microendoscope, using two fluorescent labels: proflavine and protoporphyrin IX (PpIX), both approved by Food and Drug Administration. This system, with the potential to perform a microscopic diagnosis and to monitor a photodynamic therapy (PDT) session, uses a halogen lamp and an image fiber bundle to perform subcellular image. Proflavine fluorescence indicates the nuclei of the cell, which is the reference for PpIX localization on image tissue. Preliminary results indicate the efficacy of this optical technique to detect abnormal tissues and to improve the PDT dosimetry. This was the first time, up to our knowledge, that PpIX fluorescence was microscopically observed in vivo, in real time, combined to other fluorescent marker (Proflavine), which allowed to simultaneously observe the spatial localization of the PpIX in the mucosal tissue. We believe this system is very promising tool to monitor PDT in mucosa as it happens. Further experiments have to be performed in order to validate the system for PDT monitoring.

A Role for Timely Nuclear Translocation of Clock Repressor Proteins in Setting Circadian Clock Speed

PubMed Central

Lee, Euna

2014-01-01

By means of a circadian clock system, all the living organisms on earth including human beings can anticipate the environmental rhythmic changes such as light/dark and warm/cold periods in a daily as well as in a yearly manner. Anticipating such environmental changes provide organisms with survival benefits via manifesting behavior and physiology at an advantageous time of the day and year. Cell-autonomous circadian oscillators, governed by transcriptional feedback loop composed of positive and negative elements, are organized into a hierarchical system throughout the organisms and generate an oscillatory expression of a clock gene by itself as well as clock controlled genes (ccgs) with a 24 hr periodicity. In the feedback loop, hetero-dimeric transcription factor complex induces the expression of negative regulatory proteins, which in turn represses the activity of transcription factors to inhibit their own transcription. Thus, for robust oscillatory rhythms of the expression of clock genes as well as ccgs, the precise control of subcellular localization and/or timely translocation of core clock protein are crucial. Here, we discuss how sub-cellular localization and nuclear translocation are controlled in a time-specific manner focusing on the negative regulatory clock proteins. PMID:25258565

Different subcellular localization of neurotensin-receptor and neurotensin-acceptor sites in the rat brain dopaminergic system.

PubMed

Schotte, A; Rostène, W; Laduron, P M

1988-04-01

The subcellular localization of neurotensin-receptor sites (NT2 sites) and neurotensin-acceptor sites (NT1 sites) was studied in rat caudate-putamen by isopycnic centrifugation in sucrose density gradients. [3H]Neurotensin binding to NT2 sites occurred as a major peak at higher sucrose densities, colocalized with [3H]dopamine uptake, and as a small peak at a lower density; whereas binding to NT1 sites occurred as a single large peak at an intermediate density. 6-Hydroxydopamine lesions of the median forebrain bundle resulted in a total loss of NT2 sites in the caudate-putamen but did not affect NT2 sites in the nucleus accumbens and the olfactory tubercle. NT1 sites were not affected. Kainic acid injections into the rat caudate-putamen led to a partial decrease of NT1 sites in this region 5 days later. After a few weeks they returned to normal. Therefore NT2 sites are probably associated with presynaptic nigrostriatal dopaminergic terminals in the caudate-putamen but not in the nucleus accumbens and the olfactory tubercle. A possible association of NT1 sites with glial cells is suggested.

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.

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

A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

PubMed

Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

2014-01-01

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellular localization in cultured macrophages

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

Renard, C.; Vanderhaeghe, H.J.; Claes, P.J.

beta-Lactam antibiotics do not accumulate in phagocytes, probably because of their acidic character. We therefore synthesized a basic derivative of penicillin G, namely, /sup 14/C-labeled N-(3-dimethylamino-propyl)benzylpenicillinamide (ABP), and studied its uptake and subcellular localization in J774 macrophages compared with that of /sup 14/C-labeled penicillin G. Whereas the intracellular concentration (Ci) of penicillin G remained lower than its extracellular concentration (Ce), ABP reached a Ci/Ce ratio of 4 to 5. Moreover, approximately 50% of intracellular ABP was found associated with lysosomes after isopycnic centrifugation of cell homogenates in isoosmotic Percoll or hyperosmotic sucrose gradients. The behavior of ABP was thus partlymore » consistent with the model of de Duve et al., in which they described the intralysosomal accumulation of weak organic bases in lysosomes. Although ABP is microbiologically inactive, our results show that beta-lactam antibiotics can be driven into cells by appropriate modification. Further efforts therefore may be warranted in the design of active compounds or prodrugs that may prove useful in the chemotherapy of intracellular infections.« less

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.

[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.

Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain.

PubMed

Christensen, Inga Baasch; Mogensen, Esben Nees; Damkier, Helle Hasager; Praetorius, Jeppe

2018-05-01

The choroid plexus epithelial cells (CPECs) belong to a small group of polarized cells, where the Na + -K + -ATPase is expressed in the luminal membrane. The basic polarity of the cells is, therefore, still debated. We investigated the subcellular distribution of an array of proteins known to play fundamental roles either in establishing and maintaining basic cell polarity or in the polarized delivery and recycling of plasma membrane proteins. Immunofluorescence histochemical analysis was applied to determine the subcellular localization of apical and basolateral membrane determinants. Mass spectrometry analysis of CPECs isolated by fluorescence-activated cell sorting was applied to determine the expression of specific forms of the proteins. CPECs mainly express the cell-adhesive P-cadherin, which is localized to the lateral membranes. Proteins belonging to the Crumbs and partitioning defective (Par) protein complexes were all localized to the luminal membrane domain. Par-1 and the Scribble complex were localized to the basolateral membrane domain. Lethal(2) giant larvae homolog 2 (Lgl2) labeling was preferentially observed in the luminal membrane domain. Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) was immunolocalized to the basolateral membrane domain, while phosphatidylinositol 4,5-bisphosphate (PIP 2 ) staining was most prominent in the luminal membrane domain along with the PIP 3 phosphatase, Pten. The apical target-SNARE syntaxin-3 and the basolateral target-SNARE syntaxin-4 were both localized to the apical membrane domain in CPECs, which lack cellular expression of the clathrin adaptor protein AP-1B for basolateral protein recycling. In conclusion, the CPECs are conventionally polarized, but express P-cadherin at cell-cell contacts, and Lgl2 and syntaxin-4 in the luminal plasma membrane domain.

Spontaneous Ca2+ sparks and Ca2+ homeostasis in a minimal model of permeabilized ventricular myocytes

PubMed Central

Hartman, Jana M.; Sobie, Eric A.

2010-01-01

Many issues remain unresolved concerning how local, subcellular Ca2+ signals interact with bulk cellular concentrations to maintain homeostasis in health and disease. To aid in the interpretation of data obtained in quiescent ventricular myocytes, we present here a minimal whole cell model that accounts for both localized (subcellular) and global (cellular) aspects of Ca2+ signaling. Using a minimal formulation of the distribution of local [Ca2+] associated with a large number of Ca2+-release sites, the model simulates both random spontaneous Ca2+ sparks and the changes in myoplasmic and sarcoplasmic reticulum (SR) [Ca2+] that result from the balance between stochastic release and reuptake into the SR. Ca2+-release sites are composed of clusters of two-state ryanodine receptors (RyRs) that exhibit activation by local cytosolic [Ca2+] but no inactivation or regulation by luminal Ca2+. Decreasing RyR open probability in the model causes a decrease in aggregate release flux and an increase in SR [Ca2+], regardless of whether RyR inhibition is mediated by a decrease in RyR open dwell time or an increase in RyR closed dwell time. The same balance of stochastic release and reuptake can be achieved, however, by either high-frequency/short-duration or low-frequency/long-duration Ca2+ sparks. The results are well correlated with recent experimental observations using pharmacological RyR inhibitors and clarify those aspects of the release-reuptake balance that are inherent to the coupling between local and global Ca2+ signals and those aspects that depend on molecular-level details. The model of Ca2+ sparks and homeostasis presented here can be a useful tool for understanding changes in cardiac Ca2+ release resulting from drugs, mutations, or acquired diseases. PMID:20852058

Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple).

PubMed

Antony, Edna; Taybi, Tahar; Courbot, Mikaël; Mugford, Sam T; Smith, J Andrew C; Borland, Anne M

2008-01-01

In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

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

Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.

PubMed

Gustavsson, Anna-Karin; Petrov, Petar N; Lee, Maurice Y; Shechtman, Yoav; Moerner, W E

2018-02-01

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5

PubMed Central

Lucas, María; Gaspar, Andrew H.; Pallara, Chiara; Rojas, Adriana Lucely; Fernández-Recio, Juan; Machner, Matthias P.; Hierro, Aitor

2014-01-01

A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA1 activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of amino acid residues located within the VipD–Rab5 interface prevented Rab5 binding and PLA1 activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization. PMID:25114243

Singlet oxygen and ROS in a new light: low-dose subcellular photodynamic treatment enhances proliferation at the single cell level.

PubMed

Blázquez-Castro, Alfonso; Breitenbach, Thomas; Ogilby, Peter R

2014-09-01

Two-photon excitation of a sensitizer with a focused laser beam was used to create a spatially-localized subcellular population of reactive oxygen species, ROS, in single HeLa cells. The sensitizer used was protoporphyrin IX, PpIX, endogenously derived from 5-aminolevulinic acid delivered to the cells. Although we infer that singlet oxygen, O2(a(1)Δg), is one ROS produced upon irradiation of PpIX under these conditions, it is possible that the superoxide ion, O2(-˙), may also play a role in this system. With a "high" dose of PpIX-sensitized ROS, the expected death of the cell was observed. However, under "low dose" conditions, clear signs of cell proliferation were observed. The present results facilitate studies of ROS-mediated signalling in imaging-based single cell experiments.

Cellular compartmentalization of secondary metabolism

PubMed Central

Kistler, H. Corby; Broz, Karen

2015-01-01

Fungal secondary metabolism is often considered apart from the essential housekeeping functions of the cell. However, there are clear links between fundamental cellular metabolism and the biochemical pathways leading to secondary metabolite synthesis. Besides utilizing key biochemical precursors shared with the most essential processes of the cell (e.g., amino acids, acetyl CoA, NADPH), enzymes for secondary metabolite synthesis are compartmentalized at conserved subcellular sites that position pathway enzymes to use these common biochemical precursors. Co-compartmentalization of secondary metabolism pathway enzymes also may function to channel precursors, promote pathway efficiency and sequester pathway intermediates and products from the rest of the cell. In this review we discuss the compartmentalization of three well-studied fungal secondary metabolite biosynthetic pathways for penicillin G, aflatoxin and deoxynivalenol, and summarize evidence used to infer subcellular localization. We also discuss how these metabolites potentially are trafficked within the cell and may be exported. PMID:25709603

Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas

PubMed Central

Chen, Kevin G.; Valencia, Julio C.; Lai, Barry; Zhang, Guofeng; Paterson, Jill K.; Rouzaud, François; Berens, Werner; Wincovitch, Stephen M.; Garfield, Susan H.; Leapman, Richard D.; Hearing, Vincent J.; Gottesman, Michael M.

2006-01-01

Multidrug resistance mechanisms underlying the intractability of malignant melanomas remain largely unknown. In this study, we demonstrate that the development of multidrug resistance in melanomas involves subcellular sequestration of intracellular cytotoxic drugs such as cis-diaminedichloroplatinum II (cisplatin; CDDP). CDDP is initially sequestered in subcellular organelles such as melanosomes, which significantly reduces its nuclear localization when compared with nonmelanoma/KB-3-1 epidermoid carcinoma cells. The melanosomal accumulation of CDDP remarkably modulates melanogenesis through a pronounced increase in tyrosinase activity. The altered melanogenesis manifested an ≈8-fold increase in both intracellular pigmentation and extracellular transport of melanosomes containing CDDP. Thus, our experiments provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export. Preventing melanosomal sequestration of cytotoxic drugs by inhibiting the functions of melanosomes may have great potential as an approach to improving the chemosensitivity of melanoma cells. PMID:16777967

Measurement of subcellular texture by optical Gabor-like filtering with a digital micromirror device

PubMed Central

Pasternack, Robert M.; Qian, Zhen; Zheng, Jing-Yi; Metaxas, Dimitris N.; White, Eileen; Boustany, Nada N.

2010-01-01

We demonstrate an optical Fourier processing method to quantify object texture arising from subcellular feature orientation within unstained living cells. Using a digital micromirror device as a Fourier spatial filter, we measured cellular responses to two-dimensional optical Gabor-like filters optimized to sense orientation of nonspherical particles, such as mitochondria, with a width around 0.45 μm. Our method showed significantly rounder structures within apoptosis-defective cells lacking the proapoptotic mitochondrial effectors Bax and Bak, when compared with Bax/Bak expressing cells functional for apoptosis, consistent with reported differences in mitochondrial shape in these cells. By decoupling spatial frequency resolution from image resolution, this method enables rapid analysis of nonspherical submicrometer scatterers in an under-sampled large field of view and yields spatially localized morphometric parameters that improve the quantitative assessment of biological function. PMID:18830354

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.

Primary structure and subcellular localization of two fimbrial subunit-like proteins involved in the biosynthesis of K99 fibrillae.

PubMed

Roosendaal, E; Jacobs, A A; Rathman, P; Sondermeyer, C; Stegehuis, F; Oudega, B; de Graaf, F K

1987-09-01

Analysis of the nucleotide sequence of the distal part of the fan gene cluster encoding the proteins involved in the biosynthesis of the fibrillar adhesin, K99, revealed the presence of two structural genes, fanG and fanH. The amino acid sequence of the gene products (FanG and FanH) showed significant homology to the amino acid sequence of the fibrillar subunit protein (FanC). Introduction of a site-specific frameshift mutation in fanG or fanH resulted in a simultaneous decrease in fibrillae production and adhesive capacity. Analysis of subcellular fractions showed that, in contrast to the K99 fibrillar subunit (FanC), both the FanH and the FanG protein were loosely associated with the outer membrane, possibly on the periplasmic side, but were not components of the fimbriae themselves.

Subcellular localization of anthracyclines in cultured rat cardiomyoblasts as possible predictors of cardiotoxicity.

PubMed

Studzian, Kazimierz; Kik, Krzysztof; Lukawska, Malgorzata; Oszczapowicz, Irena; Strek, Malgorzata; Szmigiero, Leszek

2015-10-01

In this study, we compared the cellular uptake, intracellular localization and cytotoxicity of two groups of anthracycline derivatives in cultured H9c2(2-1) rat cardiomyoblasts. The first group consisted of doxorubicin (DOX) and two of its derivatives containing a formamidino group (-N = CH-N<) at the C-3' position with a morpholine (DOXM) or a hexamethyleneimine (DOXH) ring. The second group consisted of daunorubicin (DRB) and its derivatives containing a morpholine (DRBM) or a hexamethyleneimine (DRBH) ring. DOXH and DRBH were taken up by cardiomyoblasts more efficiently than estimated for other tested anthracyclines. The cellular uptakes of DOXM and DRBM were reduced compared to those of the parent compounds. Applied structural modifications of DOX and DRB influenced the subcellular localization of the tested derivatives. DOX and DOXH were localized primarily in nuclei, whereas the other anthracyclines were found in the nuclei and cytoplasm. The percentages of the compounds that accumulated in the nuclei were 80.2 and 54.2 % for DOX and DOXH, respectively. The lowest nuclear accumulation values were observed for DRBM (19.9 %), DRBH (21.9 %) and DOXM (23.7 %). The ability of anthracyclines to accumulate in the nuclei correlated with their DNA binding constants (r = 0.858, P = 0.029). A correlation was found between the accumulation of the tested anthracyclines in the nuclei of cardiomyoblasts and their cardiotoxicity in vivo, which was observed in our previous study. We suggest that cytotoxicity and the anthracycline accumulation level in the nuclei of cultured cardiomyoblasts could be used for early prediction of their cardiotoxicity.

Membrane Targeting of Grb2-associated Binder-1 (Gab1) Scaffolding Protein through Src Myristoylation Sequence Substitutes for Gab1 Pleckstrin Homology Domain and Switches an Epidermal Growth Factor Response to an Invasive Morphogenic Program

PubMed Central

Maroun, Christiane R.; Naujokas, Monica A.; Park, Morag

2003-01-01

The hepatocyte growth factor receptor tyrosine kinase Met promotes cell dissociation and the inherent morphogenic program of epithelial cells. In a search for substrates downstream from Met, we have previously identified the Grb2-associated binder-1 (Gab1) as critical for the morphogenic program. Gab1 is a scaffold protein that acts to diversify the signal downstream from the Met receptor through its ability to couple with multiple signal transduction pathways. Gab1 contains a pleckstrin homology (PH) domain with specificity for phosphatidylinositol 3,4,5-trisphosphate. The phospholipid binding capacity of the Gab1 PH domain is required for the localization of Gab1 at sites of cell-cell contact in colonies of epithelial cells and for epithelial morphogenesis, suggesting that PH domain-dependent subcellular localization of Gab1 is a prerequisite for function. We have investigated the requirement for membrane localization of Gab1 for biological activity. We show that substitution of the Gab1 PH domain with the myristoylation signal from the c-Src protein is sufficient to replace the Gab1 PH domain for epithelial morphogenesis. The membrane targeting of Gab1 enhances Rac activity in the absence of stimulation and switches a nonmorphogenic noninvasive response to epidermal growth factor to a morphogenic invasive program. These results suggest that the subcellular localization of Gab1 is a critical determinant for epithelial morphogenesis and invasiveness. PMID:12686619

Functional Implications of the Subcellular Localization of Ethylene-Induced Chitinase and [beta]-1,3-Glucanase in Bean Leaves.

PubMed Central

Mauch, F.; Staehelin, L. A.

1989-01-01

Plants respond to an attack by potentially pathogenic organisms and to the plant stress hormone ethylene with an increased synthesis of hydrolases such as chitinase and [beta]-1,3-glucanase. We have studied the subcellular localization of these two enzymes in ethylene-treated bean leaves by immunogold cytochemistry and by biochemical fractionation techniques. Our micrographs indicate that chitinase and [beta]-1,3-glucanase accumulate in the vacuole of ethylene-treated leaf cells. Within the vacuole label was found predominantly over ethylene-induced electron dense protein aggregates. A second, minor site of accumulation of [beta]-1,3-glucanase was the cell wall, where label was present nearly exclusively over the middle lamella surrounding intercellular air spaces. Both kinds of antibodies labeled Golgi cisternae of ethylene-treated tissue, suggesting that the newly synthesized chitinase and [beta]-1,3-glucanase are processed in the Golgi apparatus. Biochemical fractionation studies confirmed the accumulation in high concentrations of both chitinase and [beta]-1,3-glucanase in isolated vacuoles, and demonstrated that only [beta]-1,3-glucanase, but not chitinase, was present in intercellular washing fluids collected from ethylene-treated leaves. Based on these results and earlier studies, we propose a model in which the vacuole-localized chitinase and [beta]-1,3-glucanase are used as a last line of defense to be released when the attacked host cells lyse. The cell wall-localized [beta]-1,3-glucanase, on the other hand, would be involved in recognition processes, releasing defense activating signaling molecules from the walls of invading pathogens. PMID:12359894

Stress-activated MAPKs and CRM1 regulate the subcellular localization of Net1A to control cell motility and invasion.

PubMed

Ulu, Arzu; Oh, Wonkyung; Zuo, Yan; Frost, Jeffrey A

2018-02-01

The neuroepithelial cell transforming gene 1A (Net1A, an isoform of Net1) is a RhoA subfamily guanine nucleotide exchange factor (GEF) that localizes to the nucleus in the absence of stimulation, preventing it from activating RhoA. Once relocalized in the cytosol, Net1A stimulates cell motility and extracellular matrix invasion. In the present work, we investigated mechanisms responsible for the cytosolic relocalization of Net1A. We demonstrate that inhibition of MAPK pathways blocks Net1A relocalization, with cells being most sensitive to JNK pathway inhibition. Moreover, activation of the JNK or p38 MAPK family pathway is sufficient to elicit Net1A cytosolic localization. Net1A relocalization stimulated by EGF or JNK activation requires nuclear export mediated by CRM1. JNK1 (also known as MAPK8) phosphorylates Net1A on serine 52, and alanine substitution at this site prevents Net1A relocalization caused by EGF or JNK activation. Glutamic acid substitution at this site is sufficient for Net1A relocalization and results in elevated RhoA signaling to stimulate myosin light chain 2 (MLC2, also known as MYL2) phosphorylation and F-actin accumulation. Net1A S52E expression stimulates cell motility, enables Matrigel invasion and promotes invadopodia formation. These data highlight a novel mechanism for controlling the subcellular localization of Net1A to regulate RhoA activation, cell motility, and invasion. © 2018. Published by The Company of Biologists Ltd.

Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control

PubMed Central

Omori, Yoshihiro; Chaya, Taro; Yoshida, Satoyo; Irie, Shoichi; Tsujii, Toshinori; Furukawa, Takahisa

2015-01-01

Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs). We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR), neuropeptide FF receptor 1 (NPFFR1), and neuromedin U receptor 1 (NMUR1), localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S) is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L) is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO) mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control. PMID:26053317

Design and evaluation of a microfluidic system for inhibition studies of yeast cell signaling

NASA Astrophysics Data System (ADS)

Hamngren, Charlotte; Dinér, Peter; Grøtli, Morten; Goksör, Mattias; Adiels, Caroline B.

2012-10-01

In cell signaling, different perturbations lead to different responses and using traditional biological techniques that result in averaged data may obscure important cell-to-cell variations. The aim of this study was to develop and evaluate a four-inlet microfluidic system that enables single-cell analysis by investigating the effect on Hog1 localization post a selective Hog1 inhibitor treatment during osmotic stress. Optical tweezers was used to position yeast cells in an array of desired size and density inside the microfluidic system. By changing the flow rates through the inlet channels, controlled and rapid introduction of two different perturbations over the cell array was enabled. The placement of the cells was determined by diffusion rates flow simulations. The system was evaluated by monitoring the subcellular localization of a fluorescently tagged kinase of the yeast "High Osmolarity Glycerol" (HOG) pathway, Hog1-GFP. By sequential treatment of the yeast cells with a selective Hog1 kinase inhibitor and sorbitol, the subcellular localization of Hog1-GFP was analysed on a single-cell level. The results showed impaired Hog1-GFP nuclear localization, providing evidence of a congenial design. The setup made it possible to remove and add an agent within 2 seconds, which is valuable for investigating the dynamic signal transduction pathways and cannot be done using traditional methods. We are confident that the features of the four-inlet microfluidic system will be a valuable tool and hence contribute significantly to unravel the mechanisms of the HOG pathway and similar dynamic signal transduction pathways.

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.

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.

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.

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

Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation.

PubMed

de Almeida, Sérgio F; Picarote, Gonçalo; Fleming, John V; Carmo-Fonseca, Maria; Azevedo, Jorge E; de Sousa, Maria

2007-09-21

HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.

Robust syntaxin-4 immunoreactivity in mammalian horizontal cell processes

PubMed Central

HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN HELMUT; VILA, ALEJANDRO; BRECHA, NICHOLAS C.

2009-01-01

Horizontal cells mediate inhibitory feed-forward and feedback communication in the outer retina; however, mechanisms that underlie transmitter release from mammalian horizontal cells are poorly understood. Toward determining whether the molecular machinery for exocytosis is present in horizontal cells, we investigated the localization of syntaxin-4, a SNARE protein involved in targeting vesicles to the plasma membrane, in mouse, rat, and rabbit retinae using immunocytochemistry. We report robust expression of syntaxin-4 in the outer plexiform layer of all three species. Syntaxin-4 occurred in processes and tips of horizontal cells, with regularly spaced, thicker sandwich-like structures along the processes. Double labeling with syntaxin-4 and calbindin antibodies, a horizontal cell marker, demonstrated syntaxin-4 localization to horizontal cell processes; whereas, double labeling with PKC antibodies, a rod bipolar cell (RBC) marker, showed a lack of co-localization, with syntaxin-4 immunolabeling occurring just distal to RBC dendritic tips. Syntaxin-4 immunolabeling occurred within VGLUT-1-immunoreactive photoreceptor terminals and underneath synaptic ribbons, labeled by CtBP2/RIBEYE antibodies, consistent with localization in invaginating horizontal cell tips at photoreceptor triad synapses. Vertical sections of retina immunostained for syntaxin-4 and peanut agglutinin (PNA) established that the prominent patches of syntaxin-4 immunoreactivity were adjacent to the base of cone pedicles. Horizontal sections through the OPL indicate a one-to-one co-localization of syntaxin-4 densities at likely all cone pedicles, with syntaxin-4 immunoreactivity interdigitating with PNA labeling. Pre-embedding immuno-electron microscopy confirmed the subcellular localization of syntaxin-4 labeling to lateral elements at both rod and cone triad synapses. Finally, co-localization with SNAP-25, a possible binding partner of syntaxin-4, indicated co-expression of these SNARE proteins in the same subcellular compartment of the horizontal cell. Taken together, the strong expression of these two SNARE proteins in the processes and endings of horizontal cells at rod and cone terminals suggests that horizontal cell axons and dendrites are likely sites of exocytotic activity. PMID:17640443

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

Fine spatiotemporal activity in contracting myometrium revealed by motion-corrected calcium imaging.

PubMed

Loftus, Fiona C; Shmygol, Anatoly; Richardson, Magnus J E

2014-10-15

Successful childbirth depends on the occurrence of precisely coordinated uterine contractions during labour. Calcium indicator fluorescence imaging is one of the main techniques for investigating the mechanisms governing this physiological process and its pathologies. The effective spatiotemporal resolution of calcium signals is, however, limited by the motion of contracting tissue: structures of interest in the order of microns can move over a hundred times their width during a contraction. The simultaneous changes in local intensity and tissue configuration make motion tracking a non-trivial problem in image analysis and confound many of the standard techniques. This paper presents a method that tracks local motion throughout the tissue and allows for the almost complete removal of motion artefacts. This provides a stabilized calcium signal down to a pixel resolution, which, for the data examined, is in the order of a few microns. As a byproduct of image stabilization, a complete kinematic description of the contraction-relaxation cycle is also obtained. This contains novel information about the mechanical response of the tissue, such as the identification of a characteristic length scale, in the order of 40-50 μm, below which tissue motion is homogeneous. Applied to our data, we illustrate that the method allows for analyses of calcium dynamics in contracting myometrium in unprecedented spatiotemporal detail. Additionally, we use the kinematics of tissue motion to compare calcium signals at the subcellular level and local contractile motion. The computer code used is provided in a freely modifiable form and has potential applicability to in vivo calcium imaging of neural tissue, as well as other smooth muscle tissue. © 2014 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Fine spatiotemporal activity in contracting myometrium revealed by motion-corrected calcium imaging

PubMed Central

Loftus, Fiona C; Shmygol, Anatoly; Richardson, Magnus J E

2014-01-01

Successful childbirth depends on the occurrence of precisely coordinated uterine contractions during labour. Calcium indicator fluorescence imaging is one of the main techniques for investigating the mechanisms governing this physiological process and its pathologies. The effective spatiotemporal resolution of calcium signals is, however, limited by the motion of contracting tissue: structures of interest in the order of microns can move over a hundred times their width during a contraction. The simultaneous changes in local intensity and tissue configuration make motion tracking a non-trivial problem in image analysis and confound many of the standard techniques. This paper presents a method that tracks local motion throughout the tissue and allows for the almost complete removal of motion artefacts. This provides a stabilized calcium signal down to a pixel resolution, which, for the data examined, is in the order of a few microns. As a byproduct of image stabilization, a complete kinematic description of the contraction–relaxation cycle is also obtained. This contains novel information about the mechanical response of the tissue, such as the identification of a characteristic length scale, in the order of 40–50 μm, below which tissue motion is homogeneous. Applied to our data, we illustrate that the method allows for analyses of calcium dynamics in contracting myometrium in unprecedented spatiotemporal detail. Additionally, we use the kinematics of tissue motion to compare calcium signals at the subcellular level and local contractile motion. The computer code used is provided in a freely modifiable form and has potential applicability to in vivo calcium imaging of neural tissue, as well as other smooth muscle tissue. PMID:25085893

Insights into Alternanthera mosaic virus TGB3 functions: Interactions with Nicotiana benthamiana PsbO correlate with chloroplast vesiculation and veinal necrosis caused by TGB3 overexpression

USDA-ARS?s Scientific Manuscript database

Alternanthera mosaic virus (AltMV) triple gene block 3 (TGB3) protein is involved in viral movement. AltMV TGB3 subcellular localization was previously shown to be distinct from that of Potato virus X (PVX) TGB3, and a chloroplast binding domain identified; veinal necrosis and chloroplast vesiculati...

Pseudomonas oleovorans strain KBPF-004 culture supernatants reduced seed transmission of Cucumber green mottle mosaic virus and Pepper mild mottle virus, and remodeled aggregation of 126 kDa and subcellular localization......

USDA-ARS?s Scientific Manuscript database

Efforts to control viral diseases in crop production include several types of physical or chemical treatments; antiviral extracts from a number of plants have also been examined to inhibit plant viral infection. However, treatments utilizing naturally selected microorganisms with activity against pl...

RNA:RNA interaction can enhance RNA localization in Drosophila oocytes

PubMed Central

Hartswood, Eve; Brodie, Jim; Vendra, Georgia; Davis, Ilan; Finnegan, David J.

2012-01-01

RNA localization is a key mechanism for targeting proteins to particular subcellular domains. Sequences necessary and sufficient for localization have been identified, but little is known about factors that affect its kinetics. Transcripts of gurken and the I factor, a non-LTR retrotransposon, colocalize at the nucleus in the dorso–antero corner of the Drosophila oocyte directed by localization signals, the GLS and ILS. I factor RNA localizes faster than gurken after injection into oocytes, due to a difference in the intrinsic localization ability of the GLS and ILS. The kinetics of localization of RNA containing the ILS are enhanced by the presence of a stem–loop, the A loop. This acts as an RNA:RNA interaction element in vivo and in vitro, and stimulates localization of RNA containing other localization signals. RNA:RNA interaction may be a general mechanism for modulating RNA localization and could allow an mRNA that lacks a localization signal to hitchhike on another RNA that has one. PMID:22345148

Autophagy capacity and sub-mitochondrial heterogeneity shape Bnip3-induced mitophagy regulation of apoptosis.

PubMed

Choe, Sehyo Charley; Hamacher-Brady, Anne; Brady, Nathan Ryan

2015-08-08

Mitochondria are key regulators of apoptosis. In response to stress, BH3-only proteins activate pro-apoptotic Bcl2 family proteins Bax and Bak, which induce mitochondrial outer membrane permeabilization (MOMP). While the large-scale mitochondrial release of pro-apoptotic proteins activates caspase-dependent cell death, a limited release results in sub-lethal caspase activation which promotes tumorigenesis. Mitochondrial autophagy (mitophagy) targets dysfunctional mitochondria for degradation by lysosomes, and undergoes extensive crosstalk with apoptosis signaling, but its influence on apoptosis remains undetermined. The BH3-only protein Bnip3 integrates apoptosis and mitophagy signaling at different signaling domains. Bnip3 inhibits pro-survival Bcl2 members via its BH3 domain and activates mitophagy through its LC3 Interacting Region (LIR), which is responsible for binding to autophagosomes. Previously, we have shown that Bnip3-activated mitophagy prior to apoptosis induction can reduce mitochondrial activation of caspases, suggesting that a reduction to mitochondrial levels may be pro-survival. An outstanding question is whether organelle dynamics and/or recently discovered subcellular variations of protein levels responsible for both MOMP sensitivity and crosstalk between apoptosis and mitophagy can influence the cellular apoptosis decision event. To that end, here we undertook a systems biology analysis of mitophagy-apoptosis crosstalk at the level of cellular mitochondrial populations. Based on experimental findings, we developed a multi-scale, hybrid model with an individually adaptive mitochondrial population, whose actions are determined by protein levels, embedded in an agent-based model (ABM) for simulating subcellular dynamics and local feedback via reactive oxygen species signaling. Our model, supported by experimental evidence, identified an emergent regulatory structure within canonical apoptosis signaling. We show that the extent of mitophagy is determined by levels and spatial localization of autophagy capacity, and subcellular mitochondrial protein heterogeneities. Our model identifies mechanisms and conditions that alter the mitophagy decision within mitochondrial subpopulations to an extent sufficient to shape cellular outcome to apoptotic stimuli. Overall, our modeling approach provides means to suggest new experiments and implement findings at multiple scales in order to understand how network topologies and subcellular heterogeneities can influence signaling events at individual organelle level, and hence, determine the emergence of heterogeneity in cellular decisions due the actions of the collective intra-cellular population.

A comparative proteomic strategy for subcellular proteome research: ICAT approach coupled with bioinformatics prediction to ascertain rat liver mitochondrial proteins and indication of mitochondrial localization for catalase.

PubMed

Jiang, Xiao-Sheng; Dai, Jie; Sheng, Quan-Hu; Zhang, Lei; Xia, Qi-Chang; Wu, Jia-Rui; Zeng, Rong

2005-01-01

Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of "contaminating" proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these "contaminants" represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM>1.0, while another 79 proteins have an ICAT ratio of PM:CM<1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM>1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM<1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM>1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location.

The Synaptic Function of α-Synuclein

PubMed Central

Burré, Jacqueline

2015-01-01

α-Synuclein is an abundant neuronal protein which localizes predominantly to presynaptic terminals, and is strongly linked genetically and pathologically to Parkinson’s disease and other neurodegenerative diseases. While the accumulation of α-synuclein in the form of misfolded oligomers and large aggregates defines multiple neurodegenerative diseases called “synucleinopathies”, its cellular function has remained largely unclear, and is the subject of intense investigation. In this review, I focus on the structural characteristics of α-synuclein, its cellular and subcellular localization, and discuss how this relates to its function in neurons, in particular at the neuronal synapse. PMID:26407041

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.

Perkinsus marinus superoxide dismutase 2 (PmSOD2) localizes to single-membrane subcellular compartments

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

Fernandez-Robledo, Jose A.; Schott, Eric J.; Vasta, Gerardo R.

2008-10-17

Perkinsus marinus (Phylum Perkinsozoa), a protozoan parasite of oysters, is considered one of the earliest diverging groups of the lineage leading to dinoflagellates. Perkinsus trophozoites are phagocytosed by oyster hemocytes, where they are likely exposed to reactive oxygen species. As part of its reactive oxygen detoxifying pathway, P. marinus possesses two iron-cofactored SOD (PmSOD1 and PmSOD2). Immunoflourescence analysis of P. marinus trophozoites and gene complementation in yeast revealed that PmSOD1 is targeted to the mitochondria. Surprisingly, although PmSOD2 is characterized by a bipartite N-terminus extension typical of plastid targeting, in preliminary immunofluorescence studies it was visualized as punctuate regions inmore » the cytoplasm that could not be assigned to any organelle. Here, we used immunogold electron microscopy to examine the subcellular localization PmSOD2 in P. marinus trophozoites. Gold grains were mostly associated with single-membrane vesicle-like structures, and eventually, localized to electron-dense, apparently amorphous material present in the lumen of a larger, unique compartment. The images suggested that PmSOD2 is targeted to small vesicles that fuse and/or discharge their content into a larger compartment, possibly the large vacuole typical of the mature trophozoites. In light of the in silico targeting prediction, the association of PmSOD2 with single-membrane compartments raises interesting questions regarding its organellar targeting, and the nature of a putative relic plastid in Perkinsus species.« less

Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts.

PubMed

Ring, Axel; Le Lay, Soazig; Pohl, Juergen; Verkade, Paul; Stremmel, Wolfgang

2006-04-01

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.

Characterization of ecto-nucleotidases in human oviducts with an improved approach simultaneously identifying protein expression and in situ enzyme activity.

PubMed

Villamonte, María Lina; Torrejón-Escribano, Benjamín; Rodríguez-Martínez, Aitor; Trapero, Carla; Vidal, August; Gómez de Aranda, Inmaculada; Sévigny, Jean; Matías-Guiu, Xavier; Martín-Satué, Mireia

2018-03-01

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those taking place in oviducts, including contraction, beating of cilia, and maintenance of fluid composition that, in turn, influences sperm capacitation and hyperactivation, as well as oocyte and embryo nourishing. Ecto-nucleotidases are the enzymes that regulate extracellular ATP and adenosine levels, thus playing a role in reproduction. We have optimized a convenient method for characterizing ecto-nucleotidases that simultaneously localizes the protein and its associated enzyme activity in the same tissue slice and characterizes ecto-nucleotidases in human oviducts. The technique combines immunofluorescence and in situ histochemistry, allowing precise identification of ecto-nucleotidases at a subcellular level. In oviducts, remarkably, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and NTPDase3, with the ability to hydrolyze ATP to AMP, are expressed in ciliated epithelial cells but with different subcellular localization. Ecto-5'nucleotidase/CD73 is also expressed apically in ciliated cells. CD73, together with alkaline phosphatase, also expressed apically in oviductal epithelium, complete the hydrolysis sequence by dephosphorylating AMP to adenosine. The concerted action of these enzymes would contribute to the local increase of adenosine concentration necessary for sperm capacitation. The use of this method would be an asset for testing new potential therapeutic drugs with inhibitory potential, which is of great interest presently in the field of oncology and in other clinical disciplines.

New Korean isolates of Pepper mild mottle virus (PMMoV) differ in symptom severity and subcellular localization of the 126 kDa protein

USDA-ARS?s Scientific Manuscript database

Two isolates of Pepper mild mottle virus (PMMoV) were selected from a nationwide survey of pepper fields in South Korea in 2014 and 2015, in which Cucumber mosaic virus was also detected; the two PMMoV isolates, Sangcheong 47 (S-47, KX399390) and Jeongsong 76 (J-76, KX399389), share ~99% nucleotide ...

Expression and subcellular localization of NHE3 in the human gallbladder epithelium

PubMed Central

Chen, Yongsheng; Kong, Jing; Wu, Shuodong

2014-01-01

Background: Enhanced gallbladder concentrating function is an important factor for the pathogenesis of cholesterol gallstone disease (CGD), but the mechanism is unknown. Potential candidates for regulation of gallbladder ion absorption are suggested to be Na+/H+ exchanger isoform 3 (NHE3). In this study, we investigated the expression and subcellular localization of NHE3 in both acalculous and calculous human gallbladders. Methods: Adult human gallbladder tissue was obtained from 23 patients (7 men, 16 women) who had undergone cholecystectomy. The patients were divided into two groups: Group A (acalculous group) and Group B (calculous group). Gene expression of NHE3 was quantitatively estimated by real-time PCR. Protein expression was studied by Western blotting assays. Furthermore, expression of immunoreactive NHE3 was investigated by immunohistochemistry. Results: There was no significant difference in the NHE3 mRNA expression between calculous and acalculous human gallbladders. NHE3 protein expression in gallbladders from patients with cholelithiasis is increased compared to those without gallstones. Immunohistochemistry studies prove that NHE3 is located both on the apical plasma membrane and in the intracellular pool in human GBECs. Conclusions: NHE3 may play a role in the pathogenesis of human CGD. Additional studies are required to further delineate the underlying mechanisms. PMID:25674247

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins.

PubMed

Islam, Kazi T; Shah, Dilip M; El-Mounadi, Kaoutar

2017-12-24

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro antifungal activity against a broad-spectrum of fungal pathogens and therefore have the potential to be used as antifungal agents in transgenic crops. In order to harness the full potential of plant defensins for diseases control, it is crucial to elucidate their mechanisms of action (MOA). With the advent of advanced microscopy techniques, live-cell imaging has become a powerful tool for understanding the dynamics of the antifungal MOA of plant defensins. Here, a confocal microscopy based live-cell imaging method is described using two fluorescently labeled plant defensins (MtDef4 and MtDef5) in combination with vital fluorescent dyes. This technique enables real-time visualization and analysis of the dynamic events of MtDef4 and MtDef5 internalization into fungal cells. Importantly, this assay generates a wealth of information including internalization kinetics, mode of entry and subcellular localization of these peptides. Along with other cell biological tools, these methods have provided critical insights into the dynamics and complexity of the MOA of these peptides. These tools can also be used to compare the MOA of these peptides against different fungi.

Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line

PubMed Central

Gregorczyk, Karolina P.; Wyżewski, Zbigniew; Szczepanowska, Joanna; Mielcarska, Matylda B.; Bossowska-Nowicka, Magdalena; Gieryńska, Małgorzata; Boratyńska-Jasińska, Anna; Niemiałtowski, Marek G.

2018-01-01

Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with ectromelia virus (ECTV). We used two different cell lines and a combination of immunofluorescence techniques, confocal and electron microscopy, and flow cytometry to address subcellular changes following infection. Early in infection of L929 fibroblasts and RAW 264.7 macrophages, mitochondria gathered around viral factories. Later, the mitochondrial network became fragmented, forming punctate mitochondria that co-localized with the progeny virions. ECTV-co-localized mitochondria associated with the cytoskeleton components. Mitochondrial membrane potential, mitochondrial fission–fusion, mitochondrial mass, and generation of reactive oxygen species (ROS) were severely altered later in ECTV infection leading to damage of mitochondria. These results suggest an important role of mitochondria in supplying energy for virus replication and morphogenesis. Presumably, mitochondria participate in transport of viral particles inside and outside of the cell and/or they are a source of membranes for viral envelope formation. We speculate that the observed changes in the mitochondrial network organization and physiology in ECTV-infected cells provide suitable conditions for viral replication and morphogenesis. PMID:29772718

Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line.

PubMed

Gregorczyk, Karolina P; Wyżewski, Zbigniew; Szczepanowska, Joanna; Toka, Felix N; Mielcarska, Matylda B; Bossowska-Nowicka, Magdalena; Gieryńska, Małgorzata; Boratyńska-Jasińska, Anna; Struzik, Justyna; Niemiałtowski, Marek G; Szulc-Dąbrowska, Lidia

2018-05-16

Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with ectromelia virus (ECTV). We used two different cell lines and a combination of immunofluorescence techniques, confocal and electron microscopy, and flow cytometry to address subcellular changes following infection. Early in infection of L929 fibroblasts and RAW 264.7 macrophages, mitochondria gathered around viral factories. Later, the mitochondrial network became fragmented, forming punctate mitochondria that co-localized with the progeny virions. ECTV-co-localized mitochondria associated with the cytoskeleton components. Mitochondrial membrane potential, mitochondrial fission⁻fusion, mitochondrial mass, and generation of reactive oxygen species (ROS) were severely altered later in ECTV infection leading to damage of mitochondria. These results suggest an important role of mitochondria in supplying energy for virus replication and morphogenesis. Presumably, mitochondria participate in transport of viral particles inside and outside of the cell and/or they are a source of membranes for viral envelope formation. We speculate that the observed changes in the mitochondrial network organization and physiology in ECTV-infected cells provide suitable conditions for viral replication and morphogenesis.

Antibody-based analysis reveals “filamentous vs. non-filamentous” and “cytoplasmic vs. nuclear” crosstalk of cytoskeletal proteins

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

Kumeta, Masahiro, E-mail: kumeta@lif.kyoto-u.ac.jp; Hirai, Yuya; Yoshimura, Shige H.

2013-12-10

To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do notmore » take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus. - Highlights: • A set of monoclonal antibodies were raised against nuclear scaffold proteins. • Helix-based cytoskeletal proteins were involved in nuclear scaffold. • Many cytoskeletal components shuttle into the nucleus in a CRM1-dependent manner. • Sets of antibodies distinguished distinct subcellular localization of a single isoform. • Nuclear keratin is soluble and does not form an obvious filamentous structure.« less

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.

ClubSub-P: Cluster-Based Subcellular Localization Prediction for Gram-Negative Bacteria and Archaea

PubMed Central

Paramasivam, Nagarajan; Linke, Dirk

2011-01-01

The subcellular localization (SCL) of proteins provides important clues to their function in a cell. In our efforts to predict useful vaccine targets against Gram-negative bacteria, we noticed that misannotated start codons frequently lead to wrongly assigned SCLs. This and other problems in SCL prediction, such as the relatively high false-positive and false-negative rates of some tools, can be avoided by applying multiple prediction tools to groups of homologous proteins. Here we present ClubSub-P, an online database that combines existing SCL prediction tools into a consensus pipeline from more than 600 proteomes of fully sequenced microorganisms. On top of the consensus prediction at the level of single sequences, the tool uses clusters of homologous proteins from Gram-negative bacteria and from Archaea to eliminate false-positive and false-negative predictions. ClubSub-P can assign the SCL of proteins from Gram-negative bacteria and Archaea with high precision. The database is searchable, and can easily be expanded using either new bacterial genomes or new prediction tools as they become available. This will further improve the performance of the SCL prediction, as well as the detection of misannotated start codons and other annotation errors. ClubSub-P is available online at http://toolkit.tuebingen.mpg.de/clubsubp/ PMID:22073040

Extra Large G-Protein Interactome Reveals Multiple Stress Response Function and Partner-Dependent XLG Subcellular Localization

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

Liang, Ying; Gao, Yajun; Jones, Alan M.

The three-member family of Arabidopsis extra-large G proteins (XLG1-3) defines the prototype of an atypical Ga subunit in the heterotrimeric G protein complex. Some recent evidence indicate that XLG subunits operate along with its Gbg dimer in root morphology, stress responsiveness, and cytokinin induced development, however downstream targets of activated XLG proteins in the stress pathways are rarely known. In order to assemble a set of candidate XLG-targeted proteins, a yeast two-hybrid complementation-based screen was performed using XLG protein baits to query interactions between XLG and partner protein found in glucose-treated seedlings, roots, and Arabidopsis cells in culture. Seventy twomore » interactors were identified and >60% of a test set displayed in vivo interaction with XLG proteins. Gene co-expression analysis shows that >70% of the interactors are positively correlated with the corresponding XLG partners. Gene Ontology enrichment for all the candidates indicates stress responses and posits a molecular mechanism involving a specific set of transcription factor partners to XLG. Genes encoding two of these transcription factors, SZF1 and 2, require XLG proteins for full NaCl-induced expression. Furthermore, the subcellular localization of the XLG proteins in the nucleus, endosome, and plasma membrane is dependent on the specific interacting partner.« less

Extra Large G-Protein Interactome Reveals Multiple Stress Response Function and Partner-Dependent XLG Subcellular Localization

DOE PAGES

Liang, Ying; Gao, Yajun; Jones, Alan M.

2017-06-13

The three-member family of Arabidopsis extra-large G proteins (XLG1-3) defines the prototype of an atypical Ga subunit in the heterotrimeric G protein complex. Some recent evidence indicate that XLG subunits operate along with its Gbg dimer in root morphology, stress responsiveness, and cytokinin induced development, however downstream targets of activated XLG proteins in the stress pathways are rarely known. In order to assemble a set of candidate XLG-targeted proteins, a yeast two-hybrid complementation-based screen was performed using XLG protein baits to query interactions between XLG and partner protein found in glucose-treated seedlings, roots, and Arabidopsis cells in culture. Seventy twomore » interactors were identified and >60% of a test set displayed in vivo interaction with XLG proteins. Gene co-expression analysis shows that >70% of the interactors are positively correlated with the corresponding XLG partners. Gene Ontology enrichment for all the candidates indicates stress responses and posits a molecular mechanism involving a specific set of transcription factor partners to XLG. Genes encoding two of these transcription factors, SZF1 and 2, require XLG proteins for full NaCl-induced expression. Furthermore, the subcellular localization of the XLG proteins in the nucleus, endosome, and plasma membrane is dependent on the specific interacting partner.« less

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.

Nanometre-scale thermometry in a living cell

NASA Astrophysics Data System (ADS)

Kucsko, G.; Maurer, P. C.; Yao, N. Y.; Kubo, M.; Noh, H. J.; Lo, P. K.; Park, H.; Lukin, M. D.

2013-08-01

Sensitive probing of temperature variations on nanometre scales is an outstanding challenge in many areas of modern science and technology. In particular, a thermometer capable of subdegree temperature resolution over a large range of temperatures as well as integration within a living system could provide a powerful new tool in many areas of biological, physical and chemical research. Possibilities range from the temperature-induced control of gene expression and tumour metabolism to the cell-selective treatment of disease and the study of heat dissipation in integrated circuits. By combining local light-induced heat sources with sensitive nanoscale thermometry, it may also be possible to engineer biological processes at the subcellular level. Here we demonstrate a new approach to nanoscale thermometry that uses coherent manipulation of the electronic spin associated with nitrogen-vacancy colour centres in diamond. Our technique makes it possible to detect temperature variations as small as 1.8 mK (a sensitivity of 9 mK Hz-1/2) in an ultrapure bulk diamond sample. Using nitrogen-vacancy centres in diamond nanocrystals (nanodiamonds), we directly measure the local thermal environment on length scales as short as 200 nanometres. Finally, by introducing both nanodiamonds and gold nanoparticles into a single human embryonic fibroblast, we demonstrate temperature-gradient control and mapping at the subcellular level, enabling unique potential applications in life sciences.

Co-localization of fluorescent labeled lipid nanoparticles with specifically tagged subcellular compartments by single particle tracking at low nanoparticle to cell ratios.

PubMed

Tiffany, Matthew; Szoka, Francis C

2016-11-01

We utilized quantitative high-resolution single particle tracking to study the internalization and endosomal sorting of lipid nanoparticles (LNPs) by HeLa cells in vitro to gain a better understanding of how cells process LNPs that are used for siRNA delivery. We compared the trafficking of three formulations that have been demonstrated to deliver siRNA into cells. They were composed of either a tritratable anionic lipid, formulation of cholesterol hemisuccinate (CHEMS), or a titratatable cationic lipid formulation of 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA) or a non-titratable cationic formulation lipid formulation of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). They also contained either a substantial percentage of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol and 5 mole percent 1,2-dimyristoyl-sn-glycerol-[methoxy(polyethylene glycol)-2000 (PEG-DMG). We optically measured the endosomal pH experienced by individual LNPs, observed the internalization pathways used and tracked the particles as they co-localized with fluorescent protein tags on compartment-specific proteins, during endosomal sorting to the lysosome. The data revealed significant differences in the accumulation in subcellular compartments among the three formulations, which help to explain the observed effects LNP composition exerts on in vitro delivery efficiency.

Phagocytosis of Advanced Glycation End Products (AGEs) in Macrophages Induces Cell Apoptosis.

PubMed

Gao, Yuan; Wake, Hidenori; Morioka, Yuta; Liu, Keyue; Teshigawara, Kiyoshi; Shibuya, Megumi; Zhou, Jingxiu; Mori, Shuji; Takahashi, Hideo; Nishibori, Masahiro

2017-01-01

Advanced glycation end products (AGEs) are the products of a series of nonenzymatic modifications of proteins by reducing sugars. AGEs play a pivotal role in development of diabetic complications and atherosclerosis. Accumulation of AGEs in a vessel wall may contribute to the development of vascular lesions. Although AGEs have a diverse range of bioactivities, the clearance process of AGEs from the extracellular space, including the incorporation of AGEs into specific cells, subcellular localization, and the fate of AGEs, remains unclear. In the present study, we examined the kinetics of the uptake of AGEs by mouse macrophage J774.1 cells in vitro and characterized the process. We demonstrated that AGEs bound to the surface of the cells and were also incorporated into the cytoplasm. The temperature- and time-dependent uptake of AGEs was saturable with AGE concentration and was inhibited by cytochalasin D but not chlorpromazine. We also observed the granule-like appearance of AGE immunoreactivity in subcellular localizations in macrophages. Higher concentrations of AGEs induced intracellular ROS and 4-HNE, which were associated with activation of the NF- κ B pathway and caspase-3. These results suggest that incorporation of AGEs occurred actively by endocytosis in macrophages, leading to apoptosis of these cells through NF- κ B activation.

Subcellular Localization and Rolling Circle Replication of Peach Latent Mosaic Viroid: Hallmarks of Group A Viroids

PubMed Central

Bussière, F.; Lehoux, J.; Thompson, D. A.; Skrzeczkowski, L. J.; Perreault, J.-P.

1999-01-01

We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. No multimeric conformer was detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed. PMID:10400727

Consistent prediction of GO protein localization.

PubMed

Spetale, Flavio E; Arce, Debora; Krsticevic, Flavia; Bulacio, Pilar; Tapia, Elizabeth

2018-05-17

The GO-Cellular Component (GO-CC) ontology provides a controlled vocabulary for the consistent description of the subcellular compartments or macromolecular complexes where proteins may act. Current machine learning-based methods used for the automated GO-CC annotation of proteins suffer from the inconsistency of individual GO-CC term predictions. Here, we present FGGA-CC + , a class of hierarchical graph-based classifiers for the consistent GO-CC annotation of protein coding genes at the subcellular compartment or macromolecular complex levels. Aiming to boost the accuracy of GO-CC predictions, we make use of the protein localization knowledge in the GO-Biological Process (GO-BP) annotations to boost the accuracy of GO-CC prediction. As a result, FGGA-CC + classifiers are built from annotation data in both the GO-CC and GO-BP ontologies. Due to their graph-based design, FGGA-CC + classifiers are fully interpretable and their predictions amenable to expert analysis. Promising results on protein annotation data from five model organisms were obtained. Additionally, successful validation results in the annotation of a challenging subset of tandem duplicated genes in the tomato non-model organism were accomplished. Overall, these results suggest that FGGA-CC + classifiers can indeed be useful for satisfying the huge demand of GO-CC annotation arising from ubiquitous high throughout sequencing and proteomic projects.

Phagocytosis of Advanced Glycation End Products (AGEs) in Macrophages Induces Cell Apoptosis

PubMed Central

Wake, Hidenori; Morioka, Yuta; Liu, Keyue; Shibuya, Megumi; Zhou, Jingxiu; Mori, Shuji; Takahashi, Hideo

2017-01-01

Advanced glycation end products (AGEs) are the products of a series of nonenzymatic modifications of proteins by reducing sugars. AGEs play a pivotal role in development of diabetic complications and atherosclerosis. Accumulation of AGEs in a vessel wall may contribute to the development of vascular lesions. Although AGEs have a diverse range of bioactivities, the clearance process of AGEs from the extracellular space, including the incorporation of AGEs into specific cells, subcellular localization, and the fate of AGEs, remains unclear. In the present study, we examined the kinetics of the uptake of AGEs by mouse macrophage J774.1 cells in vitro and characterized the process. We demonstrated that AGEs bound to the surface of the cells and were also incorporated into the cytoplasm. The temperature- and time-dependent uptake of AGEs was saturable with AGE concentration and was inhibited by cytochalasin D but not chlorpromazine. We also observed the granule-like appearance of AGE immunoreactivity in subcellular localizations in macrophages. Higher concentrations of AGEs induced intracellular ROS and 4-HNE, which were associated with activation of the NF-κB pathway and caspase-3. These results suggest that incorporation of AGEs occurred actively by endocytosis in macrophages, leading to apoptosis of these cells through NF-κB activation. PMID:29430285

Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38.

PubMed

Yustein, Jason T; Xia, Liang; Kahlenburg, J Michelle; Robinson, Dan; Templeton, Dennis; Kung, Hsing-Jien

2003-09-18

The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated. Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s. Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken). For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively. These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus. All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase. We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association. Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence.

A Rapid, Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis

PubMed Central

Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

2014-01-01

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale. PMID:24416168

Using distant supervised learning to identify protein subcellular localizations from full-text scientific articles.

PubMed

Zheng, Wu; Blake, Catherine

2015-10-01

Databases of curated biomedical knowledge, such as the protein-locations reflected in the UniProtKB database, provide an accurate and useful resource to researchers and decision makers. Our goal is to augment the manual efforts currently used to curate knowledge bases with automated approaches that leverage the increased availability of full-text scientific articles. This paper describes experiments that use distant supervised learning to identify protein subcellular localizations, which are important to understand protein function and to identify candidate drug targets. Experiments consider Swiss-Prot, the manually annotated subset of the UniProtKB protein knowledge base, and 43,000 full-text articles from the Journal of Biological Chemistry that contain just under 11.5 million sentences. The system achieves 0.81 precision and 0.49 recall at sentence level and an accuracy of 57% on held-out instances in a test set. Moreover, the approach identifies 8210 instances that are not in the UniProtKB knowledge base. Manual inspection of the 50 most likely relations showed that 41 (82%) were valid. These results have immediate benefit to researchers interested in protein function, and suggest that distant supervision should be explored to complement other manual data curation efforts. Copyright © 2015 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.

The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence

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

Hofbauer, Anna; Peters, Jenny; Arcalis, Elsa

2014-12-11

Naturally occurring storage proteins such as zeins are used as fusion partners for recombinant proteins because they induce the formation of ectopic storage organelles known as protein bodies (PBs) where the proteins are stabilized by intermolecular interactions and the formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER). Here, we have used different targeting sequences to determine whether ectopic PBs composed of the N-terminal portion of mature 27 kDa γ-zein added to a fluorescent protein could be induced to form elsewhere in the cell. The addition of a transit peptide for targeting to plastids causes PBmore » formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain. The subcellular localization of the PBs influences their morphology and the solubility of the stored recombinant fusion protein. Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.« less

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.

Activation of the Lbc Rho Exchange Factor Proto-Oncogene by Truncation of an Extended C Terminus That Regulates Transformation and Targeting

PubMed Central

Sterpetti, Paola; Hack, Andrew A.; Bashar, Mariam P.; Park, Brian; Cheng, Sou-De; Knoll, Joan H. M.; Urano, Takeshi; Feig, Larry A.; Toksoz, Deniz

1999-01-01

The human lbc oncogene product is a guanine nucleotide exchange factor that specifically activates the Rho small GTP binding protein, thus resulting in biologically active, GTP-bound Rho, which in turn mediates actin cytoskeletal reorganization, gene transcription, and entry into the mitotic S phase. In order to elucidate the mechanism of onco-Lbc transformation, here we report that while proto- and onco-lbc cDNAs encode identical N-terminal dbl oncogene homology (DH) and pleckstrin homology (PH) domains, proto-Lbc encodes a novel C terminus absent in the oncoprotein that includes a predicted α-helical region homologous to cyto-matrix proteins, followed by a proline-rich region. The lbc proto-oncogene maps to chromosome 15, and onco-lbc represents a fusion of the lbc proto-oncogene N terminus with a short, unrelated C-terminal sequence from chromosome 7. Both onco- and proto-Lbc can promote formation of GTP-bound Rho in vivo. Proto-Lbc transforming activity is much reduced compared to that of onco-Lbc, and a significant increase in transforming activity requires truncation of both the α-helical and proline-rich regions in the proto-Lbc C terminus. Deletion of the chromosome 7-derived C terminus of onco-Lbc does not destroy transforming activity, demonstrating that it is loss of the proto-Lbc C terminus, rather than gain of an unrelated C-terminus by onco-Lbc, that confers transforming activity. Mutations of onco-Lbc DH and PH domains demonstrate that both domains are necessary for full transforming activity. The proto-Lbc product localizes to the particulate (membrane) fraction, while the majority of the onco-Lbc product is cytosolic, and mutations of the PH domain do not affect this localization. The proto-Lbc C-terminus alone localizes predominantly to the particulate fraction, indicating that the C terminus may play a major role in the correct subcellular localization of proto-Lbc, thus providing a mechanism for regulating Lbc oncogenic potential. PMID:9891067

Histidine residues in the Na+-coupled ascorbic acid transporter-2 (SVCT2) are central regulators of SVCT2 function, modulating pH sensitivity, transporter kinetics, Na+ cooperativity, conformational stability, and subcellular localization.

PubMed

Ormazabal, Valeska; Zuñiga, Felipe A; Escobar, Elizabeth; Aylwin, Carlos; Salas-Burgos, Alexis; Godoy, Alejandro; Reyes, Alejandro M; Vera, Juan Carlos; Rivas, Coralia I

2010-11-19

Na(+)-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His(109), His(203), His(206), His(269), and His(413), are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na(+) cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His(413), localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na(+) and loss of Na(+) cooperativity, which leads to a decreased V(max) without altering the transport K(m); (ii) exofacial histidine residues His(203), His(206), and His(413) may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K(m); and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.

Nucleocytoplasmic shuttling of the rabies virus P protein requires a nuclear localization signal and a CRM1-dependent nuclear export signal

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

Pasdeloup, David; Poisson, Nicolas; Raux, Helene

2005-04-10

Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal partmore » of P (172-297). This domain contains a short lysine-rich stretch ({sup 211}KKYK{sup 214}) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to {beta}-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products.« less

Prominin-1 Localizes to the Open Rims of Outer Segment Lamellae in Xenopus laevis Rod and Cone Photoreceptors

PubMed Central

Han, Zhou; Anderson, David W.

2012-01-01

Purpose. Prominin-1 expresses in rod and cone photoreceptors. Mutations in the prominin-1 gene cause retinal degeneration in humans. In this study, the authors investigated the expression and subcellular localization of xlProminin-1 protein, the Xenopus laevis ortholog of prominin-1, in rod and cone photoreceptors of this frog. Methods. Antibodies specific for xlProminin-1 were generated. Immunoblotting was used to study the expression and posttranslational processing of xlProminin-1 protein. Immunocytochemical light and electron microscopy and transgenesis were used to study the subcellular distribution of xlProminin-1. Results. xlProminin-1 is expressed and is subject to posttranslational proteolytic processing in the retina, brain, and kidney. xlProminin-1 is differently expressed and localized in outer segments of rod and cone photoreceptors of X. laevis. Antibodies specific for the N or C termini of xlProminin-1 labeled the open rims of lamellae of cone outer segments (COS) and the open lamellae at the base of rod outer segments (ROS). By contrast, anti–peripherin-2/rds antibody, Xper5A11, labeled the closed rims of cone lamellae adjacent to the ciliary axoneme and the rims of the closed ROS disks. The extent of labeling of the basal ROS by anti–xlProminin-1 antibodies varied with the light cycle in this frog. The entire ROS was also faintly labeled by both antibodies, a result that contrasts with the current notion that prominin-1 localizes only to the basal ROS. Conclusions. These findings suggest that xlProminin-1 may serve as an anti–fusogenic factor in the regulation of disk morphogenesis and may help to maintain the open lamellar structure of basal ROS and COS disks in X. laevis photoreceptors. PMID:22076989

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.

Nutritional channels in breast cancer.

PubMed

Godoy, Alejandro; Salazar, Katherine; Figueroa, Carlos; Smith, Gary J; de Los Angeles Garcia, Maria; Nualart, Francisco J

2009-09-01

Breast cancers increase glucose uptake by increasing expression of the facilitative glucose transporters (GLUTs), mainly GLUT1. However, little is known about the relationship between GLUT1 expression and malignant potential in breast cancer. In this study, expression and subcellular localization of GLUT1 was analysed in vivo in breast cancer tissue specimens with differing malignant potential, based on the Scarff-Bloom-Richardson (SBRI, II, III) histological grading system, and in vitro in the breast cancer cell lines, MDA-MB-468 and MCF-7, and in MDA-MB-468 cells grown as xenografts in nude athymic BALB/c male mice. In situ hybridization analyses demonstrated similar levels of GLUT1 mRNA expression in tissue sections from breast cancers of all histological grades. However, GLUT1 protein was expressed at higher levels in grade SBRII cancer, compared with SBRI and SBRIII, and associated with the expression of the proliferation marker PCNA. Immunolocalization analyses in SBRII cancers demonstrated a preferential localization of GLUT1 to the portions of the cellular membrane that faced neighbouring cells and formed 'canaliculi-like structures', that we hypothesize could have a potential role as 'nutritional channels'. A similar pattern of GLUT1 localization was observed in confluent cultures of MDA-MB-468 and MCF-7, and in MDA-MB-468 cells grown as xenografts, but not in the normal breast epithelial cell line HMEC. However, no relationship between GLUT1 expression and malignant potential of human breast cancer was observed. Preferential subcellular localization of GLUT1 could represent a physiological adaptation of a subset of breast cancer cells that form infiltrative tumours with a nodular growth pattern and that therefore need a major diffusion of glucose from blood vessels.

The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton ▿

PubMed Central

Luginbuehl, Edith; Ryter, Damaris; Schranz-Zumkehr, Judith; Oberholzer, Michael; Kunz, Stefan; Seebeck, Thomas

2010-01-01

The precise subcellular localization of the components of the cyclic AMP (cAMP) signaling pathways is a crucial aspect of eukaryotic intracellular signaling. In the human pathogen Trypanosoma brucei, the strict control of cAMP levels by cAMP-specific phosphodiesterases is essential for parasite survival, both in cell culture and in the infected host. Among the five cyclic nucleotide phosphodiesterases identified in this organism, two closely related isoenzymes, T. brucei PDEB1 (TbrPDEB1) (PDEB1) and TbrPDEB2 (PDEB2) are predominantly responsible for the maintenance of cAMP levels. Despite their close sequence similarity, they are distinctly localized in the cell. PDEB1 is mostly located in the flagellum, where it forms an integral part of the flagellar skeleton. PDEB2 is mainly located in the cell body, and only a minor part of the protein localizes to the flagellum. The current study, using transfection of procyclic trypanosomes with green fluorescent protein (GFP) reporters, demonstrates that the N termini of the two enzymes are essential for determining their final subcellular localization. The first 70 amino acids of PDEB1 are sufficient to specifically direct a GFP reporter to the flagellum and to lead to its detergent-resistant integration into the flagellar skeleton. In contrast, the analogous region of PDEB2 causes the GFP reporter to reside predominantly in the cell body. Mutagenesis of selected residues in the N-terminal region of PDEB2 demonstrated that single amino acid changes are sufficient to redirect the reporter from a cell body location to stable integration into the flagellar skeleton. PMID:20693305

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

Quantitative Proteomic Profiling of Low Dose Ionizing Radiation Effects in a Human Skin Model

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

Hengel, Shawna; Aldrich, Joshua T.; Waters, Katrina M.

2014-07-29

To assess molecular responses to low doses of radiation that may be encountered during medical diagnostic procedures, nuclear accidents, or terrorist acts, a quantitative global proteomic approach was used to identify protein alterations in a reconstituted human skin tissue treated with 10 cGy of ionizing radiation. Subcellular fractionation was employed to remove highly abundant structural proteins and provide insight on radiation induced alterations in protein abundance and localization. In addition, peptides were post-fractionated using high resolution 2-dimensional liquid chromatography to increase the dynamic range of detection of protein abundance and translocation changes. Quantitative data was obtained by labeling peptides withmore » 8-plex isobaric iTRAQ tags. A total of 207 proteins were detected with statistically significant alterations in abundance and/or subcellular localization compared to sham irradiated tissues. Bioinformatics analysis of the data indicated that the top canonical pathways affected by low dose radiation are related to cellular metabolism. Among the proteins showing alterations in abundance, localization and proteolytic processing was the skin barrier protein filaggrin which is consistent with our previous observation that ionizing radiation alters profilaggrin processing with potential effects on skin barrier functions. In addition, a large number of proteases and protease regulators were affected by low dose radiation exposure indicating that altered proteolytic activity may be a hallmark of low dose radiation exposure. While several studies have demonstrated altered transcriptional regulation occurs following low dose radiation exposures, the data presented here indicates post-transcriptional regulation of protein abundance, localization, and proteolytic processing play an important role in regulating radiation responses in complex human tissues.« less

Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

PubMed

Watanabe, Shigeki; Richards, Jackson; Hollopeter, Gunther; Hobson, Robert J; Davis, Wayne M; Jorgensen, Erik M

2012-12-03

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

pLoc-mAnimal: predict subcellular localization of animal proteins with both single and multiple sites.

PubMed

Cheng, Xiang; Zhao, Shu-Guang; Lin, Wei-Zhong; Xiao, Xuan; Chou, Kuo-Chen

2017-11-15

Cells are deemed the basic unit of life. However, many important functions of cells as well as their growth and reproduction are performed via the protein molecules located at their different organelles or locations. Facing explosive growth of protein sequences, we are challenged to develop fast and effective method to annotate their subcellular localization. However, this is by no means an easy task. Particularly, mounting evidences have indicated proteins have multi-label feature meaning that they may simultaneously exist at, or move between, two or more different subcellular location sites. Unfortunately, most of the existing computational methods can only be used to deal with the single-label proteins. Although the 'iLoc-Animal' predictor developed recently is quite powerful that can be used to deal with the animal proteins with multiple locations as well, its prediction quality needs to be improved, particularly in enhancing the absolute true rate and reducing the absolute false rate. Here we propose a new predictor called 'pLoc-mAnimal', which is superior to iLoc-Animal as shown by the compelling facts. When tested by the most rigorous cross-validation on the same high-quality benchmark dataset, the absolute true success rate achieved by the new predictor is 37% higher and the absolute false rate is four times lower in comparison with the state-of-the-art predictor. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc-mAnimal/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. xxiao@gordonlifescience.org or kcchou@gordonlifescience.org. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

In Situ Spatiotemporal Mapping of Flow Fields around Seeded Stem Cells at the Subcellular Length Scale

PubMed Central

Song, Min Jae; Dean, David; Knothe Tate, Melissa L.

2010-01-01

A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV) for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD) predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms. PMID:20862249

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues.

PubMed

Molenaar, Remco J; Khurshed, Mohammed; Hira, Vashendriya V V; Van Noorden, Cornelis J F

2018-05-26

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P) + and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

iLoc-Animal: a multi-label learning classifier for predicting subcellular localization of animal proteins.

PubMed

Lin, Wei-Zhong; Fang, Jian-An; Xiao, Xuan; Chou, Kuo-Chen

2013-04-05

Predicting protein subcellular localization is a challenging problem, particularly when query proteins have multi-label features meaning that they may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing methods can only be used to deal with the single-label proteins. Actually, multi-label proteins should not be ignored because they usually bear some special function worthy of in-depth studies. By introducing the "multi-label learning" approach, a new predictor, called iLoc-Animal, has been developed that can be used to deal with the systems containing both single- and multi-label animal (metazoan except human) proteins. Meanwhile, to measure the prediction quality of a multi-label system in a rigorous way, five indices were introduced; they are "Absolute-True", "Absolute-False" (or Hamming-Loss"), "Accuracy", "Precision", and "Recall". As a demonstration, the jackknife cross-validation was performed with iLoc-Animal on a benchmark dataset of animal proteins classified into the following 20 location sites: (1) acrosome, (2) cell membrane, (3) centriole, (4) centrosome, (5) cell cortex, (6) cytoplasm, (7) cytoskeleton, (8) endoplasmic reticulum, (9) endosome, (10) extracellular, (11) Golgi apparatus, (12) lysosome, (13) mitochondrion, (14) melanosome, (15) microsome, (16) nucleus, (17) peroxisome, (18) plasma membrane, (19) spindle, and (20) synapse, where many proteins belong to two or more locations. For such a complicated system, the outcomes achieved by iLoc-Animal for all the aforementioned five indices were quite encouraging, indicating that the predictor may become a useful tool in this area. It has not escaped our notice that the multi-label approach and the rigorous measurement metrics can also be used to investigate many other multi-label problems in molecular biology. As a user-friendly web-server, iLoc-Animal is freely accessible to the public at the web-site .

In vivo imaging of microscopic structures in the rat retina

PubMed Central

Geng, Ying; Greenberg, Kenneth P.; Wolfe, Robert; Gray, Daniel C.; Hunter, Jennifer J.; Dubra, Alfredo; Flannery, John G.; Williams, David R.; Porter, Jason

2010-01-01

Purpose The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. We have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo. Methods Green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy. Results Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics (AO). AO correction reduced the total root mean square wavefront error, on average, from 0.30 μm to 0.05 μm (1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was ∼1.84 μm, approximately 82% greater than the FWHM of the diffraction-limited LSF. Conclusions With perfect aberration compensation, the in vivo resolution in the rat eye could be ∼2× greater than that in the human eye due to its large numerical aperture (∼0.43). While the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using AO to investigate the rodent eye in vivo with high resolution. PMID:19578019

Localizome: a server for identifying transmembrane topologies and TM helices of eukaryotic proteins utilizing domain information

PubMed Central

Lee, Sunghoon; Lee, Byungwook; Jang, Insoo; Kim, Sangsoo; Bhak, Jong

2006-01-01

The Localizome server predicts the transmembrane (TM) helix number and TM topology of a user-supplied eukaryotic protein and presents the result as an intuitive graphic representation. It utilizes hmmpfam to detect the presence of Pfam domains and a prediction algorithm, Phobius, to predict the TM helices. The results are combined and checked against the TM topology rules stored in a protein domain database called LocaloDom. LocaloDom is a curated database that contains TM topologies and TM helix numbers of known protein domains. It was constructed from Pfam domains combined with Swiss-Prot annotations and Phobius predictions. The Localizome server corrects the combined results of the user sequence to conform to the rules stored in LocaloDom. Compared with other programs, this server showed the highest accuracy for TM topology prediction: for soluble proteins, the accuracy and coverage were 99 and 75%, respectively, while for TM protein domain regions, they were 96 and 68%, respectively. With a graphical representation of TM topology and TM helix positions with the domain units, the Localizome server is a highly accurate and comprehensive information source for subcellular localization for soluble proteins as well as membrane proteins. The Localizome server can be found at . PMID:16845118

Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization.

PubMed

Shiheido, Hirokazu; Shimizu, Jun

2015-02-20

BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND356-58, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. Copyright © 2015 Elsevier Inc. All rights reserved.

Subcellular localization of celery mannitol dehydrogenase. A cytosolic metabolic enzyme in nuclei.

PubMed Central

Yamamoto, Y T; Zamski, E; Williamson, J D; Conkling, M A; Pharr, D M

1997-01-01

Mannitol dehydrogenase (MTD) is the first enzyme in mannitol catabolism in celery (Apium graveolens L. var dulce [Mill] Pers. cv Florida 638). Mannitol is an important photoassimilate, as well as providing plants with resistance to salt and osmotic stress. Previous work has shown that expression of the celery Mtd gene is regulated by many factors, such as hexose sugars, salt and osmotic stress, and salicylic acid. Furthermore, MTD is present in cells of sink organs, phloem cells, and mannitol-grown suspension cultures. Immunogold localization and biochemical analyses presented here demonstrate that celery MTD is localized in the cytosol and nuclei. Although the cellular density of MTD varies among different cell types, densities of nuclear and cytosolic MTD in a given cell are approximately equal. Biochemical analyses of nuclear extracts from mannitol-grown cultured cells confirmed that the nuclear-localized MTD is enzymatically active. The function(s) of nuclear-localized MTD is unknown. PMID:9414553

A20 Functional Domains Regulate Subcellular Localization and NF-Kappa B Activation

DTIC Science & Technology

2013-08-15

that the first function to be described for A20 was that of an anti -apoptotic protein (55). They based their choice of experiments and preliminary...mediated apoptosis (55). After positive selection of the resulting clones with neomycin and verification of A20 expression, they compared the...Karposi sarcoma herpesvirus (KSHV) mediated cell transformation (72). K13 can directly activate NF-κB by interacting with the IKK complex and is

A photocleavable rapamycin conjugate for spatiotemporal control of small GTPase activity.

PubMed

Umeda, Nobuhiro; Ueno, Tasuku; Pohlmeyer, Christopher; Nagano, Tetsuo; Inoue, Takanari

2011-01-12

We developed a novel method to spatiotemporally control the activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV irradiation. With this system and the spatially confined UV irradiation, we achieved subcellularly localized activation of Rac, a member of small GTPases. Our technique offers a powerful approach to studies of dynamic intracellular signaling events.

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.

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

PubMed Central

Lavenant, Gwendoline Thiery; Zavalin, Andrey I.; Caprioli, Richard M.

2013-01-01

Targeted multiplex Imaging Mass Spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This manuscript describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet. PMID:23397138

Discriminative motif discovery via simulated evolution and random under-sampling.

PubMed

Song, Tao; Gu, Hong

2014-01-01

Conserved motifs in biological sequences are closely related to their structure and functions. Recently, discriminative motif discovery methods have attracted more and more attention. However, little attention has been devoted to the data imbalance problem, which is one of the main reasons affecting the performance of the discriminative models. In this article, a simulated evolution method is applied to solve the multi-class imbalance problem at the stage of data preprocessing, and at the stage of Hidden Markov Models (HMMs) training, a random under-sampling method is introduced for the imbalance between the positive and negative datasets. It is shown that, in the task of discovering targeting motifs of nine subcellular compartments, the motifs found by our method are more conserved than the methods without considering data imbalance problem and recover the most known targeting motifs from Minimotif Miner and InterPro. Meanwhile, we use the found motifs to predict protein subcellular localization and achieve higher prediction precision and recall for the minority classes.

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

High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions

PubMed Central

Nahmani, Marc; Lanahan, Conor; DeRosier, David; Turrigiano, Gina G.

2017-01-01

Superresolution microscopy has fundamentally altered our ability to resolve subcellular proteins, but improving on these techniques to study dense structures composed of single-molecule-sized elements has been a challenge. One possible approach to enhance superresolution precision is to use cryogenic fluorescent imaging, reported to reduce fluorescent protein bleaching rates, thereby increasing the precision of superresolution imaging. Here, we describe an approach to cryogenic photoactivated localization microscopy (cPALM) that permits the use of a room-temperature high-numerical-aperture objective lens to image frozen samples in their native state. We find that cPALM increases photon yields and show that this approach can be used to enhance the effective resolution of two photoactivatable/switchable fluorophore-labeled structures in the same frozen sample. This higher resolution, two-color extension of the cPALM technique will expand the accessibility of this approach to a range of laboratories interested in more precise reconstructions of complex subcellular targets. PMID:28348224

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

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

Detection of focal adhesion kinase activation at membrane microdomains by fluorescence resonance energy transfer.

PubMed

Seong, Jihye; Ouyang, Mingxing; Kim, Taejin; Sun, Jie; Wen, Po-Chao; Lu, Shaoying; Zhuo, Yue; Llewellyn, Nicholas M; Schlaepfer, David D; Guan, Jun-Lin; Chien, Shu; Wang, Yingxiao

2011-07-26

Proper subcellular localization of focal adhesion kinase (FAK) is crucial for many cellular processes. It remains, however, unclear how FAK activity is regulated at subcellular compartments. To visualize the FAK activity at different membrane microdomains, we develop a fluorescence resonance energy transfer (FRET)-based FAK biosensor, and target it into or outside of detergent-resistant membrane (DRM) regions at the plasma membrane. Here we show that, on cell adhesion to extracellular matrix proteins or stimulation by platelet-derived growth factor (PDGF), the FRET responses of DRM-targeting FAK biosensor are stronger than that at non-DRM regions, suggesting that FAK activation can occur at DRM microdomains. Further experiments reveal that the PDGF-induced FAK activation is mediated and maintained by Src activity, whereas FAK activation on cell adhesion is independent of, and in fact essential for the Src activation. Therefore, FAK is activated at membrane microdomains with distinct activation mechanisms in response to different physiological stimuli. © 2011 Macmillan Publishers Limited. All rights reserved.

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.

Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells

PubMed Central

Lake, Michael P.; Bouchard, Louis-S.

2017-01-01

Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging. PMID:28636640

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

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

NASA Astrophysics Data System (ADS)

Thiery-Lavenant, Gwendoline; Zavalin, Andre I.; Caprioli, Richard M.

2013-04-01

Targeted multiplex imaging mass spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This article describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet.

Subcellular storage compartments of bacteriopheophorbide sensitizers

NASA Astrophysics Data System (ADS)

Moser, Joerg G.; Dembeck, U.; Hubert, M.; Spengler, Bernhard; Bayer, Rainer; Wagner, Birgit

1994-03-01

Fluorescence colocalization with the Golgi specific stain, NBD-ceramide, and the mitochondrial localizing stain, Rhodamine 123, confirmed the earlier assumption that the Golgi apparatus is one of the prominent storage compartments for bacteriopheophorbide esters in OAT 75 SCLC cells and several amelanotic melanoma cell lines (A375, Melur SP18, SkAMel 25). Furthermore, a diffuse staining of mitochondria, of non-structured cytoplasm, and an additional storage in melanine vesicles of the amelanotic melanoma cells suggests further storage compartments with quantitatively different contributions to the phototoxicity of bacteriochlorophyll-derived photosensitizers. Independent observations of early phototoxic effects on microfilamentous networks, enzymatic activities (succinate dehydrogenase, lactate dehydrogenase), and redistribution phenomena following primary uptake of the sensitizers let us assume that only a part of the 108 molecules taken up by a cell contribute directly to phototoxicity. Thus it may be asked if a proper subcellular positioning of only a few sensitizer molecules may have similar phototoxic effects as the huge amounts stored at apparently ineffective sites.

In vivo ROS and redox potential fluorescent detection in plants: Present approaches and future perspectives.

PubMed

Ortega-Villasante, Cristina; Burén, Stefan; Barón-Sola, Ángel; Martínez, Flor; Hernández, Luis E

2016-10-15

Reactive oxygen species (ROS) are metabolic by-products in aerobic organisms including plants. Endogenously produced ROS act as cellular messengers and redox regulators involved in several plant biological processes, but excessive accumulation of ROS cause oxidative stress and cell damage. Understanding ROS signalling and stress responses requires precise imaging and quantification of local, subcellular and global ROS dynamics with high selectivity, sensitivity, and spatiotemporal resolution. Several fluorescent vital dyes have been tested so far, which helped to provide relevant spatially resolved information of oxidative stress dynamics in plants subjected to harmful environmental conditions. However, certain plant characteristics, such as high background fluorescence of plant tissues in vivo and antioxidant mechanisms, can interfere with ROS detection. The development of improved small-molecule fluorescent dyes and protein-based ROS sensors targeted to subcellular compartments will enable in vivo monitoring of ROS and redox changes in photosynthetic organisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas

NASA Astrophysics Data System (ADS)

Chen, Kevin G.; Valencia, Julio C.; Lai, Barry; Zhang, Guofeng; Paterson, Jill K.; Rouzaud, François; Berens, Werner; Wincovitch, Stephen M.; Garfield, Susan H.; Leapman, Richard D.; Hearing, Vincent J.; Gottesman, Michael M.

2006-06-01

Multidrug resistance mechanisms underlying the intractability of malignant melanomas remain largely unknown. In this study, we demonstrate that the development of multidrug resistance in melanomas involves subcellular sequestration of intracellular cytotoxic drugs such as cis-diaminedichloroplatinum II (cisplatin; CDDP). CDDP is initially sequestered in subcellular organelles such as melanosomes, which significantly reduces its nuclear localization when compared with nonmelanoma/KB-3-1 epidermoid carcinoma cells. The melanosomal accumulation of CDDP remarkably modulates melanogenesis through a pronounced increase in tyrosinase activity. The altered melanogenesis manifested an 8-fold increase in both intracellular pigmentation and extracellular transport of melanosomes containing CDDP. Thus, our experiments provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export. Preventing melanosomal sequestration of cytotoxic drugs by inhibiting the functions of melanosomes may have great potential as an approach to improving the chemosensitivity of melanoma cells. cancer | melanosomes | skin | tumor therapy | multidrug resistance

Glutamate and GABA receptors and transporters in the basal ganglia: What does their subsynaptic localization reveal about their function?

PubMed Central

Galvan, Adriana; Kuwajima, Masaaki; Smith, Yoland

2006-01-01

GABA and glutamate, the main transmitters in the basal ganglia, exert their effects through ionotropic and metabotropic receptors. The dynamic activation of these receptors in response to released neurotransmitter depends, among other factors, on their precise localization in relation to corresponding synapses. The use of high resolution quantitative electron microscope immunocytochemical techniques has provided in-depth description of the subcellular and subsynaptic localization of these receptors in the CNS. In this article, we review recent findings on the ultrastructural localization of GABA and glutamate receptors and transporters in the basal ganglia, at synaptic, extrasynaptic and presynaptic sites. The anatomical evidence supports numerous potential locations for receptor-neurotransmitter interactions, and raises important questions regarding mechanisms of activation and function of synaptic versus extrasynaptic receptors in the basal ganglia. PMID:17059868

Aquaporins in Salivary Glands: From Basic Research to Clinical Applications

PubMed Central

Delporte, Christine; Bryla, Angélic; Perret, Jason

2016-01-01

Salivary glands are involved in saliva secretion that ensures proper oral health. Aquaporins are expressed in salivary glands and play a major role in saliva secretion. This review will provide an overview of the salivary gland morphology and physiology of saliva secretion, and focus on the expression, subcellular localization and role of aquaporins under physiological and pathophysiological conditions, as well as clinical applications involving aquaporins. This review is highlighting expression and localization of aquaporins in human, rat and mouse, the most studied species and is pointing out possible difference between major salivary glands, i.e., parotid, submandibular and sublingual glands. PMID:26828482

Detecting Circular RNAs by RNA Fluorescence In Situ Hybridization.

PubMed

Zirkel, Anne; Papantonis, Argyris

2018-01-01

Fluorescence in situ hybridization (FISH) coupled to high-resolution microscopy is a powerful method for analyzing the subcellular localization of RNA. However, the detection of circular RNAs (circRNAs) using microscopy is challenging because the only feature of a circRNA that can be used for the probe design is its junction. Circular RNAs are expressed at varying levels, and for their efficient monitoring by FISH, background fluorescence levels need to be kept low. Here, we describe a FISH protocol coupled to high-precision localizations using a single fluorescently labeled probe spanning the circRNA junction; this allows circRNA detection in mammalian cells with high signal-to-noise ratios.

Deep-Sea Bacterium Shewanella piezotolerans WP3 Has Two Dimethyl Sulfoxide Reductases in Distinct Subcellular Locations

PubMed Central

Xiong, Lei; Jian, Huahua

2017-01-01

ABSTRACT Dimethyl sulfoxide (DMSO) acts as a substantial sink for dimethyl sulfide (DMS) in deep waters and is therefore considered a potential electron acceptor supporting abyssal ecosystems. Shewanella piezotolerans WP3 was isolated from west Pacific deep-sea sediments, and two functional DMSO respiratory subsystems are essential for maximum growth of WP3 under in situ conditions (4°C/20 MPa). However, the relationship between these two subsystems and the electron transport pathway underlying DMSO reduction by WP3 remain unknown. In this study, both DMSO reductases (type I and type VI) in WP3 were found to be functionally independent despite their close evolutionary relationship. Moreover, immunogold labeling of DMSO reductase subunits revealed that the type I DMSO reductase was localized on the outer leaflet of the outer membrane, whereas the type VI DMSO reductase was located within the periplasmic space. CymA, a cytoplasmic membrane-bound tetraheme c-type cytochrome, served as a preferential electron transport protein for the type I and type VI DMSO reductases, in which type VI accepted electrons from CymA in a DmsE- and DmsF-independent manner. Based on these results, we proposed a core electron transport model of DMSO reduction in the deep-sea bacterium S. piezotolerans WP3. These results collectively suggest that the possession of two sets of DMSO reductases with distinct subcellular localizations may be an adaptive strategy for WP3 to achieve maximum DMSO utilization in deep-sea environments. IMPORTANCE As the dominant methylated sulfur compound in deep oceanic water, dimethyl sulfoxide (DMSO) has been suggested to play an important role in the marine biogeochemical cycle of the volatile anti-greenhouse gas dimethyl sulfide (DMS). Two sets of DMSO respiratory systems in the deep-sea bacterium Shewanella piezotolerans WP3 have previously been identified to mediate DMSO reduction under in situ conditions (4°C/20 MPa). Here, we report that the two DMSO reductases (type I and type VI) in WP3 have distinct subcellular localizations, in which type I DMSO reductase is localized to the exterior surface of the outer membrane and type VI DMSO reductase resides in the periplasmic space. A core electron transport model of DMSO reduction in WP3 was constructed based on genetic and physiological data. These results will contribute to a comprehensive understanding of the adaptation mechanisms of anaerobic respiratory systems in benthic microorganisms. PMID:28687647

The testis-specific VAD1.3/AEP1 interacts with β-actin and syntaxin 1 and directs peri-nuclear/Golgi expression with bipartite nucleus localization (BNL) sequence.

PubMed

Zuo, Yan; Gao, Jing; Yeung, William S B; Lee, Kai-Fai

2010-10-15

VAD1.3 (AEP1), a novel testis-specific gene, was first isolated from the testis of a retinol-treated vitamin-A-deficient (VAD) rat model. It is expressed at the acrosomal region of spermatids from postnatal day 25. VAD1.3 immunoreactivity is present in rat, human, monkey and porcine spermatids and spermatozoa, suggesting that VAD1.3 may play a role in acrosome formation. However, direct evidence on the detailed sub-cellular localization of the VAD1.3 protein in the acrosome and how VAD1.3 is involved in acrosome formation remains largely unknown. Here, we isolated and identified VAD1.3 interacting proteins by immunoprecipitation followed by mass spectrometry, and determined the functional motifs of VAD1.3 that were important for its specific sub-cellular location in vitro. We found that VAD1.3 bound to syntaxin 1 and β-actin proteins in vitro. Immunogold electron microscopic study localized VAD1.3 immunoreactivity to the acrosome membranes and matrix, and colocalized it with the β-actin protein. The full-length GFP-VAD (1-3601) and GFP-VAD (1-730) fusion proteins that contain the bipartite nucleus localization (BNL) signal were located in the peri-nucleus/Golgi of the transfected cells. In addition, the GFP signal colocalized with the endoplasmic reticulum marker and the syntaxin 1 protein in the transfected HeLa and GC-2spd cells. The C-terminal GFP-VAD (1770-3601) was expressed in the nucleus. Taken together, VAD1.3 interacts with β-actin and syntaxin 1 in vitro. The BNL signal may mediate the peri-nuclei localization of the protein that may interact with syntaxin 1 and β-actin for acrosome formation in spermatogenesis. Crown Copyright © 2010. Published by Elsevier Inc. All rights reserved.

Effects of sulfite on the uptake and binding of benzo[a]pyrene diol epoxide in cultured murine respiratory epithelial cells.

PubMed Central

Green, J L; Jones, B C; Reed, G A

1994-01-01

Sulfur dioxide (SO2) may act as a cocarcinogen with benzo[a]pyrene (BaP) in the respiratory tract. We have modeled this effect by examining the interactions of 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with sulfite, the physiological form of SO2, in a murine respiratory epithelial cell line (C10). We exposed C10 cells to [3H]-anti-BPDE and determined the effects of 1 and 10 mM sulfite on the uptake and subcellular localization of labeled products. Autoradiographic analysis showed that sulfite doubled the nuclear localization of anti-BPDE-derived materials after a 4-hr incubation period. The net nuclear localization of anti-BPDE-derived materials was not affected by sulfite during the first 60 min, but nuclear localization continued to increase in the sulfite-containing incubations throughout the 4-hr incubation period. Little increase in nuclear localization of anti-BPDE-derived material was noted in the incubations without sulfite after 60 min. Subcellular fractionation was performed to determine the amount of label associated with cytosolic and nuclear fractions and to determine covalent binding to protein and DNA. Sulfite produced a modest increase in the amount of [3H]-anti-BPDE-derived products bound to protein; however, binding to nuclear DNA increased by more than 200% with 10 mM sulfite. Analysis of the supernatants from the cytosolic and nuclear fractions of cells exposed to anti-BPDE and sulfite demonstrated the presence of 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10c-su lfonate (BPT-10-sulfonate). [3H]-BPT-10-sulfonate was unable to enter C10 cells, suggesting that it is formed intracellularly.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1. Figure 2. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 4. PMID:8033853

Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

PubMed

Dong, Lemeng; Miettinen, Karel; Goedbloed, Miriam; Verstappen, Francel W A; Voster, Alessandra; Jongsma, Maarten A; Memelink, Johan; van der Krol, Sander; Bouwmeester, Harro J

2013-11-01

Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.