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Sample records for affect subcellular localization

  1. Alternative splicing affects the subcellular localization of Drosha

    PubMed Central

    Link, Steffen; Grund, Stefanie E.; Diederichs, Sven

    2016-01-01

    The RNase III enzyme Drosha is a key factor in microRNA (miRNA) biogenesis and as such indispensable for cellular homeostasis and developmental processes. Together with its co-factor DGCR8, it converts the primary transcript (pri-miRNA) into the precursor hairpin (pre-miRNA) in the nucleus. While the middle and the C-terminal domain are crucial for pri-miRNA processing and DGCR8 binding, the function of the N-terminus remains cryptic. Different studies have linked this region to the subcellular localization of Drosha, stabilization and response to stress. In this study, we identify alternatively spliced Drosha transcripts that are devoid of a part of the arginine/serine-rich (RS-rich) domain and expressed in a large set of human cells. In contrast to their expected habitation, we find two isoforms also present in the cytoplasm, while the other two isoforms reside exclusively in the nucleus. Their processing activity for pri-miRNAs and the binding to co-factors remains unaltered. In multiple cell lines, the endogenous mRNA expression of the Drosha isoforms correlates with the localization of endogenous Drosha proteins. The pri-miRNA processing efficiency is not significantly different between groups of cells with or without cytoplasmic Drosha expression. In summary, we discovered novel isoforms of Drosha with differential subcellular localization pointing toward additional layers of complexity in the regulation of its activity. PMID:27185895

  2. Missense mutations in Otopetrin 1 affect subcellular localization and inhibition of purinergic signaling in vestibular supporting cells.

    PubMed

    Kim, Euysoo; Hyrc, Krzysztof L; Speck, Judith; Salles, Felipe T; Lundberg, Yunxia W; Goldberg, Mark P; Kachar, Bechara; Warchol, Mark E; Ornitz, David M

    2011-03-01

    Otopetrin 1 (Otop1) encodes a protein that is essential for the development of otoconia. Otoconia are the extracellular calcium carbonate containing crystals that are important for vestibular mechanosensory transduction of linear motion and gravity. There are two mutant alleles of Otop1 in mice, titled (tlt) and mergulhador (mlh), which result in non-syndromic otoconia agenesis and a consequent balance defect. Biochemically, Otop1 has been shown to modulate purinergic control of intracellular calcium in vestibular supporting cells, which could be one of the mechanisms by which Otop1 participates in the mineralization of otoconia. To understand how tlt and mlh mutations affect the biochemical function of Otop1, we examined the purinergic response of COS7 cells expressing mutant Otop1 proteins, and dissociated sensory epithelial cells from tlt and mlh mice. We also examined the subcellular localization of Otop1 in whole sensory epithelia from tlt and mlh mice. Here we show that tlt and mlh mutations uncouple Otop1 from inhibition of P2Y receptor function. Although the in vitro biochemical function of the Otop1 mutant proteins is normal, in vivo they behave as null alleles. We show that in supporting cells the apical membrane localization of the mutant Otop1 proteins is lost. These data suggest that the tlt and mlh mutations primarily affect the localization of Otop1, which interferes with its ability to interact with other proteins that are important for its cellular and biochemical function.

  3. 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.

  4. RNALocate: a resource for RNA subcellular localizations.

    PubMed

    Zhang, Ting; Tan, Puwen; Wang, Liqiang; Jin, Nana; Li, Yana; Zhang, Lin; Yang, Huan; Hu, Zhenyu; Zhang, Lining; Hu, Chunyu; Li, Chunhua; Qian, Kun; Zhang, Changjian; Huang, Yan; Li, Kongning; Lin, Hao; Wang, Dong

    2017-01-04

    Increasing evidence has revealed that RNA subcellular localization is a very important feature for deeply understanding RNA's biological functions after being transported into intra- or extra-cellular regions. RNALocate is a web-accessible database that aims to provide a high-quality RNA subcellular localization resource and facilitate future researches on RNA function or structure. The current version of RNALocate documents more than 37 700 manually curated RNA subcellular localization entries with experimental evidence, involving more than 21 800 RNAs with 42 subcellular localizations in 65 species, mainly including Homo sapiens, Mus musculus and Saccharomyces cerevisiae etc. Besides, RNA homology, sequence and interaction data have also been integrated into RNALocate. Users can access these data through online search, browse, blast and visualization tools. In conclusion, RNALocate will be of help in elucidating the entirety of RNA subcellular localization, and developing new prediction methods. The database is available at http://www.rna-society.org/rnalocate/.

  5. RNALocate: a resource for RNA subcellular localizations

    PubMed Central

    Zhang, Ting; Tan, Puwen; Wang, Liqiang; Jin, Nana; Li, Yana; Zhang, Lin; Yang, Huan; Hu, Zhenyu; Zhang, Lining; Hu, Chunyu; Li, Chunhua; Qian, Kun; Zhang, Changjian; Huang, Yan; Li, Kongning; Lin, Hao; Wang, Dong

    2017-01-01

    Increasing evidence has revealed that RNA subcellular localization is a very important feature for deeply understanding RNA's biological functions after being transported into intra- or extra-cellular regions. RNALocate is a web-accessible database that aims to provide a high-quality RNA subcellular localization resource and facilitate future researches on RNA function or structure. The current version of RNALocate documents more than 37 700 manually curated RNA subcellular localization entries with experimental evidence, involving more than 21 800 RNAs with 42 subcellular localizations in 65 species, mainly including Homo sapiens, Mus musculus and Saccharomyces cerevisiae etc. Besides, RNA homology, sequence and interaction data have also been integrated into RNALocate. Users can access these data through online search, browse, blast and visualization tools. In conclusion, RNALocate will be of help in elucidating the entirety of RNA subcellular localization, and developing new prediction methods. The database is available at http://www.rna-society.org/rnalocate/. PMID:27543076

  6. In silico identification of new putative pathogenic variants in the NEU1 sialidase gene affecting enzyme function and subcellular localization.

    PubMed

    Bonardi, Dario; Ravasio, Viola; Borsani, Giuseppe; d'Azzo, Alessandra; Bresciani, Roberto; Monti, Eugenio; Giacopuzzi, Edoardo

    2014-01-01

    The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes that remove the terminal sialic acid from oligosaccharide chains. Mutations in NEU1 gene are causative of sialidosis (MIM 256550), a severe lysosomal storage disorder showing autosomal recessive mode of inheritance. Sialidosis has been classified into two subtypes: sialidosis type I, a normomorphic, late-onset form, and sialidosis type II, a more severe neonatal or early-onset form. A total of 50 causative mutations are reported in HGMD database, most of which are missense variants. To further characterize the NEU1 gene and identify new functionally relevant protein isoforms, we decided to study its genetic variability in the human population using the data generated by two large sequencing projects: the 1000 Genomes Project (1000G) and the NHLBI GO Exome Sequencing Project (ESP). Together these two datasets comprise a cohort of 7595 sequenced individuals, making it possible to identify rare variants and dissect population specific ones. By integrating this approach with biochemical and cellular studies, we were able to identify new rare missense and frameshift alleles in NEU1 gene. Among the 9 candidate variants tested, only two resulted in significantly lower levels of sialidase activity (p<0.05), namely c.650T>C and c.700G>A. These two mutations give rise to the amino acid substitutions p.V217A and p.D234N, respectively. NEU1 variants including either of these two amino acid changes have 44% and 25% residual sialidase activity when compared to the wild-type enzyme, reduced protein levels and altered subcellular localization. Thus they may represent new, putative pathological mutations resulting in sialidosis type I. The in silico approach used in this study has enabled the identification of previously unknown NEU1 functional alleles that are widespread in the population and could be tested in future functional studies.

  7. Subcellular localization of the yeast proteome.

    PubMed

    Kumar, Anuj; Agarwal, Seema; Heyman, John A; Matson, Sandra; Heidtman, Matthew; Piccirillo, Stacy; Umansky, Lara; Drawid, Amar; Jansen, Ronald; Liu, Yang; Cheung, Kei-Hoi; Miller, Perry; Gerstein, Mark; Roeder, G Shirleen; Snyder, Michael

    2002-03-15

    Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass approximately 5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability--a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu.

  8. Subcellular localization of the yeast proteome

    PubMed Central

    Kumar, Anuj; Agarwal, Seema; Heyman, John A.; Matson, Sandra; Heidtman, Matthew; Piccirillo, Stacy; Umansky, Lara; Drawid, Amar; Jansen, Ronald; Liu, Yang; Cheung, Kei-Hoi; Miller, Perry; Gerstein, Mark; Roeder, G. Shirleen; Snyder, Michael

    2002-01-01

    Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass ∼5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability—a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu. PMID:11914276

  9. Tau regulates the subcellular localization of calmodulin

    SciTech Connect

    Barreda, Elena Gomez de

    2011-05-13

    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 a 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.

  10. Limited proteolysis differentially modulates the stability and subcellular localization of domains of RPGRIP1 that are distinctly affected by mutations in Leber's congenital amaurosis.

    PubMed

    Lu, Xinrong; Guruju, Mallikarjuna; Oswald, John; Ferreira, Paulo A

    2005-05-15

    The retinitis pigmentosa GTPase regulator (RPGR) protein interacts with the retinitis pigmentosa GTPase regulator interacting protein-1 (RPGRIP1). Genetic lesions in the cognate genes lead to distinct and severe human retinal dystrophies. The biological role of these proteins in retinal function and pathogenesis of retinal diseases is elusive. Here, we present the first physiological assay of the role of RPGRIP1 and mutations therein. We found that the monoallelic and homozygous mutations, DeltaE1279 and D1114G, in the RPGR-interacting domain (RID) of RPGRIP1, enhance and abolish, respectively, its interaction in vivo with RPGR without affecting the stability of RID. In contrast to RID(WT) and RID(D1114G), chemical genetics shows that the interaction of RID(DeltaE1279) with RPGR is resistant to various stress treatments such as osmotic, pH and heat-shock stimuli. Hence, RID(D1114G) and RID(DeltaE1279) constitute loss- and gain-of-function mutations. Moreover, we find that the isoforms, bRPGRIP1 and bRPGRIP1b, undergo limited proteolysis constitutively in vivo in the cytoplasm compartment. This leads to the relocation and accumulation of a small and stable N-terminal domain of approximately 7 kDa to the nucleus, whereas the cytosolic C-terminal domain of RPGRIP1 is degraded and short-lived. The RID(D1114G) and RID(DeltaE1279) mutations exhibit strong cis-acting and antagonistic biological effects on the nuclear relocation, subcellular distribution and proteolytic cleavage of RPGRIP1 and/or domains thereof. These data support distinct and spatiotemporal subcellular-specific roles to RPGRIP1. A novel RPGRIP1-mediated nucleocytoplasmic crosstalk and transport pathway regulated by RID, and hence by RPGR, emerges with implications in the molecular pathogenesis of retinopathies, and a model to other diseases.

  11. Arabidopsis acyl-CoA-binding proteins ACBP4 and ACBP5 are subcellularly localized to the cytosol and ACBP4 depletion affects membrane lipid composition.

    PubMed

    Xiao, Shi; Li, Hong-Ye; Zhang, Jiao-Ping; Chan, Suk-Wah; Chye, Mee-Len

    2008-12-01

    In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by six genes, and they display varying affinities for acyl-CoA esters. Recombinant ACBP4 and ACBP5 have been shown to bind oleoyl-CoA esters in vitro. In this study, the subcellular localizations of ACBP4 and ACBP5 were determined by biochemical fractionation followed by western blot analyses using anti-ACBP4 and anti-ACBP5 antibodies and immuno-electron microscopy. Confocal microscopy of autofluorescence-tagged ACBP4 and ACBP5, expressed transiently in onion epidermal cells and in transgenic Arabidopsis, confirmed their expression in the cytosol. Taken together, ACBP4 and ACBP5 are available in the cytosol to bind and transfer cytosolic oleoyl-CoA esters. Lipid profile analysis further revealed that an acbp4 knockout mutant showed decreases in membrane lipids (digalactosyldiacylglycerol, monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol) while acbp4-complemented lines attained levels similar to wild type, suggesting that ACBP4 plays a role in the biosynthesis of membrane lipids including galactolipids and phospholipids.

  12. Feature Fusion Based SVM Classifier for Protein Subcellular Localization Prediction.

    PubMed

    Rahman, Julia; Mondal, Md Nazrul Islam; Islam, Md Khaled Ben; Hasan, Md Al Mehedi

    2016-12-18

    For the importance of protein subcellular localization in different branches of life science and drug discovery, researchers have focused their attentions on protein subcellular localization prediction. Effective representation of features from protein sequences plays a most vital role in protein subcellular localization prediction specially in case of machine learning techniques. Single feature representation-like pseudo amino acid composition (PseAAC), physiochemical property models (PPM), and amino acid index distribution (AAID) contains insufficient information from protein sequences. To deal with such problems, we have proposed two feature fusion representations, AAIDPAAC and PPMPAAC, to work with Support Vector Machine classifiers, which fused PseAAC with PPM and AAID accordingly. We have evaluated the performance for both single and fused feature representation of a Gram-negative bacterial dataset. We have got at least 3% more actual accuracy by AAIDPAAC and 2% more locative accuracy by PPMPAAC than single feature representation.

  13. Subcellular localization of mammalian type II membrane proteins.

    PubMed

    Aturaliya, Rajith N; Fink, J Lynn; Davis, Melissa J; Teasdale, Melvena S; Hanson, Kelly A; Miranda, Kevin C; Forrest, Alistair R R; Grimmond, Sean M; Suzuki, Harukazu; Kanamori, Mutsumi; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Teasdale, Rohan D

    2006-05-01

    Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

  14. Protein subcellular localization assays using split fluorescent proteins

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    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).

  15. Axonal localization of Ca2+-dependent activator protein for secretion 2 is critical for subcellular locality of brain-derived neurotrophic factor and neurotrophin-3 release affecting proper development of postnatal mouse cerebellum.

    PubMed

    Sadakata, Tetsushi; Kakegawa, Wataru; Shinoda, Yo; Hosono, Mayu; Katoh-Semba, Ritsuko; Sekine, Yukiko; Sato, Yumi; Saruta, Chihiro; Ishizaki, Yasuki; Yuzaki, Michisuke; Kojima, Masami; Furuichi, Teiichi

    2014-01-01

    Ca2+-dependent activator protein for secretion 2 (CAPS2) is a protein that is essential for enhanced release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from cerebellar granule cells. We previously identified dex3, a rare alternative splice variant of CAPS2, which is overrepresented in patients with autism and is missing an exon 3 critical for axonal localization. We recently reported that a mouse model CAPS2Δex3/Δex3 expressing dex3 showed autistic-like behavioral phenotypes including impaired social interaction and cognition and increased anxiety in an unfamiliar environment. Here, we verified impairment in axonal, but not somato-dendritic, localization of dex3 protein in cerebellar granule cells and demonstrated cellular and physiological phenotypes in postnatal cerebellum of CAPS2Δex3/Δex3 mice. Interestingly, both BDNF and NT-3 were markedly reduced in axons of cerebellar granule cells, resulting in a significant decrease in their release. As a result, dex3 mice showed developmental deficits in dendritic arborization of Purkinje cells, vermian lobulation and fissurization, and granule cell precursor proliferation. Paired-pulse facilitation at parallel fiber-Purkinje cell synapses was also impaired. Together, our results indicate that CAPS2 plays an important role in subcellular locality (axonal vs. somato-dendritic) of enhanced BDNF and NT-3 release, which is indispensable for proper development of postnatal cerebellum.

  16. 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.

  17. TESTLoc: protein subcellular localization prediction from EST data

    PubMed Central

    2010-01-01

    Background The eukaryotic cell has an intricate architecture with compartments and substructures dedicated to particular biological processes. Knowing the subcellular location of proteins not only indicates how bio-processes are organized in different cellular compartments, but also contributes to unravelling the function of individual proteins. Computational localization prediction is possible based on sequence information alone, and has been successfully applied to proteins from virtually all subcellular compartments and all domains of life. However, we realized that current prediction tools do not perform well on partial protein sequences such as those inferred from Expressed Sequence Tag (EST) data, limiting the exploitation of the large and taxonomically most comprehensive body of sequence information from eukaryotes. Results We developed a new predictor, TESTLoc, suited for subcellular localization prediction of proteins based on their partial sequence conceptually translated from ESTs (EST-peptides). Support Vector Machine (SVM) is used as computational method and EST-peptides are represented by different features such as amino acid composition and physicochemical properties. When TESTLoc was applied to the most challenging test case (plant data), it yielded high accuracy (~85%). Conclusions TESTLoc is a localization prediction tool tailored for EST data. It provides a variety of models for the users to choose from, and is available for download at http://megasun.bch.umontreal.ca/~shenyq/TESTLoc/TESTLoc.html PMID:21078192

  18. Protein subcellular localization prediction using multiple kernel learning based support vector machine.

    PubMed

    Hasan, Md Al Mehedi; Ahmad, Shamim; Molla, Md Khademul Islam

    2017-03-28

    Predicting the subcellular locations of proteins can provide useful hints that reveal their functions, increase our understanding of the mechanisms of some diseases, and finally aid in the development of novel drugs. As the number of newly discovered proteins has been growing exponentially, which in turns, makes the subcellular localization prediction by purely laboratory tests prohibitively laborious and expensive. In this context, to tackle the challenges, computational methods are being developed as an alternative choice to aid biologists in selecting target proteins and designing related experiments. However, the success of protein subcellular localization prediction is still a complicated and challenging issue, particularly, when query proteins have multi-label characteristics, i.e., if they exist simultaneously in more than one subcellular location or if they move between two or more different subcellular locations. To date, to address this problem, several types of subcellular localization prediction methods with different levels of accuracy have been proposed. The support vector machine (SVM) has been employed to provide potential solutions to the protein subcellular localization prediction problem. However, the practicability of an SVM is affected by the challenges of selecting an appropriate kernel and selecting the parameters of the selected kernel. To address this difficulty, in this study, we aimed to develop an efficient multi-label protein subcellular localization prediction system, named as MKLoc, by introducing multiple kernel learning (MKL) based SVM. We evaluated MKLoc using a combined dataset containing 5447 single-localized proteins (originally published as part of the Höglund dataset) and 3056 multi-localized proteins (originally published as part of the DBMLoc set). Note that this dataset was used by Briesemeister et al. in their extensive comparison of multi-localization prediction systems. Finally, our experimental results indicate that

  19. Self-calibrating viscosity probes: design and subcellular localization.

    PubMed

    Dakanali, Marianna; Do, Thai H; Horn, Austin; Chongchivivat, Akaraphon; Jarusreni, Tuptim; Lichlyter, Darcy; Guizzunti, Gianni; Haidekker, Mark A; Theodorakis, Emmanuel A

    2012-07-15

    We describe the design, synthesis and fluorescence profiles of new self-calibrating viscosity dyes in which a coumarin (reference fluorophore) has been covalently linked with a molecular rotor (viscosity sensor). Characterization of their fluorescence properties was made with separate excitation of the units and through resonance energy transfer from the reference to the sensor dye. We have modified the linker and the substitution of the rotor in order to change the hydrophilicity of these probes thereby altering their subcellular localization. For instance, hydrophilic dye 12 shows a homogeneous distribution inside the cell and represents a suitable probe for viscosity measurements in the cytoplasm.

  20. Subcellular Localization of Carotenoid Biosynthesis in Synechocystis sp. PCC 6803

    PubMed Central

    Selstam, Eva; Norling, Birgitta

    2015-01-01

    The biosynthesis pathway of carotenoids in cyanobacteria is partly described. However, the subcellular localization of individual steps is so far unknown. Carotenoid analysis of different membrane subfractions in Synechocystis sp. PCC6803 shows that “light” plasma membranes have a high carotenoid/protein ratio, when compared to “heavier” plasma membranes or thylakoids. The localization of CrtQ and CrtO, two well-defined carotenoid synthesis pathway enzymes in Synechocystis, was studied by epitope tagging and western blots. Both enzymes are locally more abundant in plasma membranes than in thylakoids, implying that the plasma membrane has higher synthesis rates of β-carotene precursor molecules and echinenone. PMID:26083372

  1. Subcellular localization and regulation of coenzyme A synthase.

    PubMed

    Zhyvoloup, Alexander; Nemazanyy, Ivan; Panasyuk, Ganna; Valovka, Taras; Fenton, Tim; Rebholz, Heike; Wang, Mong-Lien; Foxon, Richard; Lyzogubov, Valeriy; Usenko, Vasylij; Kyyamova, Ramziya; Gorbenko, Olena; Matsuka, Genadiy; Filonenko, Valeriy; Gout, Ivan T

    2003-12-12

    CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.

  2. Subcellular localization of ubiquitin and ubiquitinated proteins in Arabidopsis thaliana.

    PubMed

    Beers, E P; Moreno, T N; Callis, J

    1992-08-05

    Ubiquitin is a highly conserved, 76-amino acid, eukaryotic protein. Its widely accepted role as a proteolytic cofactor depends on its unique ability to covalently ligate to other cellular proteins. While there is good evidence for the existence of such ubiquitinated proteins in the cytosolic and nuclear compartments, relatively little is known about the presence of free ubiquitin and ubiquitinated proteins in other subcellular compartments. This is especially true of higher plants, which have not previously been the subject of extensive biochemical subcellular localizations of ubiquitinated proteins. We extracted cell wall proteins and purified nuclei, vacuoles, chloroplasts, and microsomes from chlorophyllous tissues of Arabidopsis. Immunoblot analyses were used to compare the profiles of ubiquitinated proteins from purified subcellular fractions to those from unfractionated extracts. Purified nuclei contained, in addition to a complex mixture of high molecular mass ubiquitinated proteins, a strongly immunoreactive 28-kDa protein. In the apoplastic extract, we did not detect any ubiquitinated proteins enriched above the background level of those due to cytosolic contamination. Vacuoles appeared to contribute significantly to the ubiquitinated proteins present in the whole protoplast extract. At least three high molecular mass ubiquitinated proteins were unique to the vacuolar extract. Chloroplast stromal proteins did not react specifically with anti-ubiquitin antibodies. When microsomal ubiquitinated proteins were compared to those found in a whole protoplast extract, a distinct pattern was evident. Microsomal ubiquitinated proteins were not visible in the 10,000 x g supernatant used to prepare the 100,000 x g pellet, indicating that they were probably low abundance proteins in the protoplast extract.

  3. The sub-cellular localization of Sulfolobus DNA replication.

    PubMed

    Gristwood, Tamzin; Duggin, Iain G; Wagner, Michaela; Albers, Sonja V; Bell, Stephen D

    2012-07-01

    Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.

  4. Predicting gram-positive bacterial protein subcellular localization based on localization motifs.

    PubMed

    Hu, Yinxia; Li, Tonghua; Sun, Jiangming; Tang, Shengnan; Xiong, Wenwei; Li, Dapeng; Chen, Guanyan; Cong, Peisheng

    2012-09-07

    The subcellular localization of proteins is closely related to their functions. In this work, we propose a novel approach based on localization motifs to improve the accuracy of predicting subcellular localization of Gram-positive bacterial proteins. Our approach performed well on a five-fold cross validation with an overall success rate of 89.5%. Besides, the overall success rate of an independent testing dataset was 97.7%. Moreover, our approach was tested using a new experimentally-determined set of Gram-positive bacteria proteins and achieved an overall success rate of 96.3%.

  5. Tissue distribution and subcellular localization of hyaluronan synthase isoenzymes.

    PubMed

    Törrönen, Kari; Nikunen, Kaisa; Kärnä, Riikka; Tammi, Markku; Tammi, Raija; Rilla, Kirsi

    2014-01-01

    Hyaluronan synthases (HAS) are unique plasma membrane glycosyltransferases secreting this glycosaminoglycan directly to the extracellular space. The three HAS isoenzymes (HAS1, HAS2, and HAS3) expressed in mammalian cells differ in their enzymatic properties and regulation by external stimuli, but clearly distinct functions have not been established. To overview the expression of different HAS isoenzymes during embryonic development and their subcellular localization, we immunostained mouse embryonic samples and cultured cells with HAS antibodies, correlating their distribution to hyaluronan staining. Their subcellular localization was further studied by GFP-HAS fusion proteins. Intense hyaluronan staining was observed throughout the development in the tissues of mesodermal origin, like heart and cartilages, but also for example during the maturation of kidneys and stratified epithelia. In general, staining for one or several HASs correlated with hyaluronan staining. The staining of HAS2 was most widespread, both spatially and temporally, correlating with hyaluronan staining especially in early mesenchymal tissues and heart. While epithelial cells were mostly negative for HASs, stratified epithelia became HAS positive during differentiation. All HAS isoenzymes showed cytoplasmic immunoreactivity, both in tissue sections and cultured cells, while plasma membrane staining was also detected, often in cellular extensions. HAS1 had brightest signal in Golgi, HAS3 in Golgi and microvillous protrusions, whereas most of the endogenous HAS2 immunoreactivity was localized in the ER. This differential pattern was also observed with transfected GFP-HASs. The large proportion of intracellular HASs suggests that HAS forms a reserve that is transported to the plasma membrane for rapid activation of hyaluronan synthesis.

  6. 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

  7. Torso RTK controls Capicua degradation by changing its subcellular localization

    PubMed Central

    Grimm, Oliver; Zini, Victoria Sanchez; Kim, Yoosik; Casanova, Jordi; Shvartsman, Stanislav Y.; Wieschaus, Eric

    2012-01-01

    The transcriptional repressor Capicua (Cic) controls multiple aspects of Drosophila embryogenesis and has been implicated in vertebrate development and human diseases. Receptor tyrosine kinases (RTKs) can antagonize Cic-dependent gene repression, but the mechanisms responsible for this effect are not fully understood. Based on genetic and imaging studies in the early Drosophila embryo, we found that Torso RTK signaling can increase the rate of Cic degradation by changing its subcellular localization. We propose that Cic is degraded predominantly in the cytoplasm and show that Torso reduces the stability of Cic by controlling the rates of its nucleocytoplasmic transport. This model accounts for the experimentally observed spatiotemporal dynamics of Cic in the early embryo and might explain RTK-dependent control of Cic in other developmental contexts. PMID:23048183

  8. Subcellular localization of the simian virus 40 agnoprotein.

    PubMed Central

    Nomura, S; Khoury, G; Jay, G

    1983-01-01

    The intracellular distribution of the simian virus 40 agnoprotein in infected cells was determined by indirect immunofluorescence and biochemical fractionation followed by indirect immunoprecipitation. The specific antibodies used in these studies were directed against either purified agnoprotein or a synthetic oligopeptide homologous to the N-terminus of the processed protein. Both procedures showed predominant localization of the agnoprotein to the cytosol and to the perinuclear region in association with the outer nuclear membrane. A minor but detectable fraction of the protein was also found in the nucleus. The definition of its subcellular distribution, as well as its high lability in vivo and affinity for nucleic acid, provide a basis for speculation on the function of this gene product. Images PMID:6296448

  9. Subcellular localization of hepatitis E virus (HEV) replicase

    SciTech Connect

    Rehman, Shagufta; Kapur, Neeraj; Durgapal, Hemlata; Panda, Subrat Kumar

    2008-01-05

    Hepatitis E virus (HEV) is a hepatotropic virus with a single sense-strand RNA genome of {approx} 7.2 kb in length. Details of the intracellular site of HEV replication can pave further understanding of HEV biology. In-frame fusion construct of functionally active replicase-enhanced green fluorescent protein (EGFP) gene was made in eukaryotic expression vector. The functionality of replicase-EGFP fusion protein was established by its ability to synthesize negative-strand viral RNA in vivo, by strand-specific anchored RT-PCR and molecular beacon binding. Subcellular co-localization was carried out using organelle specific fluorophores and by immuno-electron microscopy. Fluorescence Resonance Energy Transfer (FRET) demonstrated the interaction of this protein with the 3' end of HEV genome. The results show localization of replicase on the endoplasmic reticulum membranes. The protein regions responsible for membrane localization was predicted and identified by use of deletion mutants. Endoplasmic reticulum was identified as the site of replicase localization and possible site of replication.

  10. Cellular and subcellular localization of Marlin-1 in the brain

    PubMed Central

    Vidal, René L; Valenzuela, José I; Luján, Rafael; Couve, Andrés

    2009-01-01

    Background Marlin-1 is a microtubule binding protein that associates specifically with the GABAB1 subunit in neurons and with members of the Janus kinase family in lymphoid cells. In addition, it binds the molecular motor kinesin-I and nucleic acids, preferentially single stranded RNA. Marlin-1 is expressed mainly in the central nervous system but little is known regarding its cellular and subcellular distribution in the brain. Results Here we have studied the localization of Marlin-1 in the rodent brain and cultured neurons combining immunohistochemistry, immunofluorescence and pre-embedding electron microscopy. We demonstrate that Marlin-1 is enriched in restricted areas of the brain including olfactory bulb, cerebral cortex, hippocampus and cerebellum. Marlin-1 is abundant in dendrites and axons of GABAergic and non-GABAergic hippocampal neurons. At the ultrastructural level, Marlin-1 is present in the cytoplasm and the nucleus of CA1 neurons in the hippocampus. In the cytoplasm it associates to microtubules in the dendritic shaft and occasionally with the Golgi apparatus, the endoplasmic reticulum (ER) and dendritic spines. In the nucleus, clusters of Marlin-1 associate to euchromatin. Conclusion Our results demonstrate that Marlin-1 is expressed in discrete areas of the brain. They also confirm the microtubule association at the ultrastructural level in neurons. Together with the abundance of the protein in dendrites and axons they are consistent with the emerging role of Marlin-1 as an intracellular protein linking the cytoskeleton and transport. Our study constitutes the first detailed description of the cellular and subcellular distribution of Marlin-1 in the brain. As such, it will set the basis for future studies on the functional implications of Marlin-1 in protein trafficking. PMID:19386132

  11. 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

  12. Cancer-Related Functions and Subcellular Localizations of Septins

    PubMed Central

    Poüs, Christian; Klipfel, Laurence; Baillet, Anita

    2016-01-01

    Since the initial discovery of septin family GTPases, the understanding of their molecular organization and cellular roles keeps being refined. Septins have been involved in many physiological processes and the misregulation of specific septin gene expression has been implicated in diverse human pathologies, including neurological disorders and cancer. In this minireview, we focus on the importance of the subunit composition and subcellular localization of septins relevant to tumor initiation, progression, and metastasis. We especially underline the importance of septin polymer composition and of their association with the plasma membrane, actin, or microtubules in cell functions involved in cancer and in resistance to cancer therapies. Through their scaffolding role, their function in membrane compartmentalization or through their protective function against protein degradation, septins also emerge as critical organizers of membrane-associated proteins and of signaling pathways implicated in cancer-associated angiogenesis, apoptosis, polarity, migration, proliferation, and in metastasis. Also, the question as to which of the free monomers, hetero-oligomers, or filaments is the functional form of mammalian septins is raised and the control over their spatial and temporal localization is discussed. The increasing amount of crosstalks identified between septins and cellular signaling mediators reinforces the exciting possibility that septins could be new targets in anti-cancer therapies or in therapeutic strategies to limit drug resistance. PMID:27878118

  13. Expression and subcellular localization of ORC1 in Leishmania major

    SciTech Connect

    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 levels 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.

  14. A Balance Between Two Nuclear Localization Sequences and a Nuclear Export Sequence Governs Extradenticle Subcellular Localization

    PubMed Central

    Stevens, Katherine E.; Mann, Richard S.

    2007-01-01

    During animal development, transcription factor activities are modulated by several means, including subcellular localization. The Hox cofactor Extradenticle (Exd) has a dynamic subcellular localization, such that Exd is cytoplasmic by default, but is nuclear when complexed with another homeodomain protein, Homothorax (Hth). These observations raise the question of whether dimerization with Hth simply induces Exd's nuclear localization or, alternatively, if Hth is also necessary for Exd activity. To address this question, we analyzed the nuclear transport signals in Exd, including a divergent nuclear export signal (NES) and two nuclear localization signals (NLSs). We show that, although these signals are weak compared to canonical signals, they balance each other in Exd. We also provide evidence that Exd contains an NLS mask that contributes to its cytoplasmic localization. With these signals characterized, we generated forms of Exd that are nuclear localized in the absence of Hth. Surprisingly, although these Exd forms are functional, they do not phenocopy Hth overexpression. These findings suggest that Hth is required for Exd activity, not simply for inducing its nuclear localization. PMID:17277370

  15. Memo interacts with c-Src to control Estrogen Receptor alpha sub-cellular localization.

    PubMed

    Frei, Anna; MacDonald, Gwen; Lund, Ingrid; Gustafsson, Jan-Åke; Hynes, Nancy E; Nalvarte, Ivan

    2016-08-30

    Understanding the complex interaction between growth factor and steroid hormone signaling pathways in breast cancer is key to identifying suitable therapeutic strategies to avoid progression and therapy resistance. The interaction between these two pathways is of paramount importance for the development of endocrine resistance. Nevertheless, the molecular mechanisms behind their crosstalk are still largely obscure. We previously reported that Memo is a small redox-active protein that controls heregulin-mediated migration of breast cancer cells. Here we report that Memo sits at the intersection between heregulin and estrogen signaling, and that Memo controls Estrogen Receptor alpha (ERα) sub-cellular localization, phosphorylation, and function downstream of heregulin and estrogen in breast cancer cells. Memo facilitates ERα and c-Src interaction, ERα Y537 phosphorylation, and has the ability to control ERα extra-nuclear localization. Thus, we identify Memo as an important key mediator between the heregulin and estrogen signaling pathways, which affects both breast cancer cell migration and proliferation.

  16. Hypoxia elicits broad and systematic changes in protein subcellular localization

    PubMed Central

    Henke, Robert Michael; Dastidar, Ranita Ghosh; Shah, Ajit; Cadinu, Daniela; Yao, Xiao; Hooda, Jagmohan

    2011-01-01

    Oxygen provides a crucial energy source in eukaryotic cells. Hence, eukaryotes ranging from yeast to humans have developed sophisticated mechanisms to respond to changes in oxygen levels. Regulation of protein localization, like protein modifications, can be an effective mechanism to control protein function and activity. However, the contribution of protein localization in oxygen signaling has not been examined on a genomewide scale. Here, we examine how hypoxia affects protein distribution on a genomewide scale in the model eukaryote, the yeast Saccharomyces cerevisiae. We demonstrate, by live cell imaging, that hypoxia alters the cellular distribution of 203 proteins in yeast. These hypoxia-redistributed proteins include an array of proteins with important functions in various organelles. Many of them are nuclear and are components of key regulatory complexes, such as transcriptional regulatory and chromatin remodeling complexes. Under hypoxia, these proteins are synthesized and retained in the cytosol. Upon reoxygenation, they relocalize effectively to their normal cellular compartments, such as the nucleus, mitochondria, endoplasmic reticulum, and cell periphery. The resumption of the normal cellular locations of many proteins can occur even when protein synthesis is inhibited. Furthermore, we show that the changes in protein distribution induced by hypoxia follow a slower trajectory than those induced by reoxygenation. These results show that the regulation of protein localization is a common and potentially dominant mechanism underlying oxygen signaling and regulation. These results may have broad implications in understanding oxygen signaling and hypoxia responses in higher eukaryotes such as humans. PMID:21753182

  17. 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.

  18. Subcellular localization of Gram-negative bacterial proteins using sparse learning.

    PubMed

    Zheng, Zhonglong; Yang, Jie

    2010-04-01

    One of the main challenges faced by biological applications is to predict protein subcellular localization in an automatic fashion accurately. To achieve this in these applications, a wide variety of machine learning methods have been proposed in recent years. Most of them focus on finding the optimal classification scheme and less of them take the simplifying the complexity of biological system into account. Traditionally such bio-data are analyzed by first performing a feature selection before classification. Motivated by CS (Compressive Sensing), we propose a method which performs locality preserving projection with a sparseness criterion such that the feature selection and dimension reduction are merged into one analysis. The proposed sparse method decreases the complexity of biological system, while increases protein subcellular localization accuracy. Experimental results are quite encouraging, indicating that the aforementioned sparse method is quite promising in dealing with complicated biological problems, such as predicting the subcellular localization of Gram-negative bacterial proteins.

  19. Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

    PubMed Central

    Gohar, Ali Vaziri; Cao, Ruofan; Jenkins, Patrick; Li, Wenyan; Houston, Jessica P.; Houston, Kevin D.

    2013-01-01

    Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime. PMID:24010001

  20. Multi-label multi-kernel transfer learning for human protein subcellular localization.

    PubMed

    Mei, Suyu

    2012-01-01

    Recent years have witnessed much progress in computational modelling for protein subcellular localization. However, the existing sequence-based predictive models demonstrate moderate or unsatisfactory performance, and the gene ontology (GO) based models may take the risk of performance overestimation for novel proteins. Furthermore, many human proteins have multiple subcellular locations, which renders the computational modelling more complicated. Up to the present, there are far few researches specialized for predicting the subcellular localization of human proteins that may reside in multiple cellular compartments. In this paper, we propose a multi-label multi-kernel transfer learning model for human protein subcellular localization (MLMK-TLM). MLMK-TLM proposes a multi-label confusion matrix, formally formulates three multi-labelling performance measures and adapts one-against-all multi-class probabilistic outputs to multi-label learning scenario, based on which to further extends our published work GO-TLM (gene ontology based transfer learning model for protein subcellular localization) and MK-TLM (multi-kernel transfer learning based on Chou's PseAAC formulation for protein submitochondria localization) for multiplex human protein subcellular localization. With the advantages of proper homolog knowledge transfer, comprehensive survey of model performance for novel protein and multi-labelling capability, MLMK-TLM will gain more practical applicability. The experiments on human protein benchmark dataset show that MLMK-TLM significantly outperforms the baseline model and demonstrates good multi-labelling ability for novel human proteins. Some findings (predictions) are validated by the latest Swiss-Prot database. The software can be freely downloaded at http://soft.synu.edu.cn/upload/msy.rar.

  1. Identifying subcellular localizations of mammalian protein complexes based on graph theory with a random forest algorithm.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Chen, Chao; Xie, Yun; Dai, Zong; Zou, Xiao-Yong

    2013-04-05

    In the post-genome era, one of the most important and challenging tasks is to identify the subcellular localizations of protein complexes, and further elucidate their functions in human health with applications to understand disease mechanisms, diagnosis and therapy. Although various experimental approaches have been developed and employed to identify the subcellular localizations of protein complexes, the laboratory technologies fall far behind the rapid accumulation of protein complexes. Therefore, it is highly desirable to develop a computational method to rapidly and reliably identify the subcellular localizations of protein complexes. In this study, a novel method is proposed for predicting subcellular localizations of mammalian protein complexes based on graph theory with a random forest algorithm. Protein complexes are modeled as weighted graphs containing nodes and edges, where nodes represent proteins, edges represent protein-protein interactions and weights are descriptors of protein primary structures. Some topological structure features are proposed and adopted to characterize protein complexes based on graph theory. Random forest is employed to construct a model and predict subcellular localizations of protein complexes. Accuracies on a training set by a 10-fold cross-validation test for predicting plasma membrane/membrane attached, cytoplasm and nucleus are 84.78%, 71.30%, and 82.00%, respectively. And accuracies for the independent test set are 81.31%, 69.95% and 81.00%, respectively. These high prediction accuracies exhibit the state-of-the-art performance of the current method. It is anticipated that the proposed method may become a useful high-throughput tool and plays a complementary role to the existing experimental techniques in identifying subcellular localizations of mammalian protein complexes. The source code of Matlab and the dataset can be obtained freely on request from the authors.

  2. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  3. Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion.

    PubMed

    Solis, Gonzalo P; Radon, Yvonne; Sempou, Emily; Jechow, Katharina; Stuermer, Claudia A O; Málaga-Trillo, Edward

    2013-01-01

    Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.

  4. Subcellular localization of calcium deposits during zebrafish (Danio rerio) oogenesis.

    PubMed

    Golpour, Amin; Pšenička, Martin; Niksirat, Hamid

    2016-01-01

    Calcium plays prominent roles in regulating a broad range of physiological events in reproduction. The aim of this study was to describe the subcellular distribution of calcium deposits during stages of oogenesis in zebrafish using a combined oxalate-pyroantimonate technique. The oocyte development of zebrafish was categorized into four stages: primary growth, cortical-alveolus, vitellogenic, and maturation, based on morphological criteria. Calcium deposits in the primary growth stage were detected in the cytoplasm, mitochondria, nucleus, and follicular cells. At the cortical-alveolus stage, calcium particles were transported from follicular cells and deposited in the cortical alveoli. In the vitellogenic stage, some cortical alveoli were compacted and transformed from flocculent electron-lucent to electron-dense objects with the progression of the stage. Calcium deposits were transformed from larger to smaller particles, coinciding with compaction of cortical alveoli. In the maturation stage, calcium deposits in all oocyte compartments decreased, with the exception of those in mitochondria. The proportion of area covered by calcium deposits in the mitochondria and cortical alveoli of oocytes at different stages of development was significantly different (p<0.05). The extent of calcium deposits in the cortical alveoli of mature oocytes was substantially lower than in earlier stages. Basic information about calcium distribution during zebrafish oogenesis may contribute to better understanding of its role in oogenesis.

  5. 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.

  6. Identifying the singleplex and multiplex proteins based on transductive learning for protein subcellular localization prediction.

    PubMed

    Cao, Junzhe; Liu, Wenqi; He, Jianjun; Gu, Hong

    2013-07-01

    A new method is proposed to identify whether a query protein is singleplex or multiplex for improving the quality of protein subcellular localization prediction. Based on the transductive learning technique, this approach utilizes the information from the both query proteins and known proteins to estimate the subcellular location number of every query protein so that the singleplex and multiplex proteins can be recognized and distinguished. Each query protein is then dealt with by a targeted single-label or multi-label predictor to achieve a high-accuracy prediction result. We assess the performance of the proposed approach by applying it to three groups of protein sequences datasets. Simulation experiments show that the proposed approach can effectively identify the singleplex and multiplex proteins. Through a comparison, the reliably of this method for enhancing the power of predicting protein subcellular localization can also be verified.

  7. Application of green fluorescent protein-labeled assay for the study of subcellular localization of Newcastle disease virus matrix protein.

    PubMed

    Duan, Zhiqiang; Li, Qunhui; He, Liang; Zhao, Guo; Chen, Jian; Hu, Shunlin; Liu, Xiufan

    2013-12-01

    Green fluorescent protein (GFP) used as a powerful marker of gene expression in vivo has so far been applied widely in studying the localizations and functions of protein in living cells. In this study, GFP-labeled assay was used to investigate the subcellular localization of matrix (M) protein of different virulence and genotype Newcastle disease virus (NDV) strains. The M protein of ten NDV strains fused with GFP (GFP-M) all showed nuclear-and-nucleolar localization throughout transfection, whereas that of the other two strains were observed in the nucleus and nucleolus early in transfection but in the cytoplasm late in transfection. In addition, mutations to the previously defined nuclear localization signal in the GFP-M fusion protein were studied as well. Single changes at positions 262 and 263 did not affect nuclear localization of M, while changing both of these arginine residues to asparagine caused re-localization of M mainly to the cytoplasm. The GFP-M was validated as a suitable system for studying the subcellular localization of M protein and could be used to assist us in further identifying the signal sequences responsible for the nucleolar localization and cytoplasmic localization of M protein.

  8. Subcellular Localization of Galloylated Catechins in Tea Plants [Camellia sinensis (L.) O. Kuntze] Assessed via Immunohistochemistry

    PubMed Central

    Xu, Huanhuan; Wang, Ya; Chen, Yana; Zhang, Pan; Zhao, Yi; Huang, Yewei; Wang, Xuanjun; Sheng, Jun

    2016-01-01

    Galloylated catechins, as the main secondary metabolites in the tea plant, including (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, comprise approximately three-quarters of all the tea plant catechins and have stronger effects than non-galloylated catechins, both on the product quality in tea processing and the pharmacological efficacy to human beings. The subcellular localization of galloylated catechins has been the primary focus of studies that assess biosynthesis and physiological functions. Classical histochemical localization staining reagents can not specifically detect galloylated catechins; thus, their subcellular localization remains controversial. In the present study, we generated a monoclonal antibody (mAb) against galloylated catechins, which can be used for the subcellular localization of galloylated catechins in the tea plant by immunohistochemistry. Direct ELISA and ForteBio Octet Red 96 System assay indicated the mAb could recognize the galloylated catechins with high specificities and affinities. In addition, tea bud was ascertained as the optimal tissue for freezing microtomic sections for immunohistochemistry. What’s more, the high quality mAbs which exhibited excellent binding capability to galloylated catechins were utilized for the visualization of them via immunohistochemistry. Our findings demonstrated that vacuoles were the primary sites of localization of galloylated catechins at the subcellular level. PMID:27303422

  9. 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.

  10. The cellular and subcellular localization of zinc transporter 7 in the mouse spinal cord

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present work addresses the cellular and subcellular localization of the zinc transporter 7 (ZNT7, SLC30a7) protein and the distribution of zinc ions (Zn2+) in the mouse spinal cord. Our results indicated that the ZNT7 immunoreactive neurons were widely distributed in the Rexed’s laminae of the g...

  11. Fluorescent tags influence the enzymatic activity and subcellular localization of procaspase-1.

    PubMed

    Heymann, Michael C; Rabe, Sabrina; Ruß, Susanne; Kapplusch, Franz; Schulze, Felix; Stein, Robert; Winkler, Stefan; Hedrich, Christian M; Rösen-Wolff, Angela; Hofmann, Sigrun R

    2015-10-01

    Subcellular localization studies and life cell imaging approaches usually benefit from fusion-reporter proteins, such as enhanced green fluorescent protein (EGFP) and mCherry to the proteins of interest. However, such manipulations have several risks, including protein misfolding, altered protein shuttling, or functional impairment when compared to the wild-type proteins. Here, we demonstrate altered subcellular distribution and function of the pro-inflammatory enzyme procaspase-1 as a result of fusion with the reporter protein mCherry. Our observations are of central importance to further investigations of subcellular behavior and possible protein-protein interactions of naturally occurring genetic variants of human procaspase-1 which have recently been linked to autoinflammatory disorders.

  12. 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.

  13. The dynamic subcellular localization of ERK: mechanisms of translocation and role in various organelles.

    PubMed

    Wainstein, Ehud; Seger, Rony

    2016-04-01

    The dynamic subcellular localization of ERK in resting and stimulated cells plays an important role in its regulation. In resting cells, ERK localizes in the cytoplasm, and upon stimulation, it translocates to its target substrates and organelles. ERK signaling initiated from different places in resting cells has distinct outcomes. In this review, we summarize the mechanisms of ERK1/2 translocation to the nucleus and mitochondria, and of ERK1c to the Golgi. We also show that ERK1/2 translocation to the nucleus is a useful anti cancer target. Unraveling the complex subcellular localization of ERK and its dynamic changes upon stimulation provides a better understanding of the regulation of ERK signaling and may result in the development of new strategies to combat ERK-related diseases.

  14. Subcellular Localization of Rice Leaf Aryl Acylamidase Activity 1

    PubMed Central

    Gaynor, John J.; Still, Cecil C.

    1983-01-01

    The intracellular localization of aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13) in rice (Oryza sativa L. var Starbonnet) leaves was investigated. The enzyme hydrolyzes and detoxifies the herbicide propanil (3,4-dichloropropionanilide) thereby accounting for immunity of the rice plant to herbicidal action. Fractionation of mesophyll protoplasts by differential centrifugation yielded the highest specific activity of amidase in the crude mitochondrial fraction. Further separation of density gradients of the silica sol Percoll also indicated that this enzyme was mitochondrial. By the use of biochemical markers, the purified mitochondrial fraction was shown to be substantially free of contamination from nuclei, chloroplasts, golgi, and plasma membranes. Subfractionation of the purified mitochondria suggests that this enzyme is located on the outer membrane. PMID:16662987

  15. Altered subcellular localization of ornithine decarboxylase in Alzheimer's disease brain

    SciTech Connect

    Nilsson, Tatjana . E-mail: Tatjana.Nilsson@ki.se; Bogdanovic, Nenad; Volkman, Inga; Winblad, Bengt; Folkesson, Ronnie; Benedikz, Eirikur

    2006-06-02

    The amyloid precursor protein can through ligand-mimicking induce expression of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine biosynthesis. We report here the regional distribution and cellular localization of ODC immunoreactivity in Alzheimer's disease (AD) brains. In frontal cortex and hippocampus of control cases, the most pronounced ODC immunoreactivity was found in the nucleus. In possible and definite AD the immunoreactivity had shifted to the cytoplasm. In cerebellum of control cases, ODC staining was found in a small portion of Purkinje cells, mostly in the nucleus. In AD, both possible and definite, the number of stained Purkinje cells increased significantly and immunoreactivity was shifted to the cytoplasm, even though it was still prominent in the nucleus. In conclusion, our study reveals an early shift of the ODC immunoreactivity in AD from the nuclear compartment towards the cytoplasm.

  16. Subcellular localization of the homocitrate synthase in Penicillium chrysogenum.

    PubMed

    Bañuelos, O; Casqueiro, J; Steidl, S; Gutiérrez, S; Brakhage, A; Martín, J F

    2002-01-01

    There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.

  17. SLocX: Predicting Subcellular Localization of Arabidopsis Proteins Leveraging Gene Expression Data.

    PubMed

    Ryngajllo, Malgorzata; Childs, Liam; Lohse, Marc; Giorgi, Federico M; Lude, Anja; Selbig, Joachim; Usadel, Björn

    2011-01-01

    Despite the growing volume of experimentally validated knowledge about the subcellular localization of plant proteins, a well performing in silico prediction tool is still a necessity. Existing tools, which employ information derived from protein sequence alone, offer limited accuracy and/or rely on full sequence availability. We explored whether gene expression profiling data can be harnessed to enhance prediction performance. To achieve this, we trained several support vector machines to predict the subcellular localization of Arabidopsis thaliana proteins using sequence derived information, expression behavior, or a combination of these data and compared their predictive performance through a cross-validation test. We show that gene expression carries information about the subcellular localization not available in sequence information, yielding dramatic benefits for plastid localization prediction, and some notable improvements for other compartments such as the mitochondrion, the Golgi, and the plasma membrane. Based on these results, we constructed a novel subcellular localization prediction engine, SLocX, combining gene expression profiling data with protein sequence-based information. We then validated the results of this engine using an independent test set of annotated proteins and a transient expression of GFP fusion proteins. Here, we present the prediction framework and a website of predicted localizations for Arabidopsis. The relatively good accuracy of our prediction engine, even in cases where only partial protein sequence is available (e.g., in sequences lacking the N-terminal region), offers a promising opportunity for similar application to non-sequenced or poorly annotated plant species. Although the prediction scope of our method is currently limited by the availability of expression information on the ATH1 array, we believe that the advances in measuring gene expression technology will make our method applicable for all Arabidopsis proteins.

  18. Many Local Pattern Texture Features: Which Is Better for Image-Based Multilabel Human Protein Subcellular Localization Classification?

    PubMed Central

    Xu, Ying-Ying; Shen, Hong-Bin

    2014-01-01

    Human protein subcellular location prediction can provide critical knowledge for understanding a protein's function. Since significant progress has been made on digital microscopy, automated image-based protein subcellular location classification is urgently needed. In this paper, we aim to investigate more representative image features that can be effectively used for dealing with the multilabel subcellular image samples. We prepared a large multilabel immunohistochemistry (IHC) image benchmark from the Human Protein Atlas database and tested the performance of different local texture features, including completed local binary pattern, local tetra pattern, and the standard local binary pattern feature. According to our experimental results from binary relevance multilabel machine learning models, the completed local binary pattern, and local tetra pattern are more discriminative for describing IHC images when compared to the traditional local binary pattern descriptor. The combination of these two novel local pattern features and the conventional global texture features is also studied. The enhanced performance of final binary relevance classification model trained on the combined feature space demonstrates that different features are complementary to each other and thus capable of improving the accuracy of classification. PMID:25050396

  19. Iron in seeds – loading pathways and subcellular localization

    PubMed Central

    Grillet, Louis; Mari, Stéphane; Schmidt, Wolfgang

    2014-01-01

    Iron (Fe) is one of the most abundant elements on earth, but its limited bioavailability poses a major constraint for agriculture and constitutes a serious problem in human health. Due to an improved understanding of the mechanisms that control Fe homeostasis in plants, major advances toward engineering biofortified crops have been made during the past decade. Examples of successful biofortification strategies are, however, still scarce and the process of Fe loading into seeds is far from being well understood in most crop species. In particular in grains where the embryo represents the main storage compartment such as legumes, increasing the seed Fe content remains a challenging task. This review aims at placing the recently identified actors in Fe transport into the unsolved puzzle of grain filling, taking the differences of Fe distribution between various species into consideration. We summarize the current knowledge on Fe transport between symplasmic and apoplasmic compartments, and provide models for Fe trafficking and localization in different seed types that may help to develop high seed Fe germplasms. PMID:24427161

  20. Characterization and subcellular localization of aminopeptidases in senescing barley leaves

    NASA Technical Reports Server (NTRS)

    Thayer, S. S.; Choe, H. T.; Rausser, S.; Huffaker, R. C.

    1988-01-01

    Four aminopeptidases (APs) were separated using native polyacrylamide gel electrophoresis of cell-free extracts and the stromal fractions of isolated chloroplasts prepared from primary barley (Hordeum vulgare L., var Numar) leaves. Activities were identified using a series of aminoacyl-beta-naphthylamide derivatives as substrates. AP1, 2, and 3 were found in the stromal fraction of isolated chloroplasts with respective molecular masses of 66.7, 56.5, and 54.6 kilodaltons. AP4 was found only in the cytoplasmic fraction. No AP activity was found in vacuoles of these leaves. It was found that 50% of the L-Leu-beta-naphthylamide and 25% of the L-Arg-beta-naphthylamide activities were localized in the chloroplasts. Several AP activities were associated with the membranes of the thylakoid fraction of isolated chloroplasts. AP1, 2, and 4 reacted against a broad range of substrates, whereas AP3 hydrolyzed only L-Arg-beta-naphthylamide. Only AP2 hydrolyzed L-Val-beta-naphthylamide. Since AP2 and AP3 were the only ones reacting against Val-beta-naphthylamide and Arg-beta-naphthylamide, respectively, several protease inhibitors were tested against these substrates using a stromal fraction from isolated chloroplasts as the source of the two APs. Both APs were sensitive to both metallo and sulfhydryl type inhibitors. Although AP activity decreased as leaves senesced, no new APs appeared on gels during senescence and none disappeared.

  1. Specific subcellular localization of siRNAs delivered by lipoplex in MCF-7 breast cancer cells.

    PubMed

    Lavigne, Carole; Thierry, Alain R

    2007-10-01

    In order to better understand the mechanism of delivery of siRNAs by lipid-based vectors, we investigated the subcellular distribution of siRNAs directed against cyclin D1 delivered by the DLS system in the breast cancer cell line MCF-7. Cells were treated with cyclopentenone or 17beta-estradiol to modulate the level of expression of cyclin D1 mRNA. We qualitatively observed that siRNA localized to specific cytoplasmic compartments in the periphery of the nucleus in granular-like structures that do not correspond to early endosomal vesicles. In cells treated with either cyclopentenone or 17beta-estradiol cellular distribution of siRNAs was not affected but variations in the amount of siRNAs present in cells were found. We suggest these variations might be associated with the effects of cyclopentenone and 17beta-estradiol in cyclin D1 gene expression. Low cytotoxicity and highly cellular uptake of lipoplexes was observed in the presence of serum indicating that the DLS system could be a useful tool for siRNA vectorization in vitro and in vivo.

  2. Non-Catalytic RISCs and Kinetics Determine Mammalian siRNA Sub-Cellular Localization.

    PubMed

    Ji, Fengmin; Liu, Lianyun; Tien, Ya-Hsin; Peng, Yi-Hsien; Lee, Hoong-Chien

    2015-01-01

    Small interfering RNAs (siRNAs) are fundamental to the regulation of cell function. Much is known about its gene interfering mechanism, but a kinetic description of it is still lacking. Here, we derived a set of reaction-diffusion equations for multiple RNA-induced silencing complex (RISC) pathways that give quantitative temporal and spatial descriptions of the siRNA process in mammalian cell, and are able to correctly describe all salient experimentally observed patterns of sub-cellular siRNA localization, including those that, at first glance, appear irreconcilable. These results suggest siRNA sub-cellular localization mainly concerns the non-catalytic RISC-target complex, and is caused by the selectiveness of RISC-target interaction and the permeability of the nuclear membrane to siRNA strands but not to RISC-target complexes. Our method is expected to be useful in devising RNAi based cell regulation strategies.

  3. 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.

  4. 'Unite and conquer': enhanced prediction of protein subcellular localization by integrating multiple specialized tools

    PubMed Central

    Shen, Yao Qing; Burger, Gertraud

    2007-01-01

    Background Knowing the subcellular location of proteins provides clues to their function as well as the interconnectivity of biological processes. Dozens of tools are available for predicting protein location in the eukaryotic cell. Each tool performs well on certain data sets, but their predictions often disagree for a given protein. Since the individual tools each have particular strengths, we set out to integrate them in a way that optimally exploits their potential. The method we present here is applicable to various subcellular locations, but tailored for predicting whether or not a protein is localized in mitochondria. Knowledge of the mitochondrial proteome is relevant to understanding the role of this organelle in global cellular processes. Results In order to develop a method for enhanced prediction of subcellular localization, we integrated the outputs of available localization prediction tools by several strategies, and tested the performance of each strategy with known mitochondrial proteins. The accuracy obtained (up to 92%) surpasses by far the individual tools. The method of integration proved crucial to the performance. For the prediction of mitochondrion-located proteins, integration via a two-layer decision tree clearly outperforms simpler methods, as it allows emphasis of biologically relevant features such as the mitochondrial targeting peptide and transmembrane domains. Conclusion We developed an approach that enhances the prediction accuracy of mitochondrial proteins by uniting the strength of specialized tools. The combination of machine-learning based integration with biological expert knowledge leads to improved performance. This approach also alleviates the conundrum of how to choose between conflicting predictions. Our approach is easy to implement, and applicable to predicting subcellular locations other than mitochondria, as well as other biological features. For a trial of our approach, we provide a webservice for mitochondrial protein

  5. Alteration of the glucocorticoid receptor subcellular localization by non steroidal compounds.

    PubMed

    Prima, V; Depoix, C; Masselot, B; Formstecher, P; Lefebvre, P

    2000-01-01

    The glucocorticoid receptor (GR) engages transient or stable interactions with chaperones (hsp90, hsp70), co-chaperones (p60/hop, hsp40) and several other polypeptides such as immunophilins (Cyp40, FKBP59) and p23 to achieve a high affinity ligand binding state. This complex dissociates in response to hormonal stimuli and holo-GR translocates into the nucleus, where it regulates the activity of glucocorticoid-sensitive genes. GR activity is controlled through its ligand binding domain by steroids displaying either agonistic or antagonistic activity. An alternative approach to modulate GR activity is to target receptor-associated proteins (RAPs), and several non steroidal compounds binding to RAPs affect GR transcriptional activity. We have studied the effect of such drugs on the intracellular localization of a EGFP-GR fusion protein, which has wild type GR pharmacological properties. Agonist and antagonist binding induced nuclear translocation of GR, whereas rifampicin was found to be inactive in our system. Immunosuppressants FK506 and cyclosporin A were able to induce partial nuclear translocation of GR, suggesting that potentiation of glucocorticoid action by these compounds may also proceed through enhanced GR nuclear transfer. Short treatment of cells with the hsp90 inhibitor geldanamycin (GA) did not prevent nuclear translocation of GR. However, longer treatments, in parrallel to the inhibition of GR transcriptional activity, strongly perturbed GR subcellular localization concomitantly to the disruption of the actin network, and caused GR aggregation and down-regulation. The GA-induced transcriptional shutdown was also observed for other nuclear receptors which do not interact stably with hsp90. Thus RAP-binding compounds may exert their effects at least in part through perturbation of the GR cytosol to nucleus partitioning, and identify these proteins as valuable therapeutic targets to control nuclear receptor activity.

  6. Subcellular Localization of RPB5-Mediating Protein and Its Putative Functional Partner

    PubMed Central

    Delgermaa, Luvsanjav; Hayashi, Naoyuki; Dorjsuren, Dorjbal; Nomura, Takahiro; Thuy, Le Thi-Thu; Murakami, Seishi

    2004-01-01

    We previously identified a novel cellular protein, RPB5-mediating protein (RMP), that retains corepressor activity and functionally antagonizes transcriptional modulation via hepatitis B virus X protein. The subcellular localization of RMP was examined using green fluorescent protein-fused protein forms. We found that a nuclear localization signal (NLS) and a coiled-coil (CC) domain functioning as a cytoplasmic localization signal (CLS) are important for the subcellular localization of RMP. The CLS apparently acts dominantly, since RMP was mostly localized in the cytoplasm with weak and diffuse signals in the nucleus, and the NLS was indispensable for the nuclear localization of RMP only in the absence of the CLS. Using a yeast two-hybrid method, we isolated a putative corepressor, DNA methyltransferase 1-associating protein (DMAP1), which was found to bind to the CC domain of RMP. DMAP1 facilitated the nuclear localization of RMP and the corepressor activity of RMP in a dose-dependent manner by interacting with the CC domain of RMP. These results are discussed in light of a recent paper showing a novel evolutionarily conserved role of URI in the TOR signaling pathway. PMID:15367675

  7. Subcellular localization of the heparin-neutralizing factor in blood platelets.

    PubMed Central

    Da Prada, M; Jakábová, M; Lüscher, E F; Pletscher, A; Richards, J G

    1976-01-01

    1. The distribution of the heparin-neutralizing factor (platelet factor 4, PF4) in subcellular organelles of blood platelets of rabbits and man was investigated. 2. In both species the organelles storing 5-hydroxytryptamine (5-HT storage organelles) contained only trivial amounts of PF4. 3. In contrast, the content of PF4 was highest in the subcellular fractions rich in alpha-granules. 4. In conclusion, PF4 is probably localized in the alpha-granules and therefore the platelets contain at least two types of organelles (5-HT organelles and alpha-granules) capable of releasing their contents in response to the same stimuli, such as exposure to collagen, thrombin, etc. Images Plate 1 Plate 2 PMID:950602

  8. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family

    PubMed Central

    Tanz, Sandra K.; Castleden, Ian; Hooper, Cornelia M.; Small, Ian; Millar, A. Harvey

    2014-01-01

    Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions. The SUBA3 database is a free web-service at http://suba.plantenergy.uwa.edu.au that helps users to explore reported experimental data and predictions concerning proteins encoded by gene families and to define the experiments required to locate these homologous sets of proteins. Here we show how SUBA3 can be used to explore the subcellular location of the Deg protease family of ATP-independent serine endopeptidases (Deg1–Deg16). Combined data integration and new experiments refined location information for Deg1 and Deg9, confirmed Deg2, Deg5, and Deg8 in plastids and Deg 15 in peroxisomes and provide substantial experimental evidence for mitochondrial localized Deg proteases. Two of these, Deg3 and Deg10, additionally localized to the plastid, revealing novel dual-targeted Deg proteases in the plastid and the mitochondrion. SUBA3 is continually updated to ensure that researchers can use the latest published data when planning the experimental steps remaining to localize gene family functions. PMID:25161662

  9. Transformation of tobacco plants by Yali PPO-GFP fusion gene and observation of subcellular localization.

    PubMed

    Qi, Jing; Li, Gui-Qin; Dong, Zhen; Zhou, Wei

    2016-01-01

    To explore the subcellular localization of Polyphenol oxidase (PPO) from Pyrus bretschneideri, the 1779 bp cDNA of PPO gene excluding the termination codon TAA was cloned and fused with GFP to construct a binary vector pBI121-PPO-GFP. Then, the binary vector was transformed into Nicotiana tabacum by the tumefanciens-mediated method. Using confocal laser scanning microscopy, green fluorescent signals were localized in chloroplasts of the transformed Nicotiana tabacum cell, suggesting that the Polyphenol oxidase from Pyrus bretschneideri was a chloroplast protein.

  10. Subcellular Localization and Functional Analysis of the Arabidopsis GTPase RabE1[W][OA

    PubMed Central

    Speth, Elena Bray; Imboden, Lori; Hauck, Paula; He, Sheng Yang

    2009-01-01

    Membrane trafficking plays a fundamental role in eukaryotic cell biology. Of the numerous known or predicted protein components of the plant cell trafficking system, only a relatively small subset have been characterized with respect to their biological roles in plant growth, development, and response to stresses. In this study, we investigated the subcellular localization and function of an Arabidopsis (Arabidopsis thaliana) small GTPase belonging to the RabE family. RabE proteins are phylogenetically related to well-characterized regulators of polarized vesicle transport from the Golgi apparatus to the plasma membrane in animal and yeast cells. The RabE family of GTPases has also been proposed to be a putative host target of AvrPto, an effector protein produced by the plant pathogen Pseudomonas syringae, based on yeast two-hybrid analysis. We generated transgenic Arabidopsis plants that constitutively expressed one of the five RabE proteins (RabE1d) fused to green fluorescent protein (GFP). GFP-RabE1d and endogenous RabE proteins were found to be associated with the Golgi apparatus and the plasma membrane in Arabidopsis leaf cells. RabE down-regulation, due to cosuppression in transgenic plants, resulted in drastically altered leaf morphology and reduced plant size, providing experimental evidence for an important role of RabE GTPases in regulating plant growth. RabE down-regulation did not affect plant susceptibility to pathogenic P. syringae bacteria; conversely, expression of the constitutively active RabE1d-Q74L enhanced plant defenses, conferring resistance to P. syringae infection. PMID:19233904

  11. Subcellular localization of Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

    PubMed

    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-microm 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

  12. 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

  13. Analysis of subcellular localization of auxin carriers PIN, AUX/LAX and PGP in Sorghum bicolor

    PubMed Central

    Wang, SuiKang; Shen, ChenJia; Zhang, SaiNa; Xu, YanXia; Jiang, DeAn; Qi, YanHua

    2011-01-01

    Auxin transport at least correlates to the three gene families: efflux carriers PIN-formed (PIN), p-glycoprotein (PGP), and influx carrier auxin resistant 1/like aux1(AUX/LAX) in Arabidopsis thaliana. In monocotyledon Sorghum bicolor, the biological function of these genes retains unclear. Our previous study reported that the member analysis, organ-specific expression and expression profiles of the auxin transporter PIN, PGP and AUX/LAX gene families in Sorghum bicolor under IAA, brassinosteroid, polar auxin transport inhibitors and abiotic stresses. Here we further supply the prediction of subcellular localization of SbPIN, SbLAX and SbPGP proteins and discuss the potential relationship between the subcellular localization and stress response. The predicted results showed that the most of SbPIN, SbLAX and SbPGP proteins are localized to the plasma membrane, except few localized to vacuolar membrane and endoplasmic reticulum. This data set provides novel information for investigation of auxin transporters in Sorghum bicolor. PMID:22112459

  14. Ubiquitous distribution and different subcellular localization of sorbitol dehydrogenase in fruit and leaf of apple.

    PubMed

    Wang, Xiu-Ling; Xu, Yan-Hong; Peng, Chang-Cao; Fan, Ren-Chun; Gao, Xin-Qi

    2009-01-01

    NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), a key enzyme in sorbitol metabolism, plays an important role in regulating sink strength and determining the quality of apple fruit. Understanding the tissue and subcellular localization of NAD-SDH is helpful for understanding sorbitol metabolism in the apple. In this study, two NAD-SDH cDNA sequences were isolated from apple fruits (Malus domestica Borkh cv. Starkrimson) and named MdSDH5 and MdSDH6. Immunohistochemical analysis revealed that NAD-SDH is distributed in both the flesh and the vascular tissue of the fruit, and the vascular tissue and mesophyll tissue in the young and old leaves, indicating that it is a ubiquitous protein expressed in both sink and source organs. Immunogold electron microscopy analysis demonstrated that NAD-SDH is localized mainly in the cytoplasm and chloroplast of the fruit and leaves. The chloroplast localization of NAD-SDH was confirmed by the transient expression of MdSDH5-GFP and MdSDH6-GFP in the mesophyll protoplast of Arabidopsis. NAD-SDH was also found in electron opaque deposits of vacuoles in young and mature leaves. These data show that NAD-SDH has different subcellular localizations in fruit and leaves, indicating that it might play a different role in sorbitol metabolism in different tissues of apple.

  15. Subcellular localization of RNA degrading proteins and protein complexes in prokaryotes.

    PubMed

    Evguenieva-Hackenberg, Elena; Roppelt, Verena; Lassek, Christian; Klug, Gabriele

    2011-01-01

    The archaeal exosome is a prokaryotic protein complex with RNA processing and degrading activities. Recently it was shown that the exosome is localized at the periphery of the cell in the thermoacidophilic archaeon Sulfolobus solfataricus. This localization is most likely mediated by the archaeal DnaG protein and depends on (direct or indirect) hydrophobic interactions with the membrane. A localization of RNA degrading proteins and protein complexes was also demonstrated in several bacteria. In bacteria a subcellular localization was also shown for substrates of these proteins and protein complexes, i.e. chromosomally encoded mRNAs and a small RNA. Thus, despite the missing compartmentalization, a spatial organization of RNA processing and degradation exists in prokaryotic cells. Recent data suggest that the spatial organization contributes to the temporal regulation of these processes.

  16. Subcellular Localization of Thiol-Capped CdTe Quantum Dots in Living Cells

    NASA Astrophysics Data System (ADS)

    Zhang, Yu; Mi, Lan; Xiong, Rongling; Wang, Pei-Nan; Chen, Ji-Yao; Yang, Wuli; Wang, Changchun; Peng, Qian

    2009-07-01

    Internalization and dynamic subcellular distribution of thiol-capped CdTe quantum dots (QDs) in living cells were studied by means of laser scanning confocal microscopy. These unfunctionalized QDs were well internalized into human hepatocellular carcinoma and rat basophilic leukemia cells in vitro. Co-localizations of QDs with lysosomes and Golgi complexes were observed, indicating that in addition to the well-known endosome-lysosome endocytosis pathway, the Golgi complex is also a main destination of the endocytosed QDs. The movement of the endocytosed QDs toward the Golgi complex in the perinuclear region of the cell was demonstrated.

  17. Expression and tissue and subcellular localization of anthocyanidin synthase (ANS) in grapevine.

    PubMed

    Wang, Huiling; Wang, Wei; Li, Hui; Zhang, Ping; Zhan, Jicheng; Huang, Weidong

    2011-04-01

    Anthocyanidin synthase (ANS) is one of the key enzymes in the biosynthesis of both anthocyanins and proanthocyanidins in grapevine. Although substantial researches have investigated ANS gene expression and regulation at the transcriptional level, little is yet known about protein expression and distribution in grapevine. Here, the expression and tissue and subcellular localization of ANS in different Cabernet sauvignon grapevine tissues were investigated by using the techniques of Western blotting, immunohistochemical localization, immuno-electron microscopy, and confocal microscopy. The results showed that the ANS was expressed in the grape berries, leaves, stems, petioles, and leaf buds. In grape berry skin and flesh, ANS expression is developmental dependent. Immunohistochemical analysis revealed that ANS is primarily distributed in the exocarp, mesocarp, and seed of the fruit; in palisade and spongy tissues of the leaves; in the primary phloem and pith ray in the stems; and in the growth point and leaf primordium of the leaf buds. Furthermore, at the subcellular level, the ANS was mainly localized in the cytoplasm regardless of cell types and some ANS were also found in the nucleus in the mesocarp vascular bundle and leaf bud cells. This research will give further insight for the biosynthesis and regulation of different flavonoid compounds in grapevine.

  18. 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.

  19. Subcellular Localization of the Sigma-1 Receptor in Retinal Neurons — an Electron Microscopy Study

    PubMed Central

    Mavlyutov, Timur A.; Epstein, Miles; Guo, Lian-Wang

    2015-01-01

    The Sigma-1 receptor (S1R) is known to play a protective role in the central nervous system including the retina. A major barrier for understanding the underlying mechanism is an ambiguity of S1R subcellular localizations. We thus conducted the first electron microscopy (EM) study of S1R subcellular distribution in the mouse retina. Immuno-EM imaging showed previously under-appreciated S1R presence in photoreceptor cells. Unlike in other cell types in previous reports, in photoreceptor cells S1R was found in the nuclear envelope but not localized in the endoplasmic reticulum (ER), raising a possibility of S1R-mediated modulatory mechanisms different than conventionally thought. While in bipolar cells S1R was detected only in the nuclear envelope, in ganglion cells S1R was identified predominantly in the nuclear envelope and found in the ER as well. A predominant localization of S1R in the nuclear envelope in all three retinal neurons implicates a potential role of S1R in modulating nuclear activities. Moreover, its absence in the plasma membrane and presence in the subsurface ER cisternae that are juxtaposed to the plasma membrane in ganglion cells may lend mechanistic insights generally important for frequently reported S1R modulations of ion channels in neurons. PMID:26033680

  20. Hum-PLoc: a novel ensemble classifier for predicting human protein subcellular localization.

    PubMed

    Chou, Kuo-Chen; Shen, Hong-Bin

    2006-08-18

    Predicting subcellular localization of human proteins is a challenging problem, especially when unknown query proteins do not have significant homology to proteins of known subcellular locations and when more locations need to be covered. To tackle the challenge, protein samples are expressed by hybridizing the gene ontology (GO) database and amphiphilic pseudo amino acid composition (PseAA). Based on such a representation frame, a novel ensemble classifier, called "Hum-PLoc", was developed by fusing many basic individual classifiers through a voting system. The "engine" of these basic classifiers was operated by the KNN (K-nearest neighbor) rule. As a demonstration, tests were performed with the ensemble classifier for human proteins among the following 12 locations: (1) centriole; (2) cytoplasm; (3) cytoskeleton; (4) endoplasmic reticulum; (5) extracell; (6) Golgi apparatus; (7) lysosome; (8) microsome; (9) mitochondrion; (10) nucleus; (11) peroxisome; (12) plasma membrane. To get rid of redundancy and homology bias, none of the proteins investigated here had > or = 25% sequence identity to any other in a same subcellular location. The overall success rates thus obtained via the jackknife cross-validation test and independent dataset test were 81.1% and 85.0%, respectively, which are more than 50% higher than those obtained by the other existing methods on the same stringent datasets. Furthermore, an incisive and compelling analysis was given to elucidate that the overwhelmingly high success rate obtained by the new predictor is by no means due to a trivial utilization of the GO annotations. This is because, for those proteins with "subcellular location unknown" annotation in Swiss-Prot database, most (more than 99%) of their corresponding GO numbers in GO database are also annotated with "cellular component unknown". The information and clues for predicting subcellular locations of proteins are actually buried into a series of tedious GO numbers, just like they

  1. Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions.

    PubMed

    Guzman, Raul E; Miranda-Laferte, Erick; Franzen, Arne; Fahlke, Christoph

    2015-10-23

    ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl(-) currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected.

  2. Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions*

    PubMed Central

    Guzman, Raul E.; Miranda-Laferte, Erick; Franzen, Arne; Fahlke, Christoph

    2015-01-01

    ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl− currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected. PMID:26342074

  3. Identification and subcellular localization of two solanesyl diphosphate synthases from Arabidopsis thaliana.

    PubMed

    Jun, Luo; Saiki, Ryoichi; Tatsumi, Kei; Nakagawa, Tsuyoshi; Kawamukai, Makoto

    2004-12-01

    Two solanesyl diphosphate synthases, designated SPS1 and SPS2, which are responsible for the synthesis of the isoprenoid side chain of either plastoquinone or ubiquinone in Arabidopsis thaliana, were identified. Heterologous expression of either SPS1 or SPS2 allowed the generation of UQ-9 in a decaprenyl diphosphate synthase-defective strain of fission yeast and also in wild-type Escherichia coli. SPS1-GFP was found to localize in the ER while SPS2-GFP localized in the plastid of tobacco BY-2 cells. These two different subcellular localizations are thought to be the reflection of their roles in solanesyl diphosphate synthesis in two different parts: presumably SPS1 and SPS2 for the side chains of ubiquinone and plastoquinone, respectively.

  4. Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells.

    PubMed

    von Arnim, Albrecht

    2007-02-01

    INTRODUCTIONRecombinant tags (i.e., reporter proteins) offer an excellent alternative to antibodies for determining the subcellular localization of proteins. The most user-friendly tags are the ß-glucuronidase (GUS) reporter enzyme from Escherichia coli and fluorescent proteins derived primarily from the green fluorescent protein (GFP) of the jellyfish Aequorea victoria. GUS is useful primarily as a tag to address nuclear localization, whereas GFP is more versatile. Moreover, GFP is detectable directly in living cells, whereas GUS is only detected indirectly by staining of fixed tissue. This may lead to artifacts or it may obscure problems with protein solubility. In this protocol, protein localization is routinely assayed after particle-mediated transient transformation of onion epidermal cells. With this method it can be determined rapidly whether a given fusion protein is active, and preliminary targeting data can be obtained.

  5. Cloning, characterization and subcellular localization of Nuclear LIM interactor interacting factor gene from Leishmania donovani.

    PubMed

    Ravinder, R; Goyal, N

    2017-05-05

    LIM domains are zinc-binding motifs that mediate protein-protein interactions and are found in a wide variety of cytoplasmic and nuclear proteins. The nuclear LIM domain family members have a number of different functions including transcription factors, gene regulation, cell fate determination, organization of the cytoskeleton and tumour formation exerting their function through various LIM domain interacting protein partners/cofactors. Nuclear LIM domain interacting proteins/factors have not been reported in any protozoan parasites including Leishmania. Here, we report for the first time cloning, characterization and subcellular localization of nuclear LIM interactor-interacting factor (NLI) like protein from Leishmania donovani, the causative agent of Indian Kala-azar. Primary sequence analysis of LdNLI revealed presence of characteristic features of nuclear LIM interactor-interacting factor. However, leishmanial NLI represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. The sub-cellular distribution of LdNLI revealed the discreet localization in nucleus and kinetoplast only, suggesting that the gene may have a role in parasite gene expression.

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

    PubMed Central

    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. PMID:25941807

  7. 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.

  8. Short-time movement of E. coli chromosomal loci depends on coordinate and subcellular localization.

    PubMed

    Javer, Avelino; Long, Zhicheng; Nugent, Eileen; Grisi, Marco; Siriwatwetchakul, Kamin; Dorfman, Kevin D; Cicuta, Pietro; Cosentino Lagomarsino, Marco

    2013-01-01

    In bacteria, chromosomal architecture shows strong spatial and temporal organization, and regulates key cellular functions, such as transcription. Tracking the motion of chromosomal loci at short timescales provides information related to both the physical state of the nucleo-protein complex and its local environment, independent of large-scale motions related to genome segregation. Here we investigate the short-time (0.1-10 s) dynamics of fluorescently labelled chromosomal loci in Escherichia coli at different growth rates. At these timescales, we observe for the first time a dependence of the loci's apparent diffusion on both their subcellular localization and chromosomal coordinate, and we provide evidence that the properties of the chromosome are similar in the tested growth conditions. Our results indicate that either non-equilibrium fluctuations due to enzyme activity or the organization of the genome as a polymer-protein complex vary as a function of the distance from the origin of replication.

  9. Dlg5 Regulates Dendritic Spine Formation and Synaptogenesis by Controlling Subcellular N-Cadherin Localization

    PubMed Central

    Wang, Shih-Hsiu J.; Celic, Ivana; Choi, Se-Young; Riccomagno, Martin; Wang, Qiang; Sun, Lu O.; Mitchell, Sarah P.; Vasioukhin, Valera; Huganir, Richard L.

    2014-01-01

    Most excitatory synapses in the mammalian brain are formed on dendritic spines, and spine density has a profound impact on synaptic transmission, integration, and plasticity. Membrane-associated guanylate kinase (MAGUK) proteins are intracellular scaffolding proteins with well established roles in synapse function. However, whether MAGUK proteins are required for the formation of dendritic spines in vivo is unclear. We isolated a novel disc large-5 (Dlg5) allele in mice, Dlg5LP, which harbors a missense mutation in the DLG5 SH3 domain, greatly attenuating its ability to interact with the DLG5 GUK domain. We show here that DLG5 is a MAGUK protein that regulates spine formation, synaptogenesis, and synaptic transmission in cortical neurons. DLG5 regulates synaptogenesis by enhancing the cell surface localization of N-cadherin, revealing a key molecular mechanism for regulating the subcellular localization of this cell adhesion molecule during synaptogenesis. PMID:25232112

  10. Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants.

    PubMed

    Corpas, Francisco J; Barroso, Juan B; Carreras, Alfonso; Quirós, Miguel; León, Ana M; Romero-Puertas, María C; Esteban, Francisco J; Valderrama, Raquel; Palma, José M; Sandalio, Luisa M; Gómez, Manuel; del Río, Luis A

    2004-09-01

    The cellular and subcellular localization of endogenous nitric oxide (NO.) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO. was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO. generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)(2) and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO. was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO., with g = 2.05 and a(N) = 12.8 G, was detected in peroxisomes. By fluorometry, NO. was also found in these organelles, and the level measured of NO. was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO. from l-Arg (nitric oxide synthase [NOS]-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO. mg(-1) protein min(-1); was strictly dependent on NADPH, calmodulin, and BH(4); and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO. could be involved in the process of senescence of pea leaves.

  11. Predicting protein subcellular localization based on information content of gene ontology terms.

    PubMed

    Zhang, Shu-Bo; Tang, Qiang-Rong

    2016-12-01

    Predicting the location where a protein resides within a cell is important in cell biology. Computational approaches to this issue have attracted more and more attentions from the community of biomedicine. Among the protein features used to predict the subcellular localization of proteins, the feature derived from Gene Ontology (GO) has been shown to be superior to others. However, most of the sights in this field are set on the presence or absence of some predefined GO terms. We proposed a method to derive information from the intrinsic structure of the GO graph. The feature vector was constructed with each element in it representing the information content of the GO term annotating to a protein investigated, and the support vector machines was used as classifier to test our extracted features. Evaluation experiments were conducted on three protein datasets and the results show that our method can enhance eukaryotic and human subcellular location prediction accuracy by up to 1.1% better than previous studies that also used GO-based features. Especially in the scenario where the cellular component annotation is absent, our method can achieved satisfied results with an overall accuracy of more than 87%.

  12. Protein expression and subcellular localization of the general purine transporter UapC from Aspergillus nidulans.

    PubMed

    Valdez-Taubas, J; Diallinas, G; Scazzocchio, C; Rosa, A L

    2000-07-01

    The uapC gene of Aspergillus nidulans belongs to a family of nucleobase-specific transporters conserved in prokaryotic and eucaryotic organisms. We report the use of immunological and green fluorescent protein based strategies to study protein expression and subcellular distribution of UapC. A chimeric protein containing a plant-adapted green fluorescent protein (sGFP) fused to the C-terminus of UapC was shown to be functional in vivo, as it complements a triple mutant (i.e., uapC(-) uapA(-) azgA(-)) unable to grow on uric acid as the sole nitrogen source. UapC-GFP is located in the plasma membrane and, secondarily, in internal structures observed as fluorescent dots. A strong correlation was found between cellular levels of UapC-GFP fluorescence and known patterns of uapC gene expression. This work represents the first in vivo study of protein expression and subcellular localization of a filamentous fungal nucleobase transporter.

  13. Organ, cellular, and subcellular localization of brain-specific anion transporter BSAT1.

    PubMed

    Baklaushev, V P; Kardashova, K Sh; Gurina, O I; Yusubaliyeva, G M; Zorkina, Ya A; Chekhonin, V P

    2013-08-01

    Organ, cellular, and subcellular localization of brain-specific anion transporter BSAT1 was studied in rats using antibodies to the extracellular fragment (451-557 a.a). The antibodies were shown to recognize the antigen predominantly localized in the nervous tissue, tumors of glial origin, and primordial ovarian follicles. The absence of BSAT1 immunofluorescence signal in kidney and liver sections and accumulation of (125)I labeled antibodies to BSAT1 in these organs indicate that these antibodies do not cross-react with the most common isoforms of OATP expressed in these organs. Analysis of the cellular localization suggests that in the brain, BSAT1 is localized predominantly in astrocytes, but not in endothelial cells, as was previously reported. Laser scanning confocal microscopy with a set of relevant trackers revealed membrane localization of BSAT1. Taking into account the data on the of localization, we can conclude that antibodies to BSAT1 451-557 can be used for basic research of the transport of thyroxin and prostaglandins across the blood brain barrier and for testing the systems for targeted transport of diagnostic preparations and drugs across the blood brain barrier, e.g. to astroglial tumors.

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

    SciTech Connect

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela

    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. 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.

  15. Classification of protein motifs based on subcellular localization uncovers evolutionary relationships at both sequence and functional levels

    PubMed Central

    2013-01-01

    Background Most proteins have evolved in specific cellular compartments that limit their functions and potential interactions. On the other hand, motifs define amino acid arrangements conserved between protein family members and represent powerful tools for assigning function to protein sequences. The ideal motif would identify all members of a protein family but in practice many motifs identify both family members and unrelated proteins, referred to as True Positive (TP) and False Positive (FP) sequences, respectively. Results To address the relationship between protein motifs, protein function and cellular localization, we systematically assigned subcellular localization data to motif sequences from the comprehensive PROSITE sequence motif database. Using this data we analyzed relationships between localization and function. We find that TPs and FPs have a strong tendency to localize in different compartments. When multiple localizations are considered, TPs are usually distributed between related cellular compartments. We also identified cases where FPs are concentrated in particular subcellular regions, indicating possible functional or evolutionary relationships with TP sequences of the same motif. Conclusions Our findings suggest that the systematic examination of subcellular localization has the potential to uncover evolutionary and functional relationships between motif-containing sequences. We believe that this type of analysis complements existing motif annotations and could aid in their interpretation. Our results shed light on the evolution of cellular organelles and potentially establish the basis for new subcellular localization and function prediction algorithms. PMID:23865897

  16. Numb directs the subcellular localization of EAAT3 through binding the YxNxxF motif.

    PubMed

    Su, Jin-Feng; Wei, Jian; Li, Pei-Shan; Miao, Hong-Hua; Ma, Yong-Chao; Qu, Yu-Xiu; Xu, Jie; Qin, Jie; Li, Bo-Liang; Song, Bao-Liang; Xu, Zheng-Ping; Luo, Jie

    2016-08-15

    Excitatory amino acid transporter type 3 (EAAT3, also known as SLC1A1) is a high-affinity, Na(+)-dependent glutamate carrier that localizes primarily within the cell and at the apical plasma membrane. Although previous studies have reported proteins and sequence regions involved in EAAT3 trafficking, the detailed molecular mechanism by which EAAT3 is distributed to the correct location still remains elusive. Here, we identify that the YVNGGF sequence in the C-terminus of EAAT3 is responsible for its intracellular localization and apical sorting in rat hepatoma cells CRL1601 and Madin-Darby canine kidney (MDCK) cells, respectively. We further demonstrate that Numb, a clathrin adaptor protein, directly binds the YVNGGF motif and regulates the localization of EAAT3. Mutation of Y503, N505 and F508 within the YVNGGF motif to alanine residues or silencing Numb by use of small interfering RNA (siRNA) results in the aberrant localization of EAAT3. Moreover, both Numb and the YVNGGF motif mediate EAAT3 endocytosis in CRL1601 cells. In summary, our study suggests that Numb is a pivotal adaptor protein that mediates the subcellular localization of EAAT3 through binding the YxNxxF (where x stands for any amino acid) motif.

  17. 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.

  18. 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

  19. The deubiquitinating enzyme USP17 is essential for GTPase subcellular localization and cell motility

    PubMed Central

    de la Vega, Michelle; Kelvin, Alyson A.; Dunican, Dara J.; McFarlane, Cheryl; Burrows, James F.; Jaworski, Jakub; Stevenson, Nigel J.; Dib, Karim; Rappoport, Joshua Z.; Scott, Christopher J.; Long, Aideen; Johnston, James A.

    2011-01-01

    Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cellular processes, but few have been demonstrated to control cell motility. Here, we show that ubiquitin-specific protease 17 (USP17) is rapidly and transiently induced in response to chemokines SDF-1/CXCL12 and IL-8/CXCL8 in both primary cells and cell lines, and that its depletion completely blocks chemokine-induced cell migration and cytoskeletal rearrangements. Using live cell imaging, we demonstrate that USP17 is required for both elongated and amoeboid motility, in addition to chemotaxis. USP17 has previously been reported to disrupt Ras localization and we now find that USP17 depletion blocks chemokine-induced subcellular relocalization of GTPases Cdc42, Rac and RhoA, which are GTPases essential for cell motility. Collectively, these results demonstrate that USP17 has a critical role in cell migration and may be a useful drug target for both inflammatory and metastatic disease. PMID:21448158

  20. Inheritance and subcellular localization of triose-phosphate isomerase in dwarf mountain pine (Pinus mugo).

    PubMed

    Odrzykoski, I J

    2001-01-01

    Several trees with expected heterozygous phenotype for triose-phosphate isomerase (TPI) were discovered in a population of dwarf mountain pine (Pinus mugo Turra) from southern Poland. As the inheritance of this enzyme in pines has not been reported, segregation of allelic variants was tested in eight trees with putative heterozygous phenotypes for two loci, TpiA and TPIB: Linkage between these and some other isozyme loci were studied and evidence for linkage has been found between TpiA and PgdA (r = 0.10) and between TpiB and DiaD (r = 0.36), but in single trees only. The subcellular localization of TPI isozymes was determined by comparing isoenzymes from the total extract with those found in fraction enriched in plastids, prepared by differential gradient centrifugation of cellular organelles. The more slowly migrating TPI-B isozyme is located in plastids.

  1. Regulation of mammalian microRNA processing and function by cellular signaling and subcellular localization

    PubMed Central

    Smalheiser, Neil R.

    2008-01-01

    For many microRNAs, in many normal tissues and in cancer cells, the cellular levels of mature microRNAs are not simply determined by transcription of microRNA genes. This mini-review will discuss how microRNA biogenesis and function can be regulated by various nuclear and cytoplasmic processing events, including emerging evidence that microRNA pathway components can be selectively regulated by control of their subcellular localization and by modifications that occur during dynamic cellular signaling. Finally, I will briefly summarize studies of microRNAs in synaptic fractions of adult mouse forebrain, which may serve as a model for other cell types as well. PMID:18433727

  2. Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells.

    PubMed

    Drastichova, Zdenka; Novotny, Jiri

    2012-08-01

    Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors. At least some types of G-protein α subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists. However, the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol. Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of G(q/11)α protein and to determine their localization in isolated fractions and stability in naïve and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G(11)α protein. We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the G(q/11) protein, which were found to be membrane-bound. Both of these complexes dissociated after prolonged treatment with TRH. Still other G(q/11)α protein complexes of lower molecular weight were determined in the cytosol. These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h) hormone treatment. These results support the notion that a portion of G(q/11)α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor. At the same time, these findings indicate that the redistribution of G(q/11)α is brought about by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the G(q/11)α protein.

  3. RSLpred: an integrative system for predicting subcellular localization of rice proteins combining compositional and evolutionary information.

    PubMed

    Kaundal, Rakesh; Raghava, Gajendra P S

    2009-05-01

    The attainment of complete map-based sequence for rice (Oryza sativa) is clearly a major milestone for the research community. Identifying the localization of encoded proteins is the key to understanding their functional characteristics and facilitating their purification. Our proposed method, RSLpred, is an effort in this direction for genome-scale subcellular prediction of encoded rice proteins. First, the support vector machine (SVM)-based modules have been developed using traditional amino acid-, dipeptide- (i+1) and four parts-amino acid composition and achieved an overall accuracy of 81.43, 80.88 and 81.10%, respectively. Secondly, a similarity search-based module has been developed using position-specific iterated-basic local alignment search tool and achieved 68.35% accuracy. Another module developed using evolutionary information of a protein sequence extracted from position-specific scoring matrix achieved an accuracy of 87.10%. In this study, a large number of modules have been developed using various encoding schemes like higher-order dipeptide composition, N- and C-terminal, splitted amino acid composition and the hybrid information. In order to benchmark RSLpred, it was tested on an independent set of rice proteins where it outperformed widely used prediction methods such as TargetP, Wolf-PSORT, PA-SUB, Plant-Ploc and ESLpred. To assist the plant research community, an online web tool 'RSLpred' has been developed for subcellular prediction of query rice proteins, which is freely accessible at http://www.imtech.res.in/raghava/rslpred.

  4. A study of SeqA subcellular localization in Escherichia coli using photo-activated localization microscopy.

    PubMed

    Mika, Jacek T; Vanhecke, Aster; Dedecker, Peter; Swings, Toon; Vangindertael, Jeroen; Van den Bergh, Bram; Michiels, Jan; Hofkens, Johan

    2015-01-01

    Escherichia coli (E. coli) cells replicate their genome once per cell cycle to pass on genetic information to the daughter cells. The SeqA protein binds the origin of replication, oriC, after DNA replication initiation and sequesters it from new initiations in order to prevent overinitiation. Conventional fluorescence microscopy studies of SeqA localization in bacterial cells have shown that the protein is localized to discrete foci. In this study we have used photo-activated localization microscopy (PALM) to determine the localization of SeqA molecules, tagged with fluorescent proteins, with a localization precision of 20-30 nm with the aim to visualize the SeqA subcellular structures in more detail than previously possible. SeqA-PAmCherry was imaged in wild type E. coli, expressed from plasmid or genetically engineered into the bacterial genome, replacing the native seqA gene. Unsynchronized cells as well as cells with a synchronized cell cycle were imaged at various time points, in order to investigate the evolution of SeqA localization during the cell cycle. We found that SeqA indeed localized into discrete foci but these were not the only subcellular localizations of the protein. A significant amount of SeqA-PAmCherry molecules was localized outside the foci and in a fraction of cells we saw patterns indicating localization at the membrane. Using quantitative PALM, we counted protein copy numbers per cell, protein copy numbers per focus, the numbers of foci per cell and the sizes of the SeqA clusters. The data showed broad cell-to-cell variation and we did not observe a correlation between SeqA-PAmCherry protein numbers and the cell cycle under the experimental conditions of this study. The numbers of SeqA-PAmCherry molecules per focus as well as the foci sizes also showed broad distributions indicating that the foci are likely not characterized by a fixed number of molecules. We also imaged an E. coli strain devoid of the dam methylase (Δdam) and observed

  5. Protein subcellular localization prediction based on compartment-specific features and structure conservation

    PubMed Central

    Su, Emily Chia-Yu; Chiu, Hua-Sheng; Lo, Allan; Hwang, Jenn-Kang; Sung, Ting-Yi; Hsu, Wen-Lian

    2007-01-01

    Background Protein subcellular localization is crucial for genome annotation, protein function prediction, and drug discovery. Determination of subcellular localization using experimental approaches is time-consuming; thus, computational approaches become highly desirable. Extensive studies of localization prediction have led to the development of several methods including composition-based and homology-based methods. However, their performance might be significantly degraded if homologous sequences are not detected. Moreover, methods that integrate various features could suffer from the problem of low coverage in high-throughput proteomic analyses due to the lack of information to characterize unknown proteins. Results We propose a hybrid prediction method for Gram-negative bacteria that combines a one-versus-one support vector machines (SVM) model and a structural homology approach. The SVM model comprises a number of binary classifiers, in which biological features derived from Gram-negative bacteria translocation pathways are incorporated. In the structural homology approach, we employ secondary structure alignment for structural similarity comparison and assign the known localization of the top-ranked protein as the predicted localization of a query protein. The hybrid method achieves overall accuracy of 93.7% and 93.2% using ten-fold cross-validation on the benchmark data sets. In the assessment of the evaluation data sets, our method also attains accurate prediction accuracy of 84.0%, especially when testing on sequences with a low level of homology to the training data. A three-way data split procedure is also incorporated to prevent overestimation of the predictive performance. In addition, we show that the prediction accuracy should be approximately 85% for non-redundant data sets of sequence identity less than 30%. Conclusion Our results demonstrate that biological features derived from Gram-negative bacteria translocation pathways yield a significant

  6. Insulin-induced Effects on the Subcellular Localization of AKT1, AKT2 and AS160 in Rat Skeletal Muscle

    PubMed Central

    Zheng, Xiaohua; Cartee, Gregory D.

    2016-01-01

    AKT1 and AKT2, the AKT isoforms that are highly expressed in skeletal muscle, have distinct and overlapping functions, with AKT2 more important for insulin-stimulated glucose metabolism. In adipocytes, AKT2 versus AKT1 has greater susceptibility for insulin-mediated redistribution from cytosolic to membrane localization, and insulin also causes subcellular redistribution of AKT Substrate of 160 kDa (AS160), an AKT2 substrate and crucial mediator of insulin-stimulated glucose transport. Although skeletal muscle is the major tissue for insulin-mediated glucose disposal, little is known about AKT1, AKT2 or AS160 subcellular localization in skeletal muscle. The major aim of this study was to determine insulin’s effects on the subcellular localization and phosphorylation of AKT1, AKT2 and AS160 in skeletal muscle. Rat skeletal muscles were incubated ex vivo ± insulin, and differential centrifugation was used to isolate cytosolic and membrane fractions. The results revealed that: 1) insulin increased muscle membrane localization of AKT2, but not AKT1; 2) insulin increased AKT2 phosphorylation in the cytosol and membrane fractions; 3) insulin increased AS160 localization to the cytosol and membranes; and 4) insulin increased AS160 phosphorylation in the cytosol, but not membranes. These results demonstrate distinctive insulin effects on the subcellular redistribution of AKT2 and its substrate AS160 in skeletal muscle. PMID:27966646

  7. Loss of Expression and Function of SOCS3 Is an Early Event in HNSCC: Altered Subcellular Localization as a Possible Mechanism Involved in Proliferation, Migration and Invasion

    PubMed Central

    Rossa, Carlos; Sommer, Gunhild; Spolidorio, Luis C.; Rosenzweig, Steven A.; Watson, Dennis K.; Kirkwood, Keith L.

    2012-01-01

    Background Suppressor of cytokine signaling 3 (SOCS3) is an inducible endogenous negative regulator of signal transduction and activator of transcription 3 (STAT3). Epigenetic silencing of SOCS3 has been shown in head and neck squamous cell carcinoma (HNSCC), which is associated with increased activation of STAT3. There is scarce information on the functional role of the reduction of SOCS3 expression and no information on altered subcellular localization of SOCS3 in HNSCC. Methodology/Principal Findings We assessed endogenous SOCS3 expression in different HNSCC cell lines by RT-qPCR and western blot. Immunofluorescence and western blot were used to study the subcellular localization of endogenous SOCS3 induced by IL-6. Overexpression of SOCS3 by CMV-driven plasmids and siRNA-mediated inhibition of endogenous SOCS3 were used to verify the role of SOCS3 on tumor cell proliferation, viability, invasion and migration in vitro. In vivo relevance of SOCS3 expression in HNSCC was studied by quantitative immunohistochemistry of commercially-available tissue microarrays. Endogenous expression of SOCS3 was heterogeneous in four HNSCC cell lines and surprisingly preserved in most of these cell lines. Subcellular localization of endogenous SOCS3 in the HNSCC cell lines was predominantly nuclear as opposed to cytoplasmic in non-neoplasic epithelial cells. Overexpression of SOCS3 produced a relative increase of the protein in the cytoplasmic compartment and significantly inhibited proliferation, migration and invasion, whereas inhibition of endogenous nuclear SOCS3 did not affect these events. Analysis of tissue microarrays indicated that loss of SOCS3 is an early event in HNSCC and was correlated with tumor size and histological grade of dysplasia, but a considerable proportion of cases presented detectable expression of SOCS3. Conclusion Our data support a role for SOCS3 as a tumor suppressor gene in HNSCC with relevance on proliferation and invasion processes and suggests

  8. Subcellular localization of proteasomes and their regulatory complexes in mammalian cells.

    PubMed Central

    Brooks, P; Fuertes, G; Murray, R Z; Bose, S; Knecht, E; Rechsteiner, M C; Hendil, K B; Tanaka, K; Dyson, J; Rivett, J

    2000-01-01

    Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes. PMID:10657252

  9. Subcellular localization of human heparanase and its alternative splice variant in COS-7 cells.

    PubMed

    Sato, Mayumi; Amemiya, Kana; Hayakawa, Sumio; Munakata, Hiroshi

    2008-08-01

    Heparanase, the enzyme that degrades heparan sulfate, has been implicated to play important and characteristic roles in organogenesis, tissue organization, cell migration, and tumor metastasis. Clarification of its expression, its intracellular sorting, and its secretion is, therefore, of much importance to understand its role in cell biology. In addition to the 1.7 Kb transcript previously reported, we detected a 1.5 Kb transcript of human heparanase by RT-PCR. The smaller transcript was shown to be an alternatively spliced variant lacking exon 5, which contains the essential glutamic acid residue required for enzyme activity. When expressed in COS-7 cells this variant did not show any heparanase activity. Full-length heparanase and the exon 5-deleted splice variant were expressed in COS-7 cells and examined by confocal laser scanning microscopy. Both proteins co-localized with calnexin, a marker protein for the endoplasmic reticulum, and they co-immunoprecipitated with calnexin. Both proteins were postulated to be precursors based upon the results of SDS-PAGE analyses. Treatment with endoglycosidases revealed that all potential N-glycosylation sites in the proteins were glycosylated. Tunicamycin treatment of transfected COS-7 cells inhibited N-glycosylation but did not change the subcellular localization. These results indicate that overexpressed heparanase and its splice variant localize to the endoplasmic reticulum independent of glycosylation in COS-7 cells.

  10. Systematic study of subcellular localization of Arabidopsis PPR proteins confirms a massive targeting to organelles

    PubMed Central

    Colcombet, Jean; Lopez-Obando, Mauricio; Heurtevin, Laure; Bernard, Clément; Martin, Karine; Berthomé, Richard; Lurin, Claire

    2013-01-01

    Four hundred and fifty-eight genes coding for PentatricoPeptide Repeat (PPR) proteins are annotated in the Arabidopsis thaliana genome. Over the past 10 years, numerous reports have shown that many of these proteins function in organelles to target specific transcripts and are involved in post-transcriptional regulation. Therefore, they are thought to be important players in the coordination between nuclear and organelle genome expression. Only four of these proteins have been described to be addressed outside organelles, indicating that some PPRs could function in post-transcriptional regulations of nuclear genes. In this work, we updated and improved our current knowledge on the localization of PPR proteins of Arabidopsis within the plant cell. We particularly investigated the subcellular localization of 166 PPR proteins whose targeting predictions were ambiguous, using a combination of high-throughput cloning and microscopy. Through systematic localization experiments and data integration, we confirmed that PPR proteins are largely targeted to organelles and showed that dual targeting to both the mitochondria and plastid occurs more frequently than expected. These results allow us to speculate that dual-targeted PPR proteins could be important for the fine coordination of gene expressions in both organelles. PMID:24037373

  11. Subcellular localization and regulation of type-1C and type-5 phosphodiesterases

    SciTech Connect

    Dolci, Susanna; Belmonte, Alessia; Santone, Rocco; Giorgi, Mauro; Pellegrini, Manuela; Carosa, Eleonora; Piccione, Emilio; Lenzi, Andrea; Jannini, Emmanuele A. . E-mail: jannini@univaq.it

    2006-03-17

    We investigated the subcellular localization of PDE5 in in vitro human myometrial cells. We demonstrated for First time that PDE5 is localized in discrete cytoplasmic foci and vesicular compartments corresponding to centrosomes. We also found that PDE5 intracellular localization is not cell- or species-specific, as it is conserved in different animal and human cells. PDE5 protein levels are strongly regulated by the mitotic activity of the smooth muscle cells (SMCs), as they were increased in quiescent, contractile myometrial cultures, and conditions in which proliferation was inhibited. In contrast, PDE1C levels decreased in all conditions that inhibited proliferation. This mirrored the enzymatic activity of both PDE5 and PDE1C. Increasing cGMP intracellular levels by dbcGMP or sildenafil treatments did not block proliferation, while dbcAMP inhibited myometrial cell proliferation. Together, these results suggest that PDE5 regulation of cGMP intracellular levels is not involved in the control of SMC cycle progression, but may represent one of the markers of the contractile phenotype.

  12. Subcellular localization of adenosine kinase in mammalian cells: The long isoform of AdK is localized in the nucleus

    SciTech Connect

    Cui, Xianying Amy; Singh, Bhag; Park, Jae; Gupta, Radhey S.

    2009-10-09

    Two isoforms of adenosine kinase (AdK) have been identified in mammalian organisms with the long isoform (AdK-long) containing extra 20-21 amino acids at the N-terminus (NTS). The subcellular localizations of these isoforms are not known and they contain no identifiable targeting sequence. Immunofluorescence labeling of mammalian cells expressing either only AdK-long or both isoforms with AdK-specific antibody showed only nuclear labeling or both nucleus and cytoplasmic labeling, respectively. The AdK-long and -short isoforms fused at the C-terminus with c-myc epitope also localized in the nucleus and cytoplasm, respectively. Fusion of the AdK-long NTS to green fluorescent protein also resulted in its nuclear localization. AdK-long NTS contains a cluster of conserved amino acids (PKPKKLKVE). Replacement of KK in this sequence with either AA or AD abolished its nuclear localization capability, indicating that this cluster likely serves as a nuclear localization signal. AdK in nucleus is likely required for sustaining methylation reactions.

  13. Complete genome sequence and in planta subcellular localization of maize fine streak virus proteins.

    PubMed

    Tsai, Chi-Wei; Redinbaugh, Margaret G; Willie, Kristen J; Reed, Sharon; Goodin, Michael; Hogenhout, Saskia A

    2005-05-01

    The genome of the nucleorhabdovirus maize fine streak virus (MFSV) consists of 13,782 nucleotides of nonsegmented, negative-sense, single-stranded RNA. The antigenomic strand consisted of seven open reading frames (ORFs), and transcripts of all ORFs were detected in infected plants. ORF1, ORF6, and ORF7 had significant similarities to the nucleocapsid protein (N), glycoprotein (G), and polymerase (L) genes of other rhabdoviruses, respectively, whereas the ORF2, ORF3, ORF4, and ORF5 proteins had no significant similarities. The N (ORF1), ORF4, and ORF5 proteins localized to nuclei, consistent with the presence of nuclear localization signals (NLSs) in these proteins. ORF5 likely encodes the matrix protein (M), based on its size, the position of its NLS, and the localization of fluorescent protein fusions to the nucleus. ORF2 probably encodes the phosphoprotein (P) because, like the P protein of Sonchus yellow net virus (SYNV), it was spread throughout the cell when expressed alone but was relocalized to a subnuclear locus when coexpressed with the MFSV N protein. Unexpectedly, coexpression of the MFSV N and P proteins, but not the orthologous proteins of SYNV, resulted in accumulations of both proteins in the nucleolus. The N and P protein relocalization was specific to cognate proteins of each virus. The subcellular localizations of the MFSV ORF3 and ORF4 proteins were distinct from that of the SYNV sc4 protein, suggesting different functions. To our knowledge, this is the first comparative study of the cellular localizations of plant rhabdoviral proteins. This study indicated that plant rhabdoviruses are diverse in genome sequence and viral protein interactions.

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

    SciTech Connect

    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 in 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.

  15. Subcellular localization of enterokinase (enteropeptidase EC 3.4.21.9) in rat small intestine.

    PubMed

    Lebenthal, E; Morrissey, G W

    1977-04-27

    The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3% in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (Mr, 2 - 10(6)) and two larger peaks of free enzyme (Mr, 3 - 10(5) and 9 -10). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.

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

    SciTech Connect

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

    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 us 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.

  17. Protein-Protein Interaction Network and Subcellular Localization of the Arabidopsis Thaliana ESCRT Machinery.

    PubMed

    Richardson, Lynn G L; Howard, Alexander S M; Khuu, Nicholas; Gidda, Satinder K; McCartney, Andrew; Morphy, Brett J; Mullen, Robert T

    2011-01-01

    The endosomal sorting complex required for transport (ESCRT) consists of several multi-protein subcomplexes which assemble sequentially at the endosomal surface and function in multivesicular body (MVB) biogenesis. While ESCRT has been relatively well characterized in yeasts and mammals, comparably little is known about ESCRT in plants. Here we explored the yeast two-hybrid protein interaction network and subcellular localization of the Arabidopsis thaliana ESCRT machinery. We show that the Arabidopsis ESCRT interactome possesses a number of protein-protein interactions that are either conserved in yeasts and mammals or distinct to plants. We show also that most of the Arabidopsis ESCRT proteins examined at least partially localize to MVBs in plant cells when ectopically expressed on their own or co-expressed with other interacting ESCRT proteins, and some also induce abnormal MVB phenotypes, consistent with their proposed functional role(s) as part of the ESCRT machinery in Arabidopsis. Overall, our results help define the plant ESCRT machinery by highlighting both conserved and unique features when compared to ESCRT in other evolutionarily diverse organisms, providing a foundation for further exploration of ESCRT in plants.

  18. Subcellular localization of Suppressor of Hairless in Drosophila sense organ cells during Notch signalling.

    PubMed

    Gho, M; Lecourtois, M; Géraud, G; Posakony, J W; Schweisguth, F

    1996-06-01

    During imaginal development of Drosophila, Suppressor of Hairless [Su(H)], an evolutionarily conserved transcription factor that mediates intracellular signalling by the Notch (N) receptor, controls successive alternative cell fate decisions leading to the differentiation of multicellular sensory organs. We describe here the distribution of the Su(H) protein in the wing disc epithelium throughout development of adult sense organs. Su(H) was found to be evenly distributed in the nuclei of all imaginal disc cells during sensory organ precursor cells selection. Thus differential expression and/or subcellular localization of Su(H) is not essential for its function. Soon after division of the pIIa secondary precursor cell, Su(H) specifically accumulates in the nucleus of the future socket cell. At the onset of differentiation of the socket cell, Su(H) is also detected in the cytoplasm. In this differentiating cell, N and deltex participate in the cytoplasmic retention of Su(H). Still, Su(H) does not colocalize with N at the apical-lateral membranes. These observations suggest that N regulates in an indirect manner the cytoplasmic localization of Su(H) in the socket cell. Finally, the pIIb, shaft and socket cells are found to adopt invariant positions along the anteroposterior axis of the notum. This raises the possibility that tissue-polarity biases these N-mediated cell fate choices.

  19. Arginine Methylation of hnRNP A2 Does Not Directly Govern Its Subcellular Localization

    PubMed Central

    Nouwens, Amanda S.; Wei, Ying; Rothnagel, Joseph A.; Smith, Ross

    2013-01-01

    The hnRNP A/B paralogs A1, A2/B1 and A3 are key components of the nuclear 40S hnRNP core particles. Despite a high degree of sequence similarity, increasing evidence suggests they perform additional, functionally distinct roles in RNA metabolism. Here we identify and study the functional consequences of differential post-translational modification of hnRNPs A1, A2 and A3. We show that while arginine residues in the RGG box domain of hnRNP A1 and A3 are almost exhaustively, asymmetrically dimethylated, hnRNP A2 is dimethylated at only a single residue (Arg-254) and this modification is conserved across cell types. It has been suggested that arginine methylation regulates the nucleocytoplasmic distribution of hnRNP A/B proteins. However, we show that transfected cells expressing an A2R254A point mutant exhibit no difference in subcellular localization. Similarly, immunostaining and mass spectrometry of endogenous hnRNP A2 in transformed cells reveals a naturally-occurring pool of unmethylated protein but an exclusively nuclear pattern of localization. Our results suggest an alternative role for post-translational arginine methylation of hnRNPs and offer further evidence that the hnRNP A/B paralogs are not functionally redundant. PMID:24098712

  20. Expression Pattern and Subcellular Localization of the Ovate Protein Family in Rice

    PubMed Central

    Yu, Hui; Jiang, Wenzhu; Liu, Qing; Zhang, Hui; Piao, Mingxin; Chen, Zhengdao; Bian, Mingdi

    2015-01-01

    The Arabidopsis ovate family proteins (AtOFPs) have been shown to function as transcriptional repressors and regulate multiple aspects of plant growth and development. There are 31 genes that encode the full-length OVATE-domain containing proteins in the rice genome. In this study, the gene structure analysis revealed that OsOFPs are intron poor. Phylogenetic analysis suggested that OVATE proteins from rice, Arabidopsis and tomato can be divided into 4 groups (I–IV). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis identified OsOFPs with different tissue-specific expression patterns at all stages of development in the rice plant. Interestingly, nearly half of the total number of OsOFP family was more highly expressed during the seed developmental stage. In addition, seed developmental cis-elements were found in the promoter region of the OsOFPs. Subcellular localization analysis revealed that YFP-OsOFP fusion proteins predominantly localized in the nucleus. Our results suggest that OsOFPs may act as regulatory proteins and play pivotal roles in the growth and development of rice. PMID:25760462

  1. Stress-Responsive Expression, Subcellular Localization and Protein-Protein Interactions of the Rice Metacaspase Family.

    PubMed

    Huang, Lei; Zhang, Huijuan; Hong, Yongbo; Liu, Shixia; Li, Dayong; Song, Fengming

    2015-07-17

    Metacaspases, a class of cysteine-dependent proteases like caspases in animals, are important regulators of programmed cell death (PCD) during development and stress responses in plants. The present study was focused on comprehensive analyses of expression patterns of the rice metacaspase (OsMC) genes in response to abiotic and biotic stresses and stress-related hormones. Results indicate that members of the OsMC family displayed differential expression patterns in response to abiotic (e.g., drought, salt, cold, and heat) and biotic (e.g., infection by Magnaporthe oryzae, Xanthomonas oryzae pv. oryzae and Rhizoctonia solani) stresses and stress-related hormones such as abscisic acid, salicylic acid, jasmonic acid, and 1-amino cyclopropane-1-carboxylic acid (a precursor of ethylene), although the responsiveness to these stresses or hormones varies to some extent. Subcellular localization analyses revealed that OsMC1 was solely localized and OsMC2 was mainly localized in the nucleus. Whereas OsMC3, OsMC4, and OsMC7 were evenly distributed in the cells, OsMC5, OsMC6, and OsMC8 were localized in cytoplasm. OsMC1 interacted with OsLSD1 and OsLSD3 while OsMC3 only interacted with OsLSD1 and that the zinc finger domain in OsMC1 is responsible for the interaction activity. The systematic expression and biochemical analyses of the OsMC family provide valuable information for further functional studies on the biological roles of OsMCs in PCD that is related to abiotic and biotic stress responses.

  2. Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochrome b5 by electron microscopy: methodology and application to various subcellular fractions

    PubMed Central

    1976-01-01

    The localization of cytochrome b5 on the membranes of various subcellular organelles of rat liver was studied by a cytoimmunological procedure using anti-cytochrome b5/anti-ferritin hybrid antibodies and ferritin as label. For this study, highly purified and biochemically characterized membrane preparations were employed. Outer mitochondrial membranes were found to be heavily labeled by the hybrid antibodies whereas Golgi and plasma membranes were not marked by the reagent. Peroxisome membranes were moderately labeled by the hybrid antibodies, suggesting that they may contain some cytochrome b5. The preparation and purification of hybrid antibodies without peptic digestion is described and an analysis made of the composition of the final reagent product. PMID:791954

  3. Functional Characterization and Subcellular Localization of Poplar (Populus trichocarpa × Populus deltoides) Cinnamate 4-Hydroxylase1

    PubMed Central

    Ro, Dae Kyun; Mah, Nancy; Ellis, Brian E.; Douglas, Carl J.

    2001-01-01

    Cinnamic acid 4-hydroxylase (C4H), a member of the cytochrome P450 monooxygenase superfamily, plays a central role in phenylpropanoid metabolism and lignin biosynthesis and possibly anchors a phenylpropanoid enzyme complex to the endoplasmic reticulum (ER). A full-length cDNA encoding C4H was isolated from a hybrid poplar (Populus trichocarpa × P. deltoides) young leaf cDNA library. RNA-blot analysis detected C4H transcripts in all organs tested, but the gene was most highly expressed in developing xylem. C4H expression was also strongly induced by elicitor-treatment in poplar cell cultures. To verify the catalytic activity of the putative C4H cDNA, two constructs, C4H and C4H fused to the FLAG epitope (C4H::FLAG), were expressed in yeast. Immunoblot analysis showed that C4H was present in the microsomal fraction and microsomal preparations from strains expressing both enzymes efficiently converted cinnamic acid to p-coumaric acid with high specific activities. To investigate the subcellular localization of C4H in vivo, a chimeric C4H-green fluorescent protein (GFP) gene was engineered and stably expressed in Arabidopsis. Confocal laser microscopy analysis clearly showed that in Arabidopsis the C4H::GFP chimeric enzyme was localized to the ER. When expressed in yeast, the C4H::GFP fusion enzyme was also active but displayed significantly lower specific activity than either C4H or C4H::FLAG in in vitro and in vivo enzyme assays. These data definitively show that C4H is localized to the ER in planta. PMID:11351095

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

    SciTech Connect

    Song, Yan; Lv, Liyang; Du, Juan; Yue, Longtao; Cao, Lili

    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 localizations 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.

  5. Subcellular localization and immunological detection of proteins encoded by the vir locus of Bordetella pertussis.

    PubMed Central

    Stibitz, S; Yang, M S

    1991-01-01

    The DNA sequence of the central regulatory locus vir of Bordetella pertussis predicts that three gene products, BvgA, BvgB, and BvgC, are encoded. Features of the predicted primary structures of these proteins and their homology to other two-component systems suggest that BvgA is located in the cytoplasm, BvgB is located in the periplasm, and BvgC spans the inner membrane. We have used gene fusions to the phoA and lacZ genes of Escherichia coli to investigate the subcellular localization and membrane topology of these proteins. PhoA fusion proteins were also purified and used to raise antibodies that allowed visualization of the vir-encoded polypeptides by Western immunoblotting. Our results have largely confirmed the predictions of the DNA sequence, with the exception that BvgB and BvgC were found to constitute one larger protein that was homologous to the sensor class of two-component systems. We propose that this protein be named BvgS (for sensor) and that its gene be named bvgS. Images PMID:2066330

  6. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells

    PubMed Central

    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-01-01

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes. PMID:26431330

  7. Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils.

    PubMed Central

    Sengeløv, H; Boulay, F; Kjeldsen, L; Borregaard, N

    1994-01-01

    The subcellular localization of N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors in human neutrophils was investigated. The fMLP receptor was detected with a high-affinity, photoactivatable, radioiodinated derivative of N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK). Neutrophils were disrupted by nitrogen cavitation and fractionated on Percoll density gradients. fMLP receptors were located in the beta-band containing gelatinase and specific granules, and in the gamma-band containing plasma membrane and secretory vesicles. Plasma membranes and secretory vesicles were separated by high-voltage free-flow electrophoresis, and secretory vesicles were demonstrated to be highly enriched in fMLP receptors. The receptors found in secretory vesicles translocated fully to the plasma membrane upon stimulation with inflammatory mediators. The receptor translocation from the beta-band indicated that the receptor present there was mainly located in gelatinase granules. A 25 kDa fMLP-binding protein was found in the beta-band. Immunoprecipitation revealed that this protein was identical with NGAL (neutrophil gelatinase-associated lipocalin), a novel protein found in specific granules. In summary, we demonstrate that the compartment in human neutrophils that is mobilized most easily and fastest, the secretory vesicle, is a major reservoir of fMLP receptors. This explains the prompt and extensive upregulation of fMLP receptors on the neutrophil surface in response to inflammatory stimuli. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8172608

  8. Lesion profiling and subcellular prion localization of cervid chronic wasting disease in domestic cats.

    PubMed

    Seelig, D M; Nalls, A V; Flasik, M; Frank, V; Eaton, S; Mathiason, C K; Hoover, E A

    2015-01-01

    Chronic wasting disease (CWD) is an efficiently transmitted, fatal, and progressive prion disease of cervids with an as yet to be fully clarified host range. While outbred domestic cats (Felis catus) have recently been shown to be susceptible to experimental CWD infection, the neuropathologic features of the infection are lacking. Such information is vital to provide diagnostic power in the event of natural interspecies transmission and insights into host and strain interactions in interspecies prion infection. Using light microscopy and immunohistochemistry, we detail the topographic pattern of neural spongiosis (the "lesion profile") and the distribution of misfolded prion protein in the primary and secondary passage of feline CWD (Fel(CWD)). We also evaluated cellular and subcellular associations between misfolded prion protein (PrP(D)) and central nervous system neurons and glial cell populations. From these studies, we (1) describe the novel neuropathologic profile of Fel(CWD), which is distinct from either cervid CWD or feline spongiform encephalopathy (FSE), and (2) provide evidence of serial passage-associated interspecies prion adaptation. In addition, we demonstrate through confocal analysis the successful co-localization of PrP(D) with neurons, astrocytes, microglia, lysosomes, and synaptophysin, which, in part, implicates each of these in the neuropathology of Fel(CWD). In conclusion, this work illustrates the simultaneous role of both host and strain in the development of a unique Fel(CWD) neuropathologic profile and that such a profile can be used to discriminate between Fel(CWD) and FSE.

  9. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence.

    PubMed

    Emanuelsson, O; Nielsen, H; Brunak, S; von Heijne, G

    2000-07-21

    A neural network-based tool, TargetP, for large-scale subcellular location prediction of newly identified proteins has been developed. Using N-terminal sequence information only, it discriminates between proteins destined for the mitochondrion, the chloroplast, the secretory pathway, and "other" localizations with a success rate of 85% (plant) or 90% (non-plant) on redundancy-reduced test sets. From a TargetP analysis of the recently sequenced Arabidopsis thaliana chromosomes 2 and 4 and the Ensembl Homo sapiens protein set, we estimate that 10% of all plant proteins are mitochondrial and 14% chloroplastic, and that the abundance of secretory proteins, in both Arabidopsis and Homo, is around 10%. TargetP also predicts cleavage sites with levels of correctly predicted sites ranging from approximately 40% to 50% (chloroplastic and mitochondrial presequences) to above 70% (secretory signal peptides). TargetP is available as a web-server at http://www.cbs.dtu.dk/services/TargetP/.

  10. 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 (τm ) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter τm 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.

  11. Cloning of human PEX cDNA. Expression, subcellular localization, and endopeptidase activity.

    PubMed

    Lipman, M L; Panda, D; Bennett, H P; Henderson, J E; Shane, E; Shen, Y; Goltzman, D; Karaplis, A C

    1998-05-29

    Mutations in the PEX gene are responsible for X-linked hypophosphatemic rickets. To gain insight into the role of PEX in normal physiology we have cloned the human full-length cDNA and studied its tissue expression, subcellular localization, and peptidase activity. We show that the cDNA encodes a 749-amino acid protein structurally related to a family of neutral endopeptidases that include neprilysin as prototype. By Northern blot analysis, the size of the full-length PEX transcript is 6.5 kilobases. PEX expression, as determined by semi-quantitative polymerase chain reaction, is high in bone and in tumor tissue associated with the paraneoplastic syndrome of renal phosphate wasting. PEX is glycosylated in the presence of canine microsomal membranes and partitions exclusively in the detergent phase from Triton X-114 extractions of transiently transfected COS cells. Immunofluorescence studies in A293 cells expressing PEX tagged with a c-myc epitope show a predominant cell-surface location for the protein with its COOH-terminal domain in the extracellular compartment, substantiating the assumption that PEX, like other members of the neutral endopeptidase family, is a type II integral membrane glycoprotein. Cell membranes from cultured COS cells transiently expressing PEX efficiently degrade exogenously added parathyroid hormone-derived peptides, demonstrating for the first time that recombinant PEX can function as an endopeptidase. PEX peptidase activity may provide a convenient target for pharmacological intervention in states of altered phosphate homeostasis and in metabolic bone diseases.

  12. 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.

  13. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells.

    PubMed

    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-11-10

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes.

  14. Detection and subcellular localization of dehydrin-like proteins in quinoa (Chenopodium quinoa Willd.) embryos.

    PubMed

    Carjuzaa, P; Castellión, M; Distéfano, A J; del Vas, M; Maldonado, S

    2008-01-01

    The aim of this study was to characterize the dehydrin content in mature embryos of two quinoa cultivars, Sajama and Baer La Unión. Cultivar Sajama grows at 3600-4000 m altitude and is adapted to the very arid conditions characteristic of the salty soils of the Bolivian Altiplano, with less than 250 mm of annual rain and a minimum temperature of -1 degrees C. Cultivar Baer La Unión grows at sea-level regions of central Chile and is adapted to more humid conditions (800 to 1500 mm of annual rain), fertile soils, and temperatures above 5 degrees C. Western blot analysis of embryo tissues from plants growing under controlled greenhouse conditions clearly revealed the presence of several dehydrin bands (at molecular masses of approximately 30, 32, 50, and 55 kDa), which were common to both cultivars, although the amount of the 30 and 32 kDa bands differed. Nevertheless, when grains originated from their respective natural environments, three extra bands (at molecular masses of approximately 34, 38, and 40 kDa), which were hardly visible in Sajama, and another weak band (at a molecular mass of approximately 28 kDa) were evident in Baer La Unión. In situ immunolocalization microscopy detected dehydrin-like proteins in all axis and cotyledon tissues. At the subcellular level, dehydrins were detected in the plasma membrane, cytoplasm and nucleus. In the cytoplasm, dehydrins were found associated with mitochondria, rough endoplasmic reticulum cisternae, and proplastid membranes. The presence of dehydrins was also recognized in the matrix of protein bodies. In the nucleus, dehydrins were associated with the euchromatin. Upon examining dehydrin composition and subcellular localization in two quinoa cultivars belonging to highly contrasting environments, we conclude that most dehydrins detected here were constitutive components of the quinoa seed developmental program, but some of them (specially the 34, 38, and 40 kDa bands) may reflect quantitative molecular differences

  15. Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides

    SciTech Connect

    Sturm, A.; Johnson, K.D.; Szumilo, T.; Elbein, A.D.; Chrispeels, M.J.

    1987-11-01

    Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc/sub 3/Man/sub 9/(GlcNAc)/sub 2/, was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associates with Golgi. These include mannosidase I (removes 1-2 mannose residues from Man/sub 6-9/(GlcNAc)/sub 2/), mannosidase II (removes mannose residues from GlcNAcMan/sub 5/(GlcNAc)/sub 2/), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). The authors have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltranferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.

  16. Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans.

    PubMed

    Chang, Peng; Fan, Xueyi; Chen, Jiangye

    2015-08-01

    Candida albicans is an opportunistic fungal pathogen commonly found in humans. It has the ability to switch reversibly between three growth forms: budding yeast, pseudohypha, and hypha. The transition between yeast and hyphal growth forms is critical for the pathogenesis of C. albicans. During the yeast-to-hypha morphologic transition, gene expression is regulated by transcriptional regulators including histone modifying complexes and chromatin remodeling complexes. We previously reported that Esa1, a catalytic subunit in the histone acetyltransferase complex NuA4, is essential for the hyphal development of C. albicans. In this study, we analyzed the functional roles of Gcn5, a catalytic subunit in the histone acetyltransferase complex SAGA, in C. albicans. Gcn5 is required for the invasive and filamentous growth of C. albicans. Deletion of GCN5 impaired hyphal elongation in sensing serum and attenuated the virulence of C. albicans in a mouse systemic infection model. The C. albicans gcn5/gcn5 mutant cells also exhibited sensitivity to cell wall stress. Functional analysis showed that the HAT domain and Bromodomain in Gcn5 play distinct roles in morphogenesis and cell wall stress response of C. albicans. Our results show that the conserved residue Glu188 is crucial for the Gcn5 HAT activity and for Gcn5 function during filamentous growth. In addition, the subcellular distribution of ectopically expressed GFP-Gcn5 correlates with the different growth states of C. albicans. In stationary phase, Gcn5 accumulated in the nucleus, while during vegetative growth it localized in the cytoplasm in a morpha-independent manner. Our results suggest that the nuclear localization of Gcn5 depends on the existence of its N-terminal NLS and HAT domains.

  17. Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization

    PubMed Central

    Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

    2015-01-01

    Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in

  18. Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization.

    PubMed

    Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

    2015-01-01

    Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in

  19. Subcellular localization of the APOBEC3 proteins during mitosis and implications for genomic DNA deamination.

    PubMed

    Lackey, Lela; Law, Emily K; Brown, William L; Harris, Reuben S

    2013-03-01

    Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme's capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.

  20. High Sequence Variability, Diverse Subcellular Localizations, and Ecological Implications of Alkaline Phosphatase in Dinoflagellates and Other Eukaryotic Phytoplankton

    PubMed Central

    Lin, Xin; Zhang, Huan; Cui, Yudong; Lin, Senjie

    2012-01-01

    Alkaline phosphatase (AP) is a key enzyme for phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphorus is limited. While three major types of AP and their correspondingly diverse subcellular localization have been recognized in bacteria, little is known about AP in eukaryotic phytoplankton such as dinoflagellates. Here, we isolated a full-length AP cDNA from a latest-diverging dinoflagellate genus Alexandrium, and conducted comparative analyses with homologs from a relatively basal (Amphidinium carterae) and late-diverging (Karenia brevis) lineage of dinoflagellates as well as other eukaryotic algae. New data and previous studies indicate that AP is common in dinoflagellates and most other major eukaryotic groups of phytoplankton. AP sequences are more variable than many other genes studied in dinoflagellates, and are divergent among different eukaryotic phytoplankton lineages. Sequence comparison to the other characterized APs suggests that dinoflagellates and some other eukaryotic phytoplankton possess the putative AP as phoA type, but some other eukaryotic phytoplankton seem to have other types. Phylogenetic analyses based on AP amino acid sequences indicated that the “red-type” eukaryotic lineages formed a monophyletic group, suggesting a common origin of their APs. As different amino acid sequences have been found to predictably determine different spatial distribution in the cells, which may facilitate access to different pools of DOP, existing computational models were adopted to predict the subcellular localizations of putative AP in the three dinoflagellates and other eukaryotic phytoplankton. Results showed different subcellular localizations of APs in different dinoflagellates and other lineages. The linkage between AP sequence divergence, subcellular localization, and ecological niche differentiation requires rigorous experimental verification, and this study now provides a framework for such a future effort

  1. HIPK2 catalytic activity and subcellular localization are regulated by activation-loop Y354 autophosphorylation

    PubMed Central

    Siepi, Francesca; Gatti, Veronica; Camerini, Serena; Crescenzi, Marco; Soddu, Silvia

    2013-01-01

    HIPK2 (homeodomain-interacting protein kinase-2) binds to and phosphorylates, at Ser and Thr residues, a large number of targets involved in cell division and cell fate decision in response to different physiological or stress stimuli. Inactivation of HIPK2 has been observed in human and mouse cancers supporting its role as a tumor suppressor. Despite the biological relevance of this kinase, very little is known on how HIPK2 becomes catalytically active. Based on sequence homologies, HIPK2 has been taxonomically classified as a subfamily member of the dual-specificity tyrosine-regulated kinases (DYRKs) and the activation-loop Y354 of HIPK2 has been found phosphorylated in different cells; however, the relevance of this Y phosphorylation is presently unknown. Here, we show that HIPK2, which is extensively phosphorylated at S/T sites throughout its functional domains, becomes catalytically active by autophosphorylation at the activation-loop Y354. In particular, we found that, in analogy to DYRKs, HIPK2-Y354 phosphorylation is an autocatalytic event and its prevention, through Y354 substitution with non-phosphorylatable amino acids or by using the kinase inhibitor purvalanol A, induces a strong reduction of the HIPK2 S/T-kinase activity on different substrates. Interestingly, at variance from DYRKs, inhibition of HIPK2-Y354 phosphorylation induces a strong out-of-target Y-kinase activity in cis and a strong cytoplasmic relocalization of the kinase. Together, these results demonstrate that the catalytic activity, substrate specificity, and subcellular localization of HIPK2 are regulated by autophosphorylation of its activation-loop Y354. PMID:23485397

  2. Subcellular localization of creatine kinase in Torpedo electrocytes: association with acetylcholine receptor-rich membranes

    PubMed Central

    1985-01-01

    Creatine kinase (CK, EC 2.7.3.2) has recently been identified as the intermediate isoelectric point species (pl 6.5-6.8) of the Mr 40,000- 43,000 nonreceptor, peripheral v-proteins in Torpedo marmorata acetylcholine receptor-rich membranes (Barrantes, F. J., G. Mieskes, and T. Wallimann, 1983, Proc. Natl. Acad. Sci. USA, 80: 5440-5444). In the present study, this finding is substantiated at the cellular and subcellular level of the T. marmorata electric organ by immunofluorescence and by protein A-gold labeling of either ultrathin cryosections of electrocytes or purified receptor-membrane vesicles that use subunit-specific anti-chicken creatine kinase antibodies. The muscle form of the kinase, on the one hand, is present throughout the entire T. marmorata electrocyte except in the nuclei. The brain form of the kinase, on the other hand, is predominantly located on the ventral, innervated face of the electrocyte, where it is closely associated with both surfaces of the postsynaptic membrane, and secondarily in the synaptic vesicles at the presynaptic terminal. Labeling of the noninnervated dorsal membrane is observed at the invaginated sac system. In the case of purified acetylcholine receptor-rich membranes, antibodies specific for chicken B-CK label only one face of the isolated vesicles. No immunoreaction is observed with anti-chicken M-CK antibodies. A discussion follows on the possible implications of these localizations of creatine kinase in connection with the function of the acetylcholine receptor at the postsynaptic membrane, the Na/K ATPase at the dorsal electrocyte membrane, and the ATP-dependent transmitter release at the nerve ending. PMID:3884630

  3. Subcellular localization and trafficking of phytolongins (non-SNARE longins) in the plant secretory pathway

    PubMed Central

    de Marcos Lousa, Carine; Soubeyrand, Eric; Bolognese, Paolo; Wattelet-Boyer, Valerie; Bouyssou, Guillaume; Marais, Claireline; Boutté, Yohann; Filippini, Francesco; Moreau, Patrick

    2016-01-01

    SNARE proteins are central elements of the machinery involved in membrane fusion of eukaryotic cells. In animals and plants, SNAREs have diversified to sustain a variety of specific functions. In animals, R-SNARE proteins called brevins have diversified; in contrast, in plants, the R-SNARE proteins named longins have diversified. Recently, a new subfamily of four longins named ‘phytolongins’ (Phyl) was discovered. One intriguing aspect of Phyl proteins is the lack of the typical SNARE motif, which is replaced by another domain termed the ‘Phyl domain’. Phytolongins have a rather ubiquitous tissue expression in Arabidopsis but still await intracellular characterization. In this study, we found that the four phytolongins are distributed along the secretory pathway. While Phyl2.1 and Phyl2.2 are strictly located at the endoplasmic reticulum network, Phyl1.2 associates with the Golgi bodies, and Phyl1.1 locates mainly at the plasma membrane and partially in the Golgi bodies and post-Golgi compartments. Our results show that export of Phyl1.1 from the endoplasmic reticulum depends on the GTPase Sar1, the Sar1 guanine nucleotide exchange factor Sec12, and the SNAREs Sec22 and Memb11. In addition, we have identified the Y48F49 motif as being critical for the exit of Phyl1.1 from the endoplasmic reticulum. Our results provide the first characterization of the subcellular localization of the phytolongins, and we discuss their potential role in regulating the secretory pathway. PMID:26962210

  4. Subcellular localization and properties of mouse adrenal C19-steroid 5beta-reductase.

    PubMed Central

    Collins, W; Cameron, E H

    1975-01-01

    The localization and some characteristics of mouse adrenal C19-steroid 5 beta-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 alpha-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3alpha-, 3beta- and a small amount of 17 beta-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 beta-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 beta products first had to be oxidized to 5 beta-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 beta-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 beta-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 beta reduction of [7 alpha-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) "/- 0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration. PMID:239699

  5. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier

    NASA Astrophysics Data System (ADS)

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-06-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics.

  6. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier

    PubMed Central

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-01-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics. PMID:27323846

  7. Do arbuscular mycorrhizal fungi affect cadmium uptake kinetics, subcellular distribution and chemical forms in rice?

    PubMed

    Li, Hui; Luo, Na; Zhang, Li Jun; Zhao, Hai Ming; Li, Yan Wen; Cai, Quan Ying; Wong, Ming Hung; Mo, Ce Hui

    2016-11-15

    Rice (Oryza sativa L.) plants were inoculated with two species of arbuscular mycorrhizal fungi (AMF) - Rhizophagus intraradices (RI) and Funneliformis mosseae (FM) and grown for 60days to ensure strong colonization. Subsequently, a short-term hydroponic experiment was carried out to investigate the effects of AMF on cadmium (Cd) uptake kinetics, subcellular distribution and chemical forms in rice exposed to six Cd levels (0, 0.005, 0.01, 0.025, 0.05, 0.1mM) for three days. The results showed that the uptake kinetics of Cd fitted the Michaelis-Menten model well (R(2)>0.89). AMF significantly decreased the Cd concentrations both in shoots and roots in Cd solutions. Furthermore, the decrement of Cd concentrations by FM was significantly higher than RI treatment in roots. AMF reduced the Cd concentrations markedly in the cell wall fractions at high Cd substrate (≥0.025mM). The main subcellular fraction contributed to Cd detoxification was cell wall at low Cd substrate (<0.05mM), while vacuoles at high Cd substrate (≥0.05mM). Moreover, the concentrations and proportions of Cd in inorganic and water-soluble form also reduced by AMF colonization at high Cd substrate (≥0.05mM), both in shoots and roots. This suggested that AMF could convert Cd into inactive forms which were less toxic. Therefore, AMF could enhance rice resistance to Cd through altering subcellular distribution and chemical forms of Cd in rice.

  8. Bypassing the need for subcellular localization of a polysaccharide export-anchor complex by overexpressing its protein subunits

    PubMed Central

    Javens, June; Wan, Zhe; Hardy, Gail G.; Brun, Yves V.

    2013-01-01

    Summary Subcellular protein localization is thought to promote protein-protein interaction by increasing the effective concentration and enabling spatial coordination and proper segregation of proteins. We found that protein overexpression allowed the assembly of a productive polysaccharide biosynthesis-export-anchoring complex in the absence of polar localization in Caulobacter crescentus. Polar localization of the holdfast export protein, HfsD, depends on the presence of the other export proteins, HfsA, and HfsB, and on the polar scaffold protein PodJ. The holdfast deficiency of hfsB and podJ mutants is suppressed by the overexpression of export proteins. Restored holdfasts are randomly positioned and co-localize with a holdfast anchor protein in these strains, indicating that functional complexes can form at non-polar sites. Therefore, overexpression of export proteins surpasses a concentration threshold necessary for holdfast synthesis. Restoration of holdfast synthesis at non-polar sites reduces surface adhesion, consistent with the need to spatially coordinate the holdfast synthesis machinery with the flagellum and pili. These strains lack the cell-specific segregation of the holdfast, resulting in the presence of holdfasts in motile daughter cells. Our results highlight the fact that multiple facets of subcellular localization can be coupled to improve the phenotypic outcome of a protein assembly. PMID:23714375

  9. Cellular and subcellular localization of cholecystokinin (CCK)-1 receptors in the pancreas, gallbladder, and stomach of mice.

    PubMed

    Konno, Kohtarou; Takahashi-Iwanaga, Hiromi; Uchigashima, Motokazu; Miyasaka, Kyoko; Funakoshi, Akihiro; Watanabe, Masahiko; Iwanaga, Toshihiko

    2015-03-01

    Information concerning the cellular localization of cholecystokinin (CCK)-1 receptors has been discrepant and remained scanty at ultrastructural levels. The present immunohistochemical study at light and electron microscopic levels revealed the distinct localization of CCK1 receptors in visceral organs. Immunohistochemistry by use of a purified antibody against mouse CCK1 receptor was applied to fixed tissue sections of the pancreas, gallbladder, stomach, and intestine of mice. A silver-intensified immunogold method revealed the subcellular localization under electron microscope. The immunoreactivity for CCK1 receptors was selectively found in the basolateral membrane of pancreatic acinar cells and gastric chief cells but was absent in pancreatic islets and gastric D cells. Another intense expression in the gut was seen in the myenteric nerve plexus of the antro-duodenal region and some populations of c-Kit-expressing pacemaker cells in the duodenal musculature. The gallbladder contained smooth muscle fibers with an intense immunoreactivity of CCK1 receptors on cell surfaces. The restricted localization of CCK1 receptors on the basolateral membrane of pancreatic acinar cells and gastric chief cells, along with their absence in the islets of Langerhans and gastric D cells, provides definitive information concerning the regulatory mechanism by circulating CCK. Especially, the subcellular localization in the acinar cells completes the investigation for the detection of circulating CCK by the basolateral membrane.

  10. Skeletal muscle glycogen synthase subcellular localization: effects of insulin and PPAR-alpha agonist (K-111) administration in rhesus monkeys.

    PubMed

    Ortmeyer, Heidi K; Adall, Yohannes; Marciani, Karina R; Katsiaras, Andreas; Ryan, Alice S; Bodkin, Noni L; Hansen, Barbara C

    2005-06-01

    Insulin covalently and allosterically regulates glycogen synthase (GS) and may also cause the translocation of GS from glycogen-poor to glycogen-rich locations. We examined the possible role of subcellular localization of GS and glycogen in insulin activation of GS in skeletal muscle of six obese monkeys and determined whether 1) insulin stimulation during a hyperinsulinemic euglycemic clamp and/or peroxisome proliferator-activated receptor (PPAR)-alpha agonist treatment (K-111, 3 mg.kg(-1).day(-1); Kowa) induced translocation of GS and 2) translocation of GS was associated with insulin activation of GS. GS and glycogen were present in all fractions obtained by differential centrifugation, except for the cytosolic fraction, under both basal and insulin-stimulated conditions. We found no evidence for translocation of GS by insulin. GS total (GST) activity was strongly associated with glycogen content (r = 0.70, P < 0.001). Six weeks of treatment with K-111 increased GST activity in all fractions, except the cytosolic fraction, and mean GST activity, GS independent activity, and glycogen content were significantly higher in the insulin-stimulated samples compared with basal samples, effects not seen with vehicle. The increase in GST activity was strongly related to the increase in glycogen content during the hyperinsulinemic euglycemic clamp after K-111 administration (r = 0.74, P < 0.001). Neither GS protein expression nor GS gene expression was affected by insulin or by K-111 treatment. We conclude that 1) in vivo insulin does not cause translocation of GS from a glycogen-poor to a glycogen-rich location in primate skeletal muscle and 2) the mechanism of action of K-111 to improve insulin sensitivity includes an increase in GST activity without an increase in GS gene or protein expression.

  11. Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts.

    PubMed

    Ferri, Paola; Barbieri, Elena; Burattini, Sabrina; Guescini, Michele; D'Emilio, Alessandra; Biagiotti, Laura; Del Grande, Paolo; De Luca, Antonio; Stocchi, Vilberto; Falcieri, Elisabetta

    2009-12-15

    It is known that the MyoD family members (MyoD, Myf5, myogenin, and MRF4) play a pivotal role in the complex mechanism of skeletal muscle cell differentiation. However, fragmentary information on transcription factor-specific regulation is available and data on their post-transcriptional and post-translational behavior are still missing. In this work, we combined mRNA and protein expression analysis with their subcellular localization. Each myogenic regulator factor (MRF) revealed a specific mRNA trend and a protein quantitative analysis not overlapping, suggesting the presence of post-transcriptional mechanisms. In addition, each MRF showed a specific behavior in situ, characterized by a differentiation stage-dependent localization suggestive of a post-translational regulation also. Consistently with their transcriptional activity, immunogold electron microscopy data revealed MRFs distribution in interchromatin domains. Our results showed a MyoD and Myf5 contrasting expression profile in proliferating myoblasts, as well as myogenin and MRF4 opposite distribution in the terminally differentiated myotubes. Interestingly, MRFs expression and subcellular localization analysis during C2C12 cell differentiation stages showed two main MRFs regulation mechanisms: (i) the protein half-life regulation to modulate the differentiation stage-dependent transcriptional activity and (ii) the cytoplasmic retention, as a translocation process, to inhibit the transcriptional activity. Therefore, our results exhibit that MRFs nucleo-cytoplasmic trafficking is involved in muscle differentiation and suggest that, besides the MRFs expression level, also MRFs subcellular localization, related to their functional activity, plays a key role as a regulatory step in transcriptional control mechanisms.

  12. Subcellular Size

    PubMed Central

    Marshall, Wallace F.

    2016-01-01

    All of the same conceptual questions about size in organisms apply equally at the level of single cells. What determines the size, not only of the whole cell, but of all its parts? What ensures that subcellular components are properly proportioned relative to the whole cell? How does alteration in organelle size affect biochemical function? Answering such fundamental questions requires us to understand how the size of individual organelles and other cellular structures is determined. Knowledge of organelle biogenesis and dynamics has advanced rapidly in recent years. Does this knowledge give us enough information to formulate reasonable models for organelle size control, or are we still missing something? PMID:25957302

  13. Subcellular localization and release of human neutrophil gelatinase, confirming the existence of separate gelatinase-containing granules.

    PubMed Central

    Kjeldsen, L; Bjerrum, O W; Askaa, J; Borregaard, N

    1992-01-01

    An e.l.i.s.a. was developed using specific polyclonal rabbit antibodies against human neutrophil gelatinase. This assay, in contrast to the functional assay, is independent of activation of gelatinase, and is specific for the detection of gelatinase in both its reduced and unreduced forms. Using this assay, we were able to demonstrate a difference between the subcellular localization of gelatinase on the one hand, and the subcellular localization of vitamin B-12-binding protein, lactoferrin and cytochrome b558 on the other hand. The latter three co-localized in fractions of slightly higher density than gelatinase on a two-layer Percoll density gradient. Furthermore, the release of gelatinase exceeded the release of vitamin B-12-binding protein as well as lactoferrin by a factor of 3-6 following stimulation with formylmethionyl-leucyl-phenylalanine, leukotriene B4 and other soluble stimuli. Thus, although gelatinase has previously been found to co-localize with lactoferrin on immuno-electron microscopy, we confirm the existence of gelatinase-rich and lactoferrin- and vitamin B-12-binding-protein-poor granules, that are lighter and mobilized more easily than specific granules. These gelatinase-containing granules are not the store of cytochrome b558. Images Fig. 1. Fig. 3. Fig. 4. PMID:1332677

  14. Subcellular localization of cytoplasmic lattice-associated proteins is dependent upon fixation and processing procedures.

    PubMed

    Morency, Eric; Anguish, Lynne; Coonrod, Scott

    2011-02-16

    We and others have recently demonstrated by immuno-EM and mutation analysis that two oocyte-restricted maternal effect genes, PADI6 and MATER, localize, in part, to the oocyte cytoplasmic lattices (CPLs). During these ongoing studies, however, we found that the localization of these factors by confocal immunofluorescence (IF) analysis can vary dramatically depending upon how the oocytes and embryos are processed, with the localization pattern sometimes appearing more uniformly cytoplasmic while at other times appearing to be primarily cortical. We set out to better understand this differential staining pattern by testing a range of IF protocol parameters, changing mainly time and temperature conditions of the primary antibody solution incubation, as well as fixation methods. We found by confocal IF whole mount analysis that PADI6 and MATER localization in germinal vesicle stage oocytes is mainly cytoplasmic when the oocytes are fixed and then incubated with primary antibodies at room temperature for 1 hour, while the localization of these factors is largely limited to the cortex when the oocytes are fixed and incubated in primary antibody at 4 °C overnight. We then probed sections of fixed/embedded ovaries and isolated two-cell embryos with specific antibodies and found that, under these conditions, PADI6 and MATER were again primarily cytoplasmically localized, although the staining for these factors is slightly more cortical at the two-cell stage. Taken together, our results suggest that the localization of CPL-associated proteins by confocal IF is particularly affected by processing conditions. Further, based on our current observations, it appears that PADI6 and MATER are primarily distributed throughout the cytoplasm as opposed to the oocyte subcortex.

  15. 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

  16. 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/.

  17. HybridGO-Loc: Mining Hybrid Features on Gene Ontology for Predicting Subcellular Localization of Multi-Location Proteins

    PubMed Central

    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/. PMID:24647341

  18. Feature extraction by statistical contact potentials and wavelet transform for predicting subcellular localizations in gram negative bacterial proteins.

    PubMed

    Arango-Argoty, G A; Jaramillo-Garzón, J A; Castellanos-Domínguez, G

    2015-01-07

    Predicting the localization of a protein has become a useful practice for inferring its function. Most of the reported methods to predict subcellular localizations in Gram-negative bacterial proteins make use of standard protein representations that generally do not take into account the distribution of the amino acids and the structural information of the proteins. Here, we propose a protein representation based on the structural information contained in the pairwise statistical contact potentials. The wavelet transform decodes the information contained in the primary structure of the proteins, allowing the identification of patterns along the proteins, which are used to characterize the subcellular localizations. Then, a support vector machine classifier is trained to categorize them. Cellular compartments like periplasm and extracellular medium are difficult to predict, having a high false negative rate. The wavelet-based method achieves an overall high performance while maintaining a low false negative rate, particularly, on "periplasm" and "extracellular medium". Our results suggest the proposed protein characterization is a useful alternative to representing and predicting protein sequences over the classical and cutting edge protein depictions.

  19. Subcellular clustering of a putative c-di-GMP-dependent exopolysaccharide machinery affecting macro colony architecture in Bacillus subtilis.

    PubMed

    Bedrunka, Patricia; Graumann, Peter L

    2016-11-29

    The structure of bacterial biofilms is predominantly established through the secretion of extracellular polymeric substances (EPS). They show that Bacillus subtilis contains an operon (ydaJ-N) whose induction leads to increased Congo Red staining of biofilms and strongly altered biofilm architecture, suggesting that it mediates the production of an unknown exopolysaccharide. Supporting this idea, overproduction of YdaJKLMN leads to cell clumping during exponential growth in liquid culture, and also causes colony morphology alterations in wild type cells, as well as in a mutant background lacking the major exopolysaccharide of B. subtilis. The first gene product of the operon, YdaJ, appears to modify the overproduction effects, but is not essential for cell clumping or altered colony morphology, while the presence of the c-di-GMP receptor YdaK is required, suggesting an involvement of second messenger c-di-GMP. YdaM, YdaN and YdaK colocalize to clusters predominantly at the cell poles and are statically positioned at this subcellular site, similar to other exopolysaccharide machinery components in other bacteria. Their analysis reveals that B. subtilis contains a static subcellular assembly of an EPS machinery that affects cell aggregation and biofilm formation.

  20. Identification and subcellular localization of human rab5b, a new member of the ras-related superfamily of GTPases.

    PubMed Central

    Wilson, D B; Wilson, M P

    1992-01-01

    Members of the mammalian rab family of GTPases are associated with specific subcellular compartments, where these proteins are postulated to function in vesicular transport. By screening a human umbilical vein endothelial cell library with degenerate oligonucleotide probes, we have isolated a 1.6-kb cDNA clone encoding a 215-amino-acid protein belonging to the rab family of GTPases. This newly identified rab protein is 81% identical to human rab5, the canine counterpart of which has been localized to the plasma membrane and early endosomes. In light of this homology, we have named this new member of the GTPase superfamily "rab5b." Northern analysis using the rab5b cDNA as a probe revealed a 3.6-kb mRNA in a variety of cell types, including human umbilical vein endothelial cells, K562 erythroleukemia cells, U937 monoblastic cells, and HeLa cells. A fusion protein between glutathione-S-transferase (GST) and rab5b was expressed in bacteria and purified to homogeneity. The recombinant protein was shown to bind GTP and GDP. As is typical of other recombinant rab proteins, the rab5b-GST fusion protein displayed a low intrinsic rate of GTP hydrolysis (0.005/min). An antiserum to rab5b was prepared and used to determine the apparent molecular size and subcellular distribution of the protein. Western blotting with this antibody revealed a 25-kD protein in COS cells transfected with rab5b and in nontransfected HeLa cells. Indirect immunofluorescence and subcellular fractionation showed that rab5b localizes to the plasma membrane. We speculate that rab5b plays a role in vesicular trafficking at the plasma membrane in various cell types. Images PMID:1541686

  1. Subcellular and intranuclear localization of neptunium-237 (V) in rat liver.

    PubMed

    Paquet, F; Verry, M; Grillon, G; Landesman, C; Masse, R; Taylor, D M

    1995-08-01

    The present investigation was aimed at establishing the distribution of 237Np within the different structures of hepatocytes. Rats were contaminated experimentally by intravenous injection of 237Np (V) and the subcellular structures of the liver were separated by ultracentrifugation. Twenty-four hours after contamination, the nuclear and cytosolic fractions bound 54 and 32%, respectively, of the total radionuclide. Purification of the nuclei followed by dissociation of the protein components in medium of increasing ionic strength showed a specific binding of neptunium to the structural proteins of the nuclear matrix.

  2. 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.

  3. Subcellular localization of K+ channels in mammalian brain neurons: remarkable precision in the midst of extraordinary complexity

    PubMed Central

    Trimmer, James S.

    2015-01-01

    Potassium channels (KChs) are the most diverse ion channels, in part due to extensive combinatorial assembly of a large number of principal and auxiliary subunits into an assortment of KCh complexes. This structural and functional diversity allows KChs to play diverse roles in neuronal function. Localization of KChs within specialized neuronal compartments defines their physiological role, and also fundamentally impacts their activity, due to localized exposure to diverse cellular determinants of channel function. Recent studies in mammalian brain reveal an exquisite refinement of KCh subcellular localization. This includes axonal KChs at the initial segment, and near/within nodes of Ranvier and presynaptic terminals, dendritic KChs found at sites reflecting specific synaptic input, and KChs defining novel compartments. Painting the remarkable diversity of KChs onto the complex architecture of mammalian neurons creates an elegant picture of electrical signal processing underlying the sophisticated function of individual neuronal compartments, and ultimately neurotransmission and behavior. PMID:25611506

  4. Predicting plant protein subcellular multi-localization by Chou's PseAAC formulation based multi-label homolog knowledge transfer learning.

    PubMed

    Mei, Suyu

    2012-10-07

    Recent years have witnessed much progress in computational modeling for protein subcellular localization. However, there are far few computational models for predicting plant protein subcellular multi-localization. In this paper, we propose a multi-label multi-kernel transfer learning model for predicting multiple subcellular locations of plant proteins (MLMK-TLM). The method proposes a multi-label confusion matrix and adapts one-against-all multi-class probabilistic outputs to multi-label learning scenario, based on which we further extend our published work MK-TLM (multi-kernel transfer learning based on Chou's PseAAC formulation for protein submitochondria localization) for plant protein subcellular multi-localization. By proper homolog knowledge transfer, MLMK-TLM is applicable to novel plant protein subcellular localization in multi-label learning scenario. The experiments on plant protein benchmark dataset show that MLMK-TLM outperforms the baseline model. Unlike the existing models, MLMK-TLM also reports its misleading tendency, which is important for comprehensive survey of model's multi-labeling performance.

  5. Chemical potential constraints on the composition and subcellular localization of proteins

    NASA Astrophysics Data System (ADS)

    Dick, J. M.; Helgeson, H. C.

    2004-12-01

    The distribution and speciation of the metallome in organisms is amenable to study using thermodynamic calculations that take into account the chemical potentials obtaining in living cells. In particular, subcellular spatial gradients of the negative logarithms of the activities of the electron and proton (pe and pH, respectively) strongly influence the speciation of aqueous metals and other inorganic species, as well as aqueous organic and biomacromolecular species. Although pe-pH diagrams are commonly used to describe speciation in inorganic aqueous systems, they have not been applied to assess and quantify the relative stabilities of biomacromolecules in living organisms. Nevertheless, there is much to be gained by doing so. The purpose of the present communication is to demonstrate this by generating pe-pH and other equilibrium activity diagrams for proteins in the system C-H-N-O-S. The relative abundances of amino acid residues in the proteins considered are representative of proteins found in different subcellular locations. For example, the boundaries of the stability fields for extracellular, cytoplasmic, and nuclear proteins can be assessed and portrayed on pe-pH diagrams. By overlaying pe-pH diagrams for proteins with those for metals, one can predict the oxidation states of metals compatible with the proteins found in the different subcellular locations. The standard molal thermodynamic properties of these proteins can be estimated from group additivity algorithms that include provision for protein ionization as a function of solution pH. The temperature and pressure dependence of these properties can be computed with the aid of the revised HKF equations of state. Because quantifying the relative stabilities of proteins is a multidimensional problem, a Gibbs free energy minimization software package was used to carry out a plethora of computer experiments for specified temperatures, pressures, and bulk compositions. Plotting the results of the Gibbs free

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

    PubMed Central

    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 PMID:25953080

  7. 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

  8. Sumoylation delays the ATF7 transcription factor subcellular localization and inhibits its transcriptional activity.

    PubMed

    Hamard, Pierre-Jacques; Boyer-Guittaut, Michaël; Camuzeaux, Barbara; Dujardin, Denis; Hauss, Charlotte; Oelgeschläger, Thomas; Vigneron, Marc; Kedinger, Claude; Chatton, Bruno

    2007-01-01

    Over the past few years, small ubiquitin-like modifier (SUMO) modification has emerged as an important regulator of diverse pathways and activities including protein localization and transcriptional regulation. We identified a consensus sumoylation motif (IKEE), located within the N-terminal activation domain of the ATF7 transcription factor and thus investigated the role of this modification. ATF7 is a ubiquitously expressed transcription factor, homologous to ATF2, that binds to CRE elements within specific promoters. This protein is able to heterodimerize with Jun or Fos proteins and its transcriptional activity is mediated by interaction with TAF12, a subunit of the general transcription factor TFIID. In the present article, we demonstrate that ATF7 is sumoylated in vitro (using RanBP2 as a E3-specific ligase) and in vivo. Moreover, we show that ATF7 sumoylation affects its intranuclear localization by delaying its entry into the nucleus. Furthermore, SUMO conjugation inhibits ATF7 transactivation activity by (i) impairing its association with TAF12 and (ii) blocking its binding-to-specific sequences within target promoters.

  9. Spatial and temporal changes in Bax subcellular localization during NPe6-PDT-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Xing, Da; Chen, Wei R.; Wan, Qingling; Zhou, Feifan

    2008-02-01

    Photodynamic therapy (PDT) employing photosensiter N-aspartyl chlorin e6 (NPe6) can induce lysosome disruption and initiate the intrinsic apoptotic pathway. Bax, a member of the Bcl-2 family of proteins, is an essential regulator of apoptosis. Bax is normally found in the cytosol of healthy cells, and translocates to mitochondria in response to many apoptotic stimuli. In this study, using real-time single-cell analysis, we have investigated the kinetics of Bax distribution during NPe6-induced apoptosis in ASTC-a-1 cells. In order to monitor Bax subcellular distribution, cells were stained with GFP-Bax and Mito Tracker Red. The results show that Bax redistribution occurred at about 170 min after treated with NPe6-PDT, and then sequestered into clusters associated with the mitochondira within 30 min. Our data clearly showed the spatial and temporal changes in Bax distribution in living cells during NPe6-induced apoptosis.

  10. Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

    SciTech Connect

    Tinglu, G.; Ghosh, A.; Ghosh, B.K.

    1984-08-01

    Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G (IgG) complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold -IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C. 27 references, 6 figures, 1 table.

  11. Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases*

    PubMed Central

    Gentry, Leanna R.; Nishimura, Akiyuki; Cox, Adrienne D.; Martin, Timothy D.; Tsygankov, Denis; Nishida, Motohiro; Elston, Timothy C.; Der, Channing J.

    2015-01-01

    The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB. PMID:26216878

  12. A Comparative Antibody Analysis of Pannexin1 Expression in Four Rat Brain Regions Reveals Varying Subcellular Localizations

    PubMed Central

    Cone, Angela C.; Ambrosi, Cinzia; Scemes, Eliana; Martone, Maryann E.; Sosinsky, Gina E.

    2012-01-01

    Pannexin1 (Panx1) channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus, and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on-line viewing, and

  13. Subcellular localization of the PGE2 synthesis activity in mouse resident peritoneal macrophages

    PubMed Central

    1984-01-01

    The aim of this work was to establish, on a quantitative basis, the subcellular distribution of the enzyme system that converts arachidonic acid into prostaglandin (PG) E2 in mouse resident peritoneal (MRP) macrophages. Kinetic studies were conducted on cell-free extracts derived from cells cultivated for 1 d, using [1-14C]arachidonic acid as substrate and measuring the label in PGE2 after extraction and thin layer chromatography. The activity was synergistically enhanced by L- adrenaline and reduced glutathione, inhibited by indomethacin, and linearly related to the concentration of the cell-free extract. It was labile at 0 degrees C in the medium used for homogenization and fractionation of the cells (half-life less than 2 h). Addition of catalase (0.15 mg/ml) to the suspension medium increased the initial activity (by congruent to 70%) and the stability (half-life congruent to 6 h) of the enzyme in cytoplasmic extracts. It enabled us to establish the density distribution after isopycnic centrifugation in a linear gradient of sucrose. The sample centrifuged consisted of untreated cytoplasmic extracts, or cytoplasmic extracts treated with digitonin and Na pyrophosphate. Comparison of the centrifugation behavior of PGE2 synthesis activity with that of various enzymes used as reference for the major subcellular entities has revealed that PGE2 synthesis fairly fits the density profile of sulfatase C in each case. The conclusion is that at least the rate-limiting reaction in the conversion of arachidonic acid into PGE2 is catalyzed by an enzyme associated with the endoplasmic reticulum. PMID:6420497

  14. Convergence of the target of rapamycin and the Snf1 protein kinase pathways in the regulation of the subcellular localization of Msn2, a transcriptional activator of STRE (Stress Response Element)-regulated genes.

    PubMed

    Mayordomo, Isabel; Estruch, Francisco; Sanz, Pascual

    2002-09-20

    The subcellular localization of Msn2, a transcriptional activator of STRE (stress response element)-regulated genes, is modulated by carbon source availability. In cells growing in glucose, Msn2 is located mainly in the cytosol, whereas in carbon source-starved cells, Msn2 is located largely inside the nucleus. However, in cells lacking Reg1 (the regulatory subunit of the Reg1/Glc7 protein phosphatase complex), the regulation of subcellular distribution is absent, Msn2 being constitutively present in the cytosol. The localization defect in these mutants is specific for carbon starvation stress, and it is because of the presence of an abnormally active Snf1 protein kinase that inhibits the nuclear localization of Msn2 upon carbon starvation. Active Snf1 kinase is also able to avoid the effects of rapamycin, a drug that by inhibiting the TOR kinase pathway leads to a nuclear localization of Msn2 in wild type cells. Therefore, active Snf1 and the TOR kinase pathway may affect similar cytosolic steps in the regulation of the subcellular localization of Msn2.

  15. Mining Proteins with Non-Experimental Annotations Based on an Active Sample Selection Strategy for Predicting Protein Subcellular Localization.

    PubMed

    Cao, Junzhe; Liu, Wenqi; He, Jianjun; Gu, Hong

    2013-01-01

    Subcellular localization of a protein is important to understand proteins' functions and interactions. There are many techniques based on computational methods to predict protein subcellular locations, but it has been shown that many prediction tasks have a training data shortage problem. This paper introduces a new method to mine proteins with non-experimental annotations, which are labeled by non-experimental evidences of protein databases to overcome the training data shortage problem. A novel active sample selection strategy is designed, taking advantage of active learning technology, to actively find useful samples from the entire data pool of candidate proteins with non-experimental annotations. This approach can adequately estimate the "value" of each sample, automatically select the most valuable samples and add them into the original training set, to help to retrain the classifiers. Numerical experiments with for four popular multi-label classifiers on three benchmark datasets show that the proposed method can effectively select the valuable samples to supplement the original training set and significantly improve the performances of predicting classifiers.

  16. Mining Proteins with Non-Experimental Annotations Based on an Active Sample Selection Strategy for Predicting Protein Subcellular Localization

    PubMed Central

    Cao, Junzhe; Liu, Wenqi; He, Jianjun; Gu, Hong

    2013-01-01

    Subcellular localization of a protein is important to understand proteins’ functions and interactions. There are many techniques based on computational methods to predict protein subcellular locations, but it has been shown that many prediction tasks have a training data shortage problem. This paper introduces a new method to mine proteins with non-experimental annotations, which are labeled by non-experimental evidences of protein databases to overcome the training data shortage problem. A novel active sample selection strategy is designed, taking advantage of active learning technology, to actively find useful samples from the entire data pool of candidate proteins with non-experimental annotations. This approach can adequately estimate the “value” of each sample, automatically select the most valuable samples and add them into the original training set, to help to retrain the classifiers. Numerical experiments with for four popular multi-label classifiers on three benchmark datasets show that the proposed method can effectively select the valuable samples to supplement the original training set and significantly improve the performances of predicting classifiers. PMID:23840667

  17. Regulation of calnexin sub-cellular localization modulates endoplasmic reticulum stress-induced apoptosis in MCF-7 cells.

    PubMed

    Delom, Frédéric; Fessart, Delphine; Chevet, Eric

    2007-02-01

    The endoplasmic reticulum (ER) is the cellular compartment where proteins enter the secretory pathway, undergo post-translational modifications and acquire a correct conformation. If these functions are chronically altered, specific ER stress signals are triggered to promote cell death through the intrinsic apoptotic pathway. Here, we show that tunicamycin causes significant alteration of calnexin sub-cellular distribution in MCF-7 cells. Interestingly, this correlates with the absence of both tunicamycin-induced calnexin phosphorylation as well as tunicamycin-induced cell death. Under these conditions, calnexin-associated Bap31, an ER integral membrane protein, is subjected to a caspase-8 cleavage pattern within a specific sub-compartment of the ER. These results suggest that MCF-7 resistance to ER stress-induced apoptosis is partially mediated by the expression level of calnexin which in turn controls its sub-cellular localization, and its association with Bap31. These data may delineate a resistance mechanism to the ER stress-induced intrinsic apoptotic pathway.

  18. Sulfotransferase 2B1b in human breast: differences in subcellular localization in African American and Caucasian women.

    PubMed

    Dumas, Nicole A; He, Dongning; Frost, Andra R; Falany, Charles N

    2008-09-01

    Breast cancer (BC) is the most commonly diagnosed cancer among American women; however, the development of post-menopausal BC is significantly lower in African Americans as compared to Caucasians. Hormonal stimulation is important in BC development and differences in the conversion of dehydroepiandrosterone (DHEA) into estrogens may be involved in the lower incidence of post-menopausal BC in African American women. DHEA sulfation by sulfotransferase 2B1b (SULT2B1b) is important in regulating the conversion of DHEA into estrogens in tissues. SULT2B1b is localized in both cytosol and nuclei of some tissues including cancerous and associated-normal breast tissue. Immunohistochemical staining was used to evaluate the total expression and subcellular localization of SULT2B1b in African American and Caucasian breast tissues. Cell fractionation, immunoblot analysis and sulfation assays were used to characterize the subcellular expression and activity of SULT2B1b in BC tissues and T-47D breast adenocarcinoma cells. Immunohistochemical analysis of SULT2B1b showed that African Americans had a significantly greater amount of SULT2B1b in epithelial cells of associated-normal breast tissue as compared to Caucasians. Also, more SULT2B1b in African American associated-normal breast epithelial cells was localized in the nuclei than in Caucasians. Equivalent levels of SULT2B1b were detected in breast adenocarcinoma tissues from both African American and Caucasian women. Nuclei isolation and immunoblot analysis of both BC tissue and human T-47D breast adenocarcinoma cells demonstrated that SULT2B1b is present in nuclei and cytoplasm.

  19. Impacts of two point mutations of RPE65 from Leber's congenital amaurosis on the stability, subcellular localization and isomerohydrolase activity of RPE65.

    PubMed

    Chen, Ying; Moiseyev, Gennadiy; Takahashi, Yusuke; Ma, Jian-Xing

    2006-07-24

    RPE65, a membrane-associated protein in the retinal pigment epithelium, is the isomerohydrolase essential for regenerating 11-cis retinal, the chromophore for visual pigments. RPE65 mutations are associated with inherited retinal dystrophies. Here we report that single point mutations of RPE65, Y144D and P363T, identified in patients with Leber's congenital amaurosis (LCA), significantly decreased the stability of RPE65. Moreover, these mutations altered subcellular localization of RPE65 and abolished its isomerohydrolase activity. These observations suggest that the decreased protein stability and altered subcellular localization of RPE65 may represent a mechanism for these mutations to lead to vision loss in LCA patients.

  20. Isoform-specific subcellular localization and function of protein kinase A identified by mosaic imaging of mouse brain

    PubMed Central

    Ilouz, Ronit; Lev-Ram, Varda; Bushong, Eric A; Stiles, Travis L; Friedmann-Morvinski, Dinorah; Douglas, Christopher; Goldberg, Geoffrey; Ellisman, Mark H; Taylor, Susan S

    2017-01-01

    Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIβ or the RIIβ subunit results in distinct neuronal phenotypes. Although RIβ contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIβ is enriched in dendrites and co-localizes with MAP2, whereas RIIβ is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIβ in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIβ, but not of RIIβ, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization. DOI: http://dx.doi.org/10.7554/eLife.17681.001 PMID:28079521

  1. Autophagy and access: understanding the role of androgen receptor subcellular localization in SBMA.

    PubMed

    Montie, Heather L; Merry, Diane E

    2009-11-01

    Ridding neurons of toxic misfolded proteins is a critical feature of many neurodegenerative diseases. We have recently reported that lack of access of nuclear polyglutamine-expanded androgen receptor (AR) to the autophagic degradation pathway is a critical point in pathogenesis. When mutant AR is contained within the cytoplasm, it can be degraded by autophagy, resulting in amelioration of its toxic effects, as has been observed in other polyglutamine expansion diseases involving cytoplasmic mutant proteins. However, we have also found that pharmacological induction of autophagy protects SBMA motor neurons from the toxic effects of even nuclear localized mutant AR, albeit without affecting mutant nuclear AR levels. Thus, we have further investigated the mechanism by which autophagy elicits therapeutic benefit in cell culture. We found that endogenous autophagy only slightly alters nuclear mutant AR aggregation compared to substantial effects on cytoplasmic AR aggregation. Interestingly, pharmacological activation of mTOR-dependent autophagy did not significantly alter nuclear AR aggregation, whereas we observed that it protects SBMA motor neurons. Our findings indicate that therapeutic intervention to induce autophagy represents a potential potent benefit for SBMA, and that it likely does so by protecting SBMA motor neurons independent of a direct effect on mutant AR.

  2. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.

    PubMed

    Harris, Thurl E; Huffman, Todd A; Chi, An; Shabanowitz, Jeffrey; Hunt, Donald F; Kumar, Anil; Lawrence, John C

    2007-01-05

    Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity.

  3. Ankyrin-G Regulates Neurogenesis and Wnt Signaling by Altering the Subcellular Localization of β-catenin

    PubMed Central

    Durak, Omer; de Anda, Froylan Calderon; Singh, Karun K.; Leussis, Melanie P.; Petryshen, Tracey L.; Sklar, Pamela; Tsai, Li-Huei

    2014-01-01

    Ankyrin-G is a scaffolding protein required for the formation of the axon initial segment in neurons. Recent genome-wide association studies and whole-exome sequencing have identified ANK3, the gene coding for ankyrin-G, to be a risk gene for multiple neuropsychiatric disorders such as bipolar disorder (BD), schizophrenia, and autism spectrum disorder (ASD). Here, we describe a novel role for ankyrin-G in neural progenitor proliferation in the developing cortex. We found that ankyrin-G regulates canonical Wnt signaling by altering the subcellular localization and availability of β-catenin in proliferating cells. Ankyrin-G loss-of-function increases β-catenin levels in the nucleus, thereby promoting neural progenitor proliferation. Importantly, abnormalities in proliferation can be rescued by reducing Wnt pathway signaling. Together, these results suggest that ankyrin-G is required for proper brain development. PMID:24821222

  4. Characterization of the subcellular localization and nuclear import molecular mechanisms of herpes simplex virus 1 UL2.

    PubMed

    Cai, Mingsheng; Huang, Zebin; Liao, Zongmin; Chen, Tao; Wang, Ping; Jiang, Si; Chen, Daixiong; Peng, Tao; Bian, Yun; Hong, Gengde; Yang, Hang; Zeng, Zhancheng; Li, Xiaowei; Li, Meili

    2017-04-01

    As a crucial protein, the herpes simplex virus 1 (HSV-1) UL2 protein has been shown to take part in various stages of viral infection, nonetheless, its exact subcellular localization and transport molecular determinants are not well known thus far. In the present study, by using live cells fluorescent microscopy assay, UL2 tagged with enhanced yellow fluorescent protein was transiently expressed in live cells and showed a completely nuclear accumulation without the presence of other HSV-1 proteins. Moreover, the nuclear transport of UL2 was characterized to be assisted by multiple transport pathways through Ran-, importin α1-, α5-, α7-, β1- and transportin-1 cellular transport receptors. Consequently, these results will improve understanding of UL2-mediated biological functions in HSV-1 infection cycles.

  5. Immunocytochemical localization and subcellular site of lectin synthesis in developing wheat embryos

    SciTech Connect

    Raikhel, N.V.; Peumans, W.J.

    1986-04-01

    Appearance of wheat germ agglutinin (WGA) during wheat germ embryogenesis was studied using indirect immunocytochemical methods at the light and electron microscope levels. Developing embryos were labelled with (/sup 35/S) cysteine in pulse and pulse-chase experiments to study the synthesis and transport of the lectin to protein bodies (PB). The radical, and coleorhiza first accumulated WGA around 10 days post anthesis (DPA), while WGA was found in the epiblast and coleoptile 15 and 20 DPA respectively. At the subcellular level WGA can be seen first in a small developing PB which later fused with larger ones. WGA was distributed evenly throughout the PB. When tissue was pulse-labelled, fractionated on an isopycnic sucrose gradient and exposed to detergent, the incorporated radioactivity of released lectin coincided with the position of the endoplasmic reticulum (ER) marker enzyme NADH-cytochrome c reductase. Both radioactivity and enzyme activity shifted to a higher density in the presence of 2 mM Mg acetate, indicating that radioactive lectin was associated with the rough ER.

  6. 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.

  7. Molecular cloning, tissue expression, and subcellular localization of porcine peptidoglycan recognition proteins 3 and 4.

    PubMed

    Ueda, Wataru; Tohno, Masanori; Shimazu, Tomoyuki; Fujie, Hitomi; Aso, Hisashi; Kawai, Yasushi; Numasaki, Muneo; Saito, Tadao; Kitazawa, Haruki

    2011-09-15

    Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are present in most invertebrates and vertebrates. Mammals have four PGRPs, PGLYRP1-4. In the present study, we cloned the cDNAs encoding porcine PGLYRP3 and 4 from the esophagus of adult swine. The length of the complete open reading frames of porcine PGLYRP3 and 4 are identical and contain 1125bp encoding 374 amino acid residues. The amino acid sequences of these two proteins were more similar to their human orthologs (78.9% [PGLYRP3] and 73.9% [PGLYRP4]) than to their mouse orthologs (71.3% [PGLYRP3] and 67.9% [PGLYRP4]). Expression analysis revealed that both PGLYRP3 and 4 were more strongly expressed in digestive tract, especially the esophagus, than in immune organs such as spleen or mesenteric lymph nodes in both newborn and adult swine. To analyze the subcellular distribution of porcine PGLYRP1-4, we constructed transfectant cell lines. Western blot and flow cytometric analyses revealed that porcine PGLYRP3 and 4 are not only secreted, but also expressed on the cell surface, unlike PGLYRP1 and 2. These results should help contribute to the understanding of PGLYRP3- and 4-mediated immune responses via their recognition of intestinal microorganisms in newborn and adult swine.

  8. Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

    PubMed

    De Wolf, M; Lagrou, A; Hilderson, H J; Dierick, W

    1978-12-01

    In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.

  9. Subcellular fractionation and localization studies reveal a direct interaction of the fragile X mental retardation protein (FMRP) with nucleolin.

    PubMed

    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.

  10. Subcellular Localization of Dhurrin β-Glucosidase and Hydroxynitrile Lyase in the Mesophyll Cells of Sorghum Leaf Blades 1

    PubMed Central

    Thayer, Susan S.; Conn, Eric E.

    1981-01-01

    Studies with purified mesophyll and epidermal protoplasts and bundle sheath strands have shown that the cyanogenic glucoside dhurrin (p-hydroxy-(S)-mandelonitrile-β-d-glucoside) is localized in the epidermis of sorghum leaves whereas the enzymes involved in its degradation (dhurrin β-glucosidase and hydroxynitrile lyase) are localized in the mesophyll tissue (Kojima M, JE Poulton, SS Thayer, EE Conn 1979 Plant Physiol 63: 1022-1028). The subcellular localization of these enzymes has now been examined using linear 30 to 55% (w/w) sucrose gradients by fractionation of mesophyll protoplast components. The hydroxynitrile lyase is found in the supernatant fractions suggesting a cytoplasmic (soluble cytoplasm, microsomal or vacuolar location). The dhurrin β-glucosidase (dhurrinase) is particulate and mostly chloroplast-associated. The dhurrinase activity peak has a shoulder of activity more dense than that of the intact chloroplasts. This shoulder does not coincide with markers of any other cell fraction. In studies of chloroplasts isolated from ruptured mesophyll protoplasts by differential, low-speed centrifugation, the dhurrinase partitions in the same manner as the chloroplast marker triose phosphate dehydrogenase. Chloroplast localization of the β-glucosidase has also been shown in histochemical studies using 6-bromo-2-naphthyl-β-d-glucoside substrate coupled with fast Blue B. Images PMID:16661725

  11. Mutant analysis, protein-protein interactions and subcellular localization of the Arabidopsis B sister (ABS) protein.

    PubMed

    Kaufmann, Kerstin; Anfang, Nicole; Saedler, Heinz; Theissen, Günter

    2005-09-01

    Recently, close relatives of class B floral homeotic genes, termed B(sister) genes, have been identified in both angiosperms and gymnosperms. In contrast to the B genes themselves, B(sister) genes are exclusively expressed in female reproductive organs, especially in the envelopes or integuments surrounding the ovules. This suggests an important ancient function in ovule or seed development for B(sister) genes, which has been conserved for about 300 million years. However, investigation of the first loss-of-function mutant for a B(sister) gene (ABS/TT16 from Arabidopsis) revealed only a weak phenotype affecting endothelium formation. Here, we present an analysis of two additional mutant alleles, which corroborates this weak phenotype. Transgenic plants that ectopically express ABS show changes in the growth and identity of floral organs, suggesting that ABS can interact with floral homeotic proteins. Yeast-two-hybrid and three-hybrid analyses indicated that ABS can form dimers with SEPALLATA (SEP) floral homeotic proteins and multimeric complexes that also include the AGAMOUS-like proteins SEEDSTICK (STK) or SHATTERPROOF1/2 (SHP1, SHP2). These data suggest that the formation of multimeric transcription factor complexes might be a general phenomenon among MIKC-type MADS-domain proteins in angiosperms. Heterodimerization of ABS with SEP3 was confirmed by gel retardation assays. Fusion proteins tagged with CFP (Cyan Fluorescent Protein) and YFP (Yellow Fluorescent Protein) in Arabidopsis protoplasts showed that ABS is localized in the nucleus. Phylogenetic analysis revealed the presence of a structurally deviant, but closely related, paralogue of ABS in the Arabidopsis genome. Thus the evolutionary developmental genetics of B(sister) genes can probably only be understood as part of a complex and redundant gene network that may govern ovule formation in a conserved manner, which has yet to be fully explored.

  12. Subcellular localization of host and viral proteins associated with tobamovirus RNA replication.

    PubMed

    Hagiwara, Yuka; Komoda, Keisuke; Yamanaka, Takuya; Tamai, Atsushi; Meshi, Tetsuo; Funada, Ryo; Tsuchiya, Tomohiro; Naito, Satoshi; Ishikawa, Masayuki

    2003-01-15

    Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus-encoded replication proteins and with AtTOM2A, suggesting that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)-like structures in plant cells. In subcellular fractionation analyses, GFP-AtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low-density tonoplast-rich fractions, whereas AtTOM1-GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast-rich fractions and partially into higher-buoyant-density fractions containing membranes from several other organelles. The tobamovirus-encoded replication proteins were co-fractionated with both NtTOM1 and viral RNA-dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non-membrane-bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamoviral RNA replication complex occurs on TOM1-containing membranes and is facilitated by TOM2A.

  13. Subcellular localization and displacement by diuretics of the peripheral benzodiazepine binding site (PBS) from rat kidney

    SciTech Connect

    Lukeman, S.; Fanestil, D.

    1986-03-05

    Although the PBS has been identified in many organs, its function and cellular location are speculative. Using rapid filtration, binding of (/sup 3/H)RO 5-4864 (*RO) (.75 nM) was assessed in four subcellular fractions (.3 mg/ml) derived from depapillated rat kidney by differential centrifugation: N (450g x 2 min), O (13,000 x 10), P (105,000 x 30), and S. The binding distribution was: N-18%, O-74%, P-6%, and S-2%. Marker enzyme analysis revealed that O was enriched in mitochondria (M), lysosomes (L), peroxisomes (P), and endoplasmic reticulum (ER), but not plasma membrane, and that N contained small amounts (10-15%) of markers for the above. Repeated washing of O removed ER enzymes but preserved *RO binding. O was further fractionated with centrifugation (57,000g x 4 hr) on a linear sucrose gradient (18-65%); *RO binding then comigrated with M but not P and L markers. Centrifugation of isolated M (5500 x 10 min) on another linear sucrose gradient (37-65%) gave low and high density bands, which contained 65% and 35% of *RO binding activity, resp. *RO binding in O was specific, saturable, reversible, and inhibited by diuretics. Inhibitors with the highest potency were indacrinone (K/sub d/ = 35 ..mu..M), hydrochlorothiazide (100 ..mu..M), and ethacrynic acid (325 ..mu..M). Low potency inhibitors (K/sub d/ greater than or equal to 1 mM) included amiloride, triamterene, furosemide, bumetanide, and ozolinone.

  14. Substrate Specificity and Subcellular Localization of the Aldehyde-Alcohol Redox-coupling Reaction in Carp Cones*

    PubMed Central

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

    2013-01-01

    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. PMID:24217249

  15. Time-of-day regulates subcellular trafficking, tripartite synaptic localization and polyadenylation of the astrocytic Fabp7 mRNA

    PubMed Central

    Gerstner, Jason R.; Vanderheyden, William M.; LaVaute, Timothy; Westmark, Cara J.; Rouhana, Labib; Pack, Allan I.; Wickens, Marv; Landry, Charles F.

    2012-01-01

    The astrocyte brain fatty acid binding protein (Fabp7) has previously been shown to have a coordinated diurnal regulation of mRNA and protein throughout mouse brain, and an age-dependent decline in protein expression within synaptoneurosomal fractions. Mechanisms that control time-of-day changes in expression and trafficking Fabp7 to the perisynaptic process are not known. In this study, we confirmed an enrichment of Fabp7 mRNA and protein in the astrocytic perisynaptic compartment, and observed a diurnal change in the intracellular distribution of Fabp7 mRNA in molecular layers of hippocampus. Northern blotting revealed a coordinated time-of-day dependent oscillation for the Fabp7 mRNA poly(A) tail throughout murine brain. Cytoplasmic polyadenylation element-(CPE-) binding protein (CPEB1) regulates subcellular trafficking and translation of synaptic plasticity-related mRNAs. Here we show that Fabp7 mRNA co-immunoprecipitated with CPEB1 from primary mouse astrocyte extracts, and its 3′UTR contains phylogenetically conserved CPEs capable of regulating translation of reporter mRNAs during Xenopus oocyte maturation. Given that Fabp7 expression is confined to astrocytes and neural progenitors in adult mouse brain, the synchronized cycling pattern of Fabp7 mRNA is therefore novel of known CPE-regulated transcripts. These results implicate circadian, sleep and/or metabolic control of CPEB-mediated subcellular trafficking and localized translation of Fabp7 mRNA in the tripartite synapse of mammalian brain. PMID:22279223

  16. 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.

  17. Subcellular localizations of AS1 and AS2 suggest their common and distinct roles in plant development.

    PubMed

    Zhu, Yan; Li, Ziyu; Xu, Ben; Li, Hongda; Wang, Lingjian; Dong, Aiwu; Huang, Hai

    2008-07-01

    During leaf organogenesis, a critical step for normal leaf primordium initiation is the repression of the class 1 KNOTTED1-like homeobox (KNOX) genes. After leaf primordia are formed, they must establish polarity for normal leaf morphogenesis. Recent studies have led to the identification of a number of genes that participate in the class 1 KNOX gene repression and/or the leaf polarity establishment. ASTMMETRIC LEAVES1 and 2 (AS1 and AS2) are two of these genes, which are critical for both of these two processes. As a first step towards understanding the molecular genetic basis of the AS1-AS2 action, we determined the subcellular localizations of the two proteins in both tobacco BY2 cells and Arabidopsis plants, by fusing them to yellow/cyan fluorescent protein (YFP/CFP). Our data showed that AS1 and AS2 alone were predominantly localized in the nucleolus and the nucleoplasm, respectively. The presence of both AS1 and AS2 proteins in the same interphase cell demonstrated their co-localization in both nucleolus and nucleoplasm. In addition, AS1 alone was able to associate with the condensed chromosome in the metaphase cell. Our data suggest that AS1, AS2 and the AS1-AS2 protein complex may have distinct functions, which are all required for normal plant development.

  18. IRF-2 regulates NF-{kappa}B activity by modulating the subcellular localization of NF-{kappa}B

    SciTech Connect

    Chae, Myounghee; Kim, Kwangsoo; Park, Sun-Mi; Jang, Ik-Soon; Seo, Taegun; Kim, Dong-Min; Kim, Il-Chul; Lee, Je-Ho; Park, Junsoo

    2008-06-06

    Nuclear Factor-kappa B (NF-{kappa}B) is a transcription factor essential to the control of cell proliferation, survival, differentiation, immune response, and inflammation. Constitutive NF-{kappa}B activation has been observed in a broad variety of solid tumors and hematological malignancies, which suggests that NF-{kappa}B signaling may perform a critical role in the development of human cancers. Interferon regulatory factor-2 (IRF-2), an antagonistic transcriptional repressor of IRF-1, evidences oncogenic potential, but little is currently known regarding the mechanism underlying the oncogenic activities of IRF-2. In this study, we report that IRF-2 recruits RelA/p65 transcription factors into the nucleus via physical interaction. While the nuclear recruitment of RelA by IRF-2 augments TNF{alpha}-induced NF-{kappa}B dependent transcription, the N-terminal truncated mutant form of IRF-2 inhibits the nuclear localization of RelA, and thus interferes with NF-{kappa}B activation. Furthermore, the knockdown of IRF-2 by IRF-2 siRNA attenuates TNF{alpha}-induced NF-{kappa}B dependent transcription by inhibiting the nuclear localization of RelA. Thus, these results show that IRF-2 regulates NF-{kappa}B activity via the modulation of NF-{kappa}B subcellular localization.

  19. 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.

  20. 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.

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

    SciTech Connect

    Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki

    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 with 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.

  2. Subcellular Localization of Matrin 3 Containing Mutations Associated with ALS and Distal Myopathy

    PubMed Central

    Gallego-Iradi, M. Carolina; Clare, Alexis M.; Brown, Hilda H.; Janus, Christopher; Lewis, Jada; Borchelt, David R.

    2015-01-01

    Background Mutations in Matrin 3 [MATR3], an RNA- and DNA-binding protein normally localized to the nucleus, have been linked to amyotrophic lateral sclerosis (ALS) and distal myopathies. In the present study, we have used transient transfection of cultured cell lines to examine the impact of different disease-causing mutations on the localization of Matrin 3 within cells. Results Using CHO and human H4 neuroglioma cell models, we find that ALS/myopathy mutations do not produce profound changes in the localization of the protein. Although we did observe variable levels of Matrin 3 in the cytoplasm either by immunostaining or visualization of fluorescently-tagged protein, the majority of cells expressing either wild-type (WT) or mutant Matrin 3 showed nuclear localization of the protein. When cytoplasmic immunostaining, or fusion protein fluorescence, was seen in the cytoplasm, the stronger intensity of staining or fluorescence was usually evident in the nucleus. In ~80% of cells treated with sodium arsenite (Ars) to induce cytoplasmic stress granules, the nuclear localization of WT and F115C mutant Matrin 3 was not disturbed. Notably, over-expression of mutant Matrin 3 did not induce the formation of obvious large inclusion-like structures in either the cytoplasm or nucleus. Conclusions Our findings indicate that mutations in Matrin 3 that are associated with ALS and myopathy do not dramatically alter the normal localization of the protein or readily induce inclusion formation. PMID:26528920

  3. Subcellular localization and enzymatic properties of differentially expressed transketolase genes isolated from the desiccation tolerant resurrection plant Craterostigma plantagineum.

    PubMed

    Willige, Björn C; Kutzer, Michael; Tebartz, Felix; Bartels, Dorothea

    2009-02-01

    The desiccation tolerant resurrection plant Craterostigma plantagineum encodes three classes of transketolase transcripts, which are distinguished by their gene structures and their expression patterns. One class, represented by tkt3, is constitutively expressed and two classes, represented by tkt7 and tkt10, are upregulated upon rehydration of desiccated C. plantagineum plants. The objective of this work was to characterize the differentially expressed transketolase isoforms with respect to subcellular localization and enzymatic activity. Using GFP fusion constructs and enzymatic activity assays, we demonstrate that C. plantagineum has novel forms of transketolase which localize not to the chloroplast, but mainly to the cytoplasm and which are distinct in the enzymatic properties from the transketolase enzymes active in the Calvin cycle or oxidative pentose phosphate pathway. A transketolase preparation from rehydrated leaves was able to synthesize the unusual C8 carbon sugar octulose when glucose-6-phosphate and hydroxy-pyruvate were used as acceptor and donor molecules in in vitro assays. This suggests that a transketolase catalyzed reaction is likely to be involved in the octulose biosynthesis in C. plantagineum.

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

    PubMed

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela

    2013-09-01

    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. 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.

  5. In vivo subcellular localization of Mal de Río Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions.

    PubMed

    Maroniche, Guillermo A; Mongelli, Vanesa C; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar; del Vas, Mariana

    2012-09-01

    The in vivo subcellular localization of Mal de Río 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 P9-1 viroplasm-like structures and co-localized with P7-2, P10 and α-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.

  6. In vivo subcellular localization of Mal de Rio Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions

    SciTech Connect

    Maroniche, Guillermo A.; Mongelli, Vanesa C.; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar

    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 P9-1 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.

  7. In Vivo Recognition of Ovalbumin Expressed by Transgenic Leishmania Is Determined by Its Subcellular Localization1

    PubMed Central

    Prickett, Sara; Gray, Peter M.; Colpitts, Sara L.; Scott, Phillip; Kaye, Paul M.; Smith, Deborah F.

    2009-01-01

    The importance of the site of Ag localization within microbial pathogens for the effective generation of CD8+ T cells has been studied extensively, generally supporting the view that Ag secretion within infected target cells is required for optimal MHC class I-restricted Ag presentation. In contrast, relatively little is known about the importance of pathogen Ag localization for the activation of MHC class II-restricted CD4+ T cells, despite their clear importance for host protection. We have used the N-terminal targeting sequence of Leishmania major hydrophilic acylated surface protein B to generate stable transgenic lines expressing physiologically relevant levels of full-length OVA on the surface of metacyclic promastigotes and amastigotes. In addition, we have mutated the hydrophilic acylated surface protein B N-terminal acylation sequence to generate control transgenic lines in which OVA expression is restricted to the parasite cytosol. In vitro, splenic dendritic cells are able to present membrane-localized, but not cytosolic, OVA to OVA-specific DO.11 T cells. Strikingly and unexpectedly, surface localization of OVA is also a strict requirement for recognition by OVA-specific T cells (DO.11 and OT-II) and for the development of OVA-specific Ab responses in vivo. However, recognition of cytosolic OVA could be observed with increasing doses of infection. These data suggest that, even under in vivo conditions, where varied pathways of Ag processing are likely to operate, the site of Leishmania Ag localization is an important determinant of immunogenicity and hence an important factor when considering the likely candidacy of vaccine Ags for inducing CD4+ T cell-dependent immunity. PMID:16585577

  8. The Subcellular Localization of an Aquaporin-2 Tetramer Depends on the Stoichiometry of Phosphorylated and Nonphosphorylated Monomers

    PubMed Central

    Kamsteeg, E.J.; Heijnen, I.; van Os, C.H.; Deen, P.M.T.

    2000-01-01

    In renal principal cells, vasopressin regulates the shuttling of the aquaporin (AQP)2 water channel between intracellular vesicles and the apical plasma membrane. Vasopressin-induced phosphorylation of AQP2 at serine 256 (S256) by protein kinase A (PKA) is essential for its localization in the membrane. However, phosphorylated AQP2 (p-AQP2) has also been detected in intracellular vesicles of noninduced principal cells. As AQP2 is expressed as homotetramers, we hypothesized that the number of p-AQP2 monomers in a tetramer might be critical for the its steady state distribution. Expressed in oocytes, AQP2-S256D and AQP2-S256A mimicked p-AQP2 and non–p-AQP2, respectively, as routing and function of AQP2-S256D and wild-type AQP2 (wt-AQP2) were identical, whereas AQP2-S256A was retained intracellularly. In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers. Coinjection of different ratios of AQP2-S256A and AQP2-S256D cRNAs revealed that minimally three AQP2-S256D monomers in an AQP2 tetramer were essential for its plasma membrane localization. Therefore, our results suggest that in principal cells, minimally three monomers per AQP2 tetramer have to be phosphorylated for its steady state localization in the apical membrane. As other multisubunit channels are also regulated by phosphorylation, it is anticipated that the stoichiometry of their phosphorylated and nonphosphorylated subunits may fine-tune the activity or subcellular localization of these complexes. PMID:11076974

  9. Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

    PubMed

    Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

    2013-09-01

    Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH.

  10. Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus

    SciTech Connect

    Xiong Ruyi; Wu Jianxiang; Zhou Yijun; Zhou Xueping

    2009-04-25

    Rice stripe virus (RSV) is a single-stranded (ss) RNA virus belonging to the genus Tenuivirus. RSV is present in many East Asian countries and causes severe diseases in rice fields, especially in China. In this study, we analyzed six proteins encoded by the virus for their abilities to suppress RNA silencing in plant using a green fluorescent protein (GFP)-based transient expression assay. Our results indicate that NS3 encoded by RSV RNA3, but not other five RSV encoded proteins, can strongly suppress local GFP silencing in agroinfiltrated Nicotiana benthamiana leaves. NS3 can reverse the GFP silencing, it can also prevent long distance spread of silencing signals which have been reported to be necessary for inducing systemic silencing in host plants. The NS3 protein can significantly reduce the levels of small interfering RNAs (siRNAs) in silencing cells, and was found to bind 21-nucleotide ss-siRNA, siRNA duplex and long ssRNA but not long double-stranded (ds)-RNA. Both N and C terminal of the NS3 protein are critical for silencing suppression, and mutation of the putative nuclear localization signal decreases its local silencing suppression efficiency and blocks its systemic silencing suppression. The NS3-GFP fusion protein and NS3 were shown to accumulate predominantly in nuclei of onion, tobacco and rice cells through transient expression assay or immunocytochemistry and electron microscopy. In addition, transgenic rice and tobacco plants expressing the NS3 did not show any apparent alteration in plant growth and morphology, although NS3 was proven to be a pathogenicity determinant in the PVX heterogenous system. Taken together, our results demonstrate that RSV NS3 is a suppressor of RNA silencing in planta, possibly through sequestering siRNA molecules generated in cells that are undergoing gene silencing.

  11. Heat shock modulates the subcellular localization, stability, and activity of HIPK2.

    PubMed

    Upadhyay, Mamta; Bhadauriya, Pratibha; Ganesh, Subramaniam

    2016-04-15

    The homeodomain-interacting protein kinase-2 (HIPK2) is a highly conserved serine/threonine kinase and is involved in transcriptional regulation. HIPK2 is a highly unstable protein, and is kept at a low level under normal physiological conditions. However, exposure of cells to physiological stress - such as hypoxia, oxidative stress, or UV damage - is known to stabilize HIPK2, leading to the HIPK2-dependent activation of p53 and the cell death pathway. Therefore HIPK2 is also known as a stress kinase and as a stress-activated pro-apoptotic factor. We demonstrate here that exposure of cells to heat shock results in the stabilization of HIPK2 and the stabilization is mediated via K63-linked ubiquitination. Intriguingly, a sub-lethal heat shock (42 °C, 1 h) results in the cytoplasmic localization of HIPK2, while a lethal heat shock (45 °C, 1 h) results in its nuclear localization. Cells exposed to the lethal heat shock showed significantly higher levels of the p53 activity than those exposed to the sub-lethal thermal stress, suggesting that both the level and the nuclear localization are essential for the pro-apoptotic activity of HIPK2 and that the lethal heat shock could retain the HIPK2 in the nucleus to promote the cell death. Taken together our study underscores the importance of HIPK2 in stress mediated cell death, and that the HIPK2 is a generic stress kinase that gets activated by diverse set of physiological stressors.

  12. Construction of Global Acyl Lipid Metabolic Map by Comparative Genomics and Subcellular Localization Analysis in the Red Alga Cyanidioschyzon merolae

    PubMed Central

    Mori, Natsumi; Moriyama, Takashi; Toyoshima, Masakazu; Sato, Naoki

    2016-01-01

    Pathways of lipid metabolism have been established in land plants, such as Arabidopsis thaliana, but the information on exact pathways is still under study in microalgae. In contrast with Chlamydomonas reinhardtii, which is currently studied extensively, the pathway information in red algae is still in the state in which enzymes and pathways are estimated by analogy with the knowledge in plants. Here we attempt to construct the entire acyl lipid metabolic pathways in a model red alga, Cyanidioschyzon merolae, as an initial basis for future genetic and biochemical studies, by exploiting comparative genomics and localization analysis. First, the data of whole genome clustering by Gclust were used to identify 121 acyl lipid-related enzymes. Then, the localization of 113 of these enzymes was analyzed by GFP-based techniques. We found that most of the predictions on the subcellular localization by existing tools gave erroneous results, probably because these tools had been tuned for plants or green algae. The experimental data in the present study as well as the data reported before in our laboratory will constitute a good training set for tuning these tools. The lipid metabolic map thus constructed show that the lipid metabolic pathways in the red alga are essentially similar to those in A. thaliana, except that the number of enzymes catalyzing individual reactions is quite limited. The absence of fatty acid desaturation to produce oleic and linoleic acids within the plastid, however, highlights the central importance of desaturation and acyl editing in the endoplasmic reticulum, for the synthesis of plastid lipids as well as other cellular lipids. Additionally, some notable characteristics of lipid metabolism in C. merolae were found. For example, phosphatidylcholine is synthesized by the methylation of phosphatidylethanolamine as in yeasts. It is possible that a single 3-ketoacyl-acyl carrier protein synthase is involved in the condensation reactions of fatty acid

  13. mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging

    PubMed Central

    2013-01-01

    Background The mammalian target of rapamycin (mTOR) signalling pathway has a key role in cellular regulation and several diseases. While it is thought that Rheb GTPase regulates mTOR, acting immediately upstream, while raptor is immediately downstream of mTOR, direct interactions have yet to be verified in living cells, furthermore the localisation of Rheb has been reported to have only a cytoplasmic cellular localization. Results In this study a cytoplasmic as well as a significant sub-cellular nuclear mTOR localization was shown , utilizing green and red fluorescent protein (GFP and DsRed) fusion and highly sensitive single photon counting fluorescence lifetime imaging microscopy (FLIM) of live cells. The interaction of the mTORC1 components Rheb, mTOR and raptor, tagged with EGFP/DsRed was determined using fluorescence energy transfer-FLIM. The excited-state lifetime of EGFP-mTOR of ~2400 ps was reduced by energy transfer to ~2200 ps in the cytoplasm and to 2000 ps in the nucleus when co-expressed with DsRed-Rheb, similar results being obtained for co-expressed EGFP-mTOR and DsRed-raptor. The localization and distribution of mTOR was modified by amino acid withdrawal and re-addition but not by rapamycin. Conclusions The results illustrate the power of GFP-technology combined with FRET-FLIM imaging in the study of the interaction of signalling components in living cells, here providing evidence for a direct physical interaction between mTOR and Rheb and between mTOR and raptor in living cells for the first time. PMID:23311891

  14. Subcellular localization of ferredoxin-NADP(+) oxidoreductase in phycobilisome retaining oxygenic photosysnthetic organisms.

    PubMed

    Morsy, Fatthy Mohamed; Nakajima, Masato; Yoshida, Takayuki; Fujiwara, Tatsuki; Sakamoto, Toshio; Wada, Keishiro

    2008-01-01

    Ferredoxin-NADP(+) oxidoreductase (FNR) catalyzing the terminal step of the linear photosynthetic electron transport was purified from the cyanobacterium Spirulina platensis and the red alga Cyanidium caldarium. FNR of Spirulina consisted of three domains (CpcD-like domain, FAD-binding domain, and NADP(+)-binding domain) with a molecular mass of 46 kDa and was localized in either phycobilisomes or thylakoid membranes. The membrane-bound FNR with 46 kDa was solublized by NaCl and the solublized FNR had an apparent molecular mass of 90 kDa. FNR of Cyanidium consisted of two domains (FAD-binding domain and NADP(+)-binding domain) with a molecular mass of 33 kDa. In Cyanidium, FNR was found on thylakoid membranes, but there was no FNR on phycobilisomes. The membrane-bound FNR of Cyanidium was not solublized by NaCl, suggesting the enzyme is tightly bound in the membrane. Although both cyanobacteria and red algae are photoautotrophic organisms bearing phycobilisomes as light harvesting complexes, FNR localization and membrane-binding characteristics were different. These results suggest that FNR binding to phycobilisomes is not characteristic for all phycobilisome retaining oxygenic photosynthetic organisms, and that the rhodoplast of red algae had possibly originated from a cyanobacterium ancestor, whose FNR lacked the CpcD-like domain.

  15. Factors influencing subcellular localization of the human papillomavirus L2 minor structural protein

    SciTech Connect

    Kieback, Elisa; Mueller, Martin . E-mail: Martin.Mueller@dkfz.de

    2006-02-05

    Two structural proteins form the capsids of papillomaviruses. The major structural protein L1 is the structural determinant of the capsids and is present in 360 copies arranged in 72 pentamers. The minor structural protein L2 is estimated to be present in twelve copies per capsid. Possible roles for L2 in interaction with cell surface receptors and in virion uptake have been suggested. As previously reported, L2 localizes in subnuclear domains identified as nuclear domain 10 (ND10). As it was demonstrated that L2 is able to recruit viral and cellular proteins to ND10, a possible role for L2 as a mediator in viral assembly has been proposed. In this study, we determined factors influencing the localization of L2 at ND10. Under conditions of moderate L2 expression level and in the absence of heterologous viral components, we observed that, in contrast to previous reports, L2 is mainly distributed homogeneously throughout the nucleus. L2, however, is recruited to ND10 at a higher expression level or in the presence of viral components derived from vaccinia virus or from Semliki Forest virus. We observed that translocation of L2 to ND10 is not a concentration-dependent accumulation but rather seems to be triggered by yet unidentified cellular factors. In contrast to HPV 11 and 16 L2, the HPV 18 L2 protein seems to require L1 for efficient nuclear accumulation.

  16. Molecular cloning, functional characterization, and subcellular localization of soybean nodule dihydrolipoamide reductase.

    PubMed

    Moran, Jose F; Sun, Zhaohui; Sarath, Gautam; Arredondo-Peter, Raúl; James, Euan K; Becana, Manuel; Klucas, Robert V

    2002-01-01

    Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH.

  17. FOXP3 Subcellular Localization Predicts Recurrence in Oral Squamous Cell Carcinoma

    PubMed Central

    Weed, Donald T.; Walker, Gail; De La Fuente, Adriana C.; Nazarian, Ronen; Vella, Jennifer L.; Gomez-Fernandez, Carmen R.; Serafini, Paolo

    2013-01-01

    Forkhead box protein P3 (FOXP3) expression in tumor infiltrating CD4+T cells is generally associated with an intrinsic capacity to suppress tumor immunity. Based on this notion, different studies have evaluated the prognostic value of this maker in cancer but contradictory results have been found. Indeed, even within the same cancer population, the presence of CD4+FOXP3+T cells has been associated,with either a poor or a good prognosis, or no correlation has beenfound. Here, we demonstrate,in patients with oral squamous cell carcinoma (OSCC), that what really represents a prognostic parameter is not the overall expression of FOXP3 but its intracellular localization.While overallFOXP3 expression in tumor infiltrating CD4+T cells does not correlate with tumor recurrence, its intracellular localization within the CD4 cells does: nuclear FOXP3 (nFOXP3) is associated with tumor recurrence within 3 years, while cytoplasmicFOXP3 (cFOXP3) is associated with a lower likelihood of recurrence. Thus, we propose elevated levels of the cFOXP3/nFOXP3 ratio within tumor infiltrating CD4+ T cells as a predictor of OSCC recurrence. PMID:23977174

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

    SciTech Connect

    Renard, C.; Vanderhaeghe, H.J.; Claes, P.J.; Zenebergh, A.; Tulkens, P.M.

    1987-03-01

    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 partly 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.

  19. Endogenous spar tin, mutated in hereditary spastic paraplegia, has a complex subcellular localization suggesting diverse roles in neurons

    SciTech Connect

    Robay, Dimitri; Patel, Heema; Simpson, Michael A.; Brown, Nigel A.; Crosby, Andrew H. . E-mail: acrosby@sgul.ac.uk

    2006-09-10

    Mutation of spartin (SPG20) underlies a complicated form of hereditary spastic paraplegia, a disorder principally defined by the degeneration of upper motor neurons. Using a polyclonal antibody against spartin to gain insight into the function of the endogenous molecule, we show that the endogenous molecule is present in two main isoforms of 85 kDa and 100 kDa, and 75 kDa and 85 kDa in human and murine, respectively, with restricted subcellular localization. Immunohistochemical studies on human and mouse embryo sections and in vitro cell studies indicate that spartin is likely to possess both nuclear and cytoplasmic functions. The nuclear expression of spartin closely mirrors that of the snRNP (small nuclear ribonucleoprotein) marker {alpha}-Sm, a component of the spliceosome. Spartin is also enriched at the centrosome within mitotic structures. Notably we show that spartin protein undergoes dynamic positional changes in differentiating human SH-SY5Y cells. In undifferentiated non-neuronal cells, spartin displays a nuclear and diffuse cytosolic profile, whereas spartin transiently accumulates in the trans-Golgi network and subsequently decorates discrete puncta along neurites in terminally differentiated neuroblastic cells. Investigation of these spartin-positive vesicles reveals that a large proportion colocalizes with the synaptic vesicle marker synaptotagmin. Spartin is also enriched in synaptic-like structures and in synaptic vesicle-enriched fraction.

  20. The leukemogenic CALM/AF10 fusion protein alters the subcellular localization of the lymphoid regulator Ikaros.

    PubMed

    Greif, P A; Tizazu, B; Krause, A; Kremmer, E; Bohlander, S K

    2008-05-01

    The t(10;11)(p13;q14) translocation leads to the fusion of the CALM and AF10 genes. This translocation can be found as the sole cytogenetic abnormality in acute lymphoblastic leukemia, acute myeloid leukemia and in malignant lymphomas. The expression of CALM/AF10 in primary murine bone marrow cells results in the development of an aggressive leukemia in a murine bone marrow transplantation model. Using a yeast two-hybrid screen, we identified the lymphoid regulator Ikaros as an AF10 interacting protein. Interestingly, Ikaros is required for normal development of lymphocytes, and aberrant expression of Ikaros has been found in leukemia. In a murine model, the expression of a dominant negative isoform of Ikaros causes leukemias and lymphomas. The Ikaros interaction domain of AF10 was mapped to the leucine zipper domain of AF10, which is required for malignant transformation both by the CALM/AF10 and the MLL/AF10 fusion proteins. The interaction between AF10 and Ikaros was confirmed by GST pull down and co-immunoprecipitation. Coexpression of CALM/AF10 but not of AF10 alters the subcellular localization of Ikaros in murine fibroblasts. The transcriptional repressor activity of Ikaros is reduced by AF10. These results suggest that CALM/AF10 might interfere with normal Ikaros function, and thereby block lymphoid differentiation in CALM/AF10 positive leukemias.

  1. 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

  2. Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization

    SciTech Connect

    Lawrence, J.B.; Marselle, L.M.; Byron, K.S.; Johnson, C.V.; Sullivan, J.L.; Singer, R.H. )

    1990-07-01

    Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell.

  3. Molecular cloning, subcellular localization and characterization of two adenylate kinases from cassava, Manihot esculenta Crantz cv. KU50.

    PubMed

    Boonrueng, Channarong; Tangpranomkorn, Surachat; Yazhisai, Uthaman; Sirikantaramas, Supaart

    2016-10-01

    Adenylate kinase (ADK) is a phosphotransferase that plays an important role in cellular energy homeostasis. Many isozymes located in different subcellular compartments have been reported. In this study, we focus on the characterization of cassava (Manihot esculenta) ADKs. We found 15 ADKs that are publicly available in the African cassava genome database. We cloned two ADKs, namely MeADK1 and MeADK2, which are phylogenetically grouped together with the plastidial ADK in potato. Both MeADK1 and MeADK2 showed 66% identity in the amino acid sequences with plastidial ADK in potato. However, we demonstrated that they are localized to mitochondria using GFP fusions of MeADK1 and MeADK2. The Escherichia coli-produced recombinant MeADK1 and MeADK2 preferred forward reactions that produce ATP. They exhibited similar specific activities. The semi-quantitative RT-PCR analysis showed that MeADK1 and MeADK2 in 2-month-old leaves have similar expression patterns under a diurnal light-dark cycle. However, MeADK2 transcripts were expressed at much higher levels than MeADK1 in 5-month-old leaves and roots. Thus, we conclude that MeADK2 might play a vital role in energy homeostasis in cassava mitochondria.

  4. Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development

    PubMed Central

    Dupé, Aurélien; Dumas, Carole; Papadopoulou, Barbara

    2015-01-01

    Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in

  5. Cloning, subcellular localization and expression of CHL1, a subunit of magnesium-chelatase in soybean.

    PubMed

    Nakayama, M; Masuda, T; Sato, N; Yamagata, H; Bowler, C; Ohta, H; Shioi, Y; Takamiya, K

    1995-10-04

    Mg-insertion is the first committed step in chlorophyll synthesis and is catalyzed by Mg-chelatase. In photosynthetic bacteria, bchI gene product was suggested to be a subunit of Mg-chelatase. We isolated a bchI homolog from a soybean cDNA library and designated it as chlI. CHLI consisted of 421 amino acid residues and the sequence exhibited a high similarity to other BchI homologs. CHLI contained an ATP-binding motif found in other BchI homologs. CHLI was localized in the soluble fraction in soybean chloroplasts, suggesting that it was a stromal subunit of Mg-chelatase. chlI mRNA in cell culture (SB-P) of soybean was reversibly induced by light.

  6. Tetramine dichloro-palladium subcellular localization in the kidney: electron microprobe study.

    PubMed

    Berry, J P

    1987-01-01

    Palladium salt has been used for some time in experimental therapy protocols; with this in mind, we carried out a study of the effect of tetramine dichloro-palladium (soluble salt) upon kidney cells. Using an electron microprobe, we were able to detect the presence of palladium associated with sulfur and iron in the lysosomes of the proximal tubule cells. Our results were compared with those obtained using Cis-diaminedichloro-platinum (Cis-DDP), an anti-cancer drug used in the treatment of diverse tumors. The mechanism of intralysosomal concentration of palladium as a non soluble salt associated with sulfur appeared to be related to local sulfatase activity. Finally, iron concentration appeared to be related to the inhibition process of erythropoiesis.

  7. Anks3 alters the sub-cellular localization of the Nek7 kinase

    SciTech Connect

    Ramachandran, Haribaskar; Engel, Christina; Müller, Barbara; Dengjel, Jörn; Walz, Gerd; Yakulov, Toma A.

    2015-08-28

    Nephronophthisis (NPH) is an autosomal recessive cystic kidney disease, and a frequent cause of end-stage renal failure in children. To date, 17 NPH-associated gene products (NPHPs) have been identified. Most NPHPs participate in large multi-protein complexes that localize to the cilium and/or basal body; however, the precise composition of these complexes and their biological function remain largely unknown. We recently observed that the ankyrin repeat protein Anks3 interacts with the NPH family member Anks6. Both Anks3 and Anks6 form complexes with multiple other NPHPs, suggesting that both proteins function in similar or overlapping signaling pathways. Here, we show that Anks3, but not Anks6 interacted with the NIMA-related kinase Nek7, and was heavily modified in the presence of Nek7, resulting in an approximately 20 kD increase in molecular weight. Although mass spectrometry revealed increased serine and threonine phosphorylation of Anks3 primarily within the N-terminal ankyrin repeats also required for Nek7 interaction, the molecular weight increase occurred even in the presence of a kinase-dead Nek7 mutant, indicating that this modification was not caused by Nek7-dependent Anks3 phosphorylation. Furthermore, the Anks3 modification was specific for Nek7, and did not occur in the presence of Nek8. Importantly, Anks3 retained Nek7 in the cytoplasm, suggesting that, Nek7 triggers the modification of Anks3, which in turn prevents the nuclear localization of Nek7. - Highlights: • Anks3 interacted with Nek7 kinase, and was heavily modified in the presence of Nek7. • Anks3 N-terminal ankyrin repeats, but not SAM domain required for Nek7 interaction. • Nek7 increased Ser/Thr phosphorylation of Anks3 primarily within ankyrin domain. • Interaction with Anks3 led to cytoplasmic retention and nuclear exclusion of Nek7.

  8. Dihydrocercosporin singlet oxygen production and subcellular localization: a possible defense against cercosporin phototoxicity in Cercospora.

    PubMed

    Daub, M E; Li, M; Bilski, P; Chignell, C F

    2000-02-01

    Fungi in the genus Cercospora produce cercosporin, a potent singlet oxygen (1O2)-generating photosensitizer that plays a critical role in the ability of these fungi to parasitize plants. Although plants, mice, bacteria and many fungi are sensitive to cercosporin, Cercospora species are resistant to its toxicity. The cellular resistance of these fungi to cercosporin has been correlated with fungal cell surface reducing ability and the ability to maintain cercosporin in a chemically reduced state. As a model for reduced cercosporin we employed a reduced, acetylated derivative (hexaacetyl-dihydrocercosporin, HAC) that we tested for 1O2 production in a range of solvents. We found that as a 1O2 photosensitizer, HAC was only moderately effective in organic solvents (phi SO = 0.14-0.18) and very poor in water (phi SO = 0.02-0.04). By contrast, the 1O2 quantum yield of cercosporin itself was unaffected by solvent (phi SO = 0.84-0.97). To investigate the localization of reduced cercosporin in fungal cells, we developed a fluorescence assay using laser scanning confocal microscopy. This assay showed a uniform green fluorescence, indicative of reduced cercosporin, in the cytoplasm of hyphal cells treated with cercosporin. We hypothesize that the main protection mechanism against cercosporin phototoxicity in the fungus consists of transformation of cercosporin to a reduced state and localization of this reduced form in the aqueous compartment of the cell, thus decreasing intracellular 1O2 production to levels that can be tolerated by the fungus. In addition, we have, for the first time, directly detected 1O2 phosphorescence from fungal culture, either stained with the photosensitizer rose bengal or actively synthesizing cercosporin, demonstrating 1O2 production in vivo and from cercosporin in culture.

  9. Differential subcellular localization of cholesterol, gangliosides, and glycosaminoglycans in murine models of mucopolysaccharide storage disorders.

    PubMed

    McGlynn, Robert; Dobrenis, Kostantin; Walkley, Steven U

    2004-12-20

    The mucopolysaccharidoses (MPSs) are a complex family of lysosomal storage disorders characterized by failure to degrade heparan sulfate (HS) and/or other types of glycosaminoglycans (GAGs) secondary to the absence of specific lysosomal enzymes. An accompanying storage of glycosphingolipids (GSLs), most notably GM2 and GM3 gangliosides, has also been documented to occur in many types of MPS disease and is believed to be caused by secondary inhibition of GSL-degradative enzymes by intracellular GAG accumulation. We have documented the presence of secondary ganglioside accumulation in mouse models of several MPS disorders (types I, IIIA, IIIB, and VII) and report that this storage is accompanied by sequestration of free cholesterol in a manner similar to that observed in primary gangliosidoses. Using confocal microscopy, we evaluated the cellular distribution of cholesterol, GM2 and GM3 gangliosides, and HS in brains of mice with MPS IIIA disease. Unexpectedly, we found that although both gangliosides often accumulated in the same neurons, they were consistently located in separate populations of cytoplasmic vesicles. Additionally, GM3 ganglioside only partially co-localized with the primary storage material (HS), and cholesterol likewise only partially co-localized with the GM2 and GM3 gangliosides. These findings raise significant questions about the mechanism(s) responsible for secondary accumulation of storage materials in MPS disease. Furthermore, given that GSLs and cholesterol are constituents of membrane rafts believed critical in signal transduction events in neurons, their co-sequestration in individual neurons suggests the presence of defects in the composition, trafficking, and/or recycling of raft components and thus possible new mechanisms to explain neuronal dysfunction in MPS disorders.

  10. Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa.

    PubMed

    Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

    2010-06-01

    Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum.

  11. Effect of cadmium on the physiological parameters and the subcellular cadmium localization in the potato (Solanum tuberosum L.).

    PubMed

    Xu, Dongyu; Chen, Zhifan; Sun, Ke; Yan, Dong; Kang, Mingjie; Zhao, Ye

    2013-11-01

    The pollution of agricultural soils with cadmium (Cd) has become a serious problem worldwide. The potato (Solanum tuberosum L.) was used to investigate how different concentrations of Cd (1, 5, and 25mgkg(-1)) affected the physiological parameters and the subcellular distribution of Cd in the potato. The analyses were conducted using scanning electron microscopy coupled with energy dispersive X-ray (SEM-EDX). The results suggest that the leaf is the organ with the highest accumulation of Cd. The malondialdehyde (MDA) content increased and the chlorophyll content decreased in response to high level of Cd. The SEM-EDX microanalysis revealed that Cd was primarily deposited in the spongy and palisade tissues of the leaf. Furthermore, Cd was also detected in the cortex and the adjacent phloem and was observed inside the intercellular space, the interior surface of the plasma membrane, and on the surface of the elliptical starch granules in the tubers of the potato. Although low concentrations of Cd migrated from the root to the tuber, the accumulation of Cd in the tuber exceeded the standard for food security. Therefore, the planting of potato plants in farmland containing Cd should be seriously evaluated because Cd-containing potatoes might present high health risk to humans.

  12. Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

    SciTech Connect

    Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank

    2013-08-30

    Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

  13. Fumarate hydratase isoforms of Leishmania major: subcellular localization, structural and kinetic properties.

    PubMed

    Feliciano, Patrícia R; Gupta, Shreedhara; Dyszy, Fabio; Dias-Baruffi, Marcelo; Costa-Filho, Antonio J; Michels, Paul A M; Nonato, M Cristina

    2012-01-01

    Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs.

  14. Identification and subcellular localization analysis of two rubber elongation factor isoforms on Hevea brasiliensis rubber particles.

    PubMed

    Dai, Longjun; Nie, Zhiyi; Kang, Guijuan; Li, Yu; Zeng, Rizhong

    2017-02-01

    Rubber elongation factor (REF) is the most abundant protein found on the rubber particles or latex from Hevea brasiliensis (the Para rubber tree) and is considered to play important roles in natural rubber (cis-polyisoprene) biosynthesis. 16 BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE separations and mass spectrometric identification had revealed that two REF isoforms shared similar amino acid sequences and common C-terminal sequences. In this study, the gene sequences encoding these two REF isoforms (one is 23.6 kDa in size with 222 amino acid residues and the other is 27.3 kDa in size with 258 amino acid residues) were obtained. Their proteins were relatively enriched by sequential extraction of the rubber particle proteins and separated by 16 BAC/SDS-PAGE. The localization of these isoforms on the surfaces of rubber particles was further verified by western blotting and immunogold electron microscopy, which demonstrated that these two REF isoforms are mainly located on the surfaces of larger rubber particles and that they bind more tightly to rubber particles than the most abundant REF and SRPP (small rubber particle protein).

  15. Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization.

    PubMed

    Miele, Claudia; Paturzo, Flora; Teperino, Raffaele; Sakane, Fumio; Fiory, Francesca; Oriente, Francesco; Ungaro, Paola; Valentino, Rossella; Beguinot, Francesco; Formisano, Pietro

    2007-11-02

    Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.

  16. Tyrosine phosphorylation within the SH3 domain regulates CAS subcellular localization, cell migration, and invasiveness.

    PubMed

    Janoštiak, Radoslav; Tolde, Ondřej; Brůhová, Zuzana; Novotný, Marian; Hanks, Steven K; Rösel, Daniel; Brábek, Jan

    2011-11-01

    Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes and plays an important role in invasiveness of Src-transformed cells. A novel phosphorylation site on CAS, Tyr-12 (Y12) within the ligand-binding hydrophobic pocket of the CAS SH3 domain, was identified and found to be enriched in Src-transformed cells and invasive human carcinoma cells. To study the biological significance of CAS Y12 phosphorylation, phosphomimicking Y12E and nonphosphorylatable Y12F mutants of CAS were studied. The phosphomimicking mutation decreased interaction of the CAS SH3 domain with focal adhesion kinase (FAK) and PTP-PEST and reduced tyrosine phosphorylation of FAK. Live-cell imaging showed that green fluorescent protein-tagged CAS Y12E mutant is, in contrast to wild-type or Y12F CAS, excluded from focal adhesions but retains its localization to podosome-type adhesions. Expression of CAS-Y12F in cas-/- mouse embryonic fibroblasts resulted in hyperphosphorylation of the CAS substrate domain, and this was associated with slower turnover of focal adhesions and decreased cell migration. Moreover, expression of CAS Y12F in Src-transformed cells greatly decreased invasiveness when compared to wild-type CAS expression. These findings reveal an important role of CAS Y12 phosphorylation in the regulation of focal adhesion assembly, cell migration, and invasiveness of Src-transformed cells.

  17. Human selenophosphate synthetase 1 has five splice variants with unique interactions, subcellular localizations and expression patterns

    SciTech Connect

    Kim, Jin Young; Lee, Kwang Hee; Shim, Myoung Sup; Shin, Hyein; Xu, Xue-Ming; Carlson, Bradley A.; Hatfield, Dolph L.; Lee, Byeong Jae

    2010-06-18

    Selenophosphate synthetase 1 (SPS1) is an essential cellular gene in higher eukaryotes. Five alternative splice variants of human SPS1 (major type, {Delta}E2, {Delta}E8, +E9, +E9a) were identified wherein +E9 and +E9a make the same protein. The major type was localized in both the nuclear and plasma membranes, and the others in the cytoplasm. All variants form homodimers, and in addition, the major type forms a heterodimer with {Delta}E2, and {Delta}E8 with +E9. The level of expression of each splice variant was different in various cell lines. The expression of each alternative splice variant was regulated during the cell cycle. The levels of the major type and {Delta}E8 were gradually increased until G2/M phase and then gradually decreased. {Delta}E2 expression peaked at mid-S phase and then gradually decreased. However, +E9/+E9a expression decreased gradually after cell cycle arrest. The possible involvement of SPS1 splice variants in cell cycle regulation is discussed.

  18. USP8 promotes smoothened signaling by preventing its ubiquitination and changing its subcellular localization.

    PubMed

    Xia, Ruohan; Jia, Hongge; Fan, Junkai; Liu, Yajuan; Jia, Jianhang

    2012-01-01

    The seven transmembrane protein Smoothened (Smo) is a critical component of the Hedgehog (Hh) signaling pathway and is regulated by phosphorylation, dimerization, and cell-surface accumulation upon Hh stimulation. However, it is not clear how Hh regulates Smo accumulation on the cell surface or how Hh regulates the intracellular trafficking of Smo. In addition, little is known about whether ubiquitination is involved in Smo regulation. In this study, we demonstrate that Smo is multi-monoubiquitinated and that Smo ubiquitination is inhibited by Hh and by phosphorylation. Using an in vivo RNAi screen, we identified ubiquitin-specific protease 8 (USP8) as a deubiquitinase that down-regulates Smo ubiquitination. Inactivation of USP8 increases Smo ubiquitination and attenuates Hh-induced Smo accumulation, leading to decreased Hh signaling activity. Moreover, overexpression of USP8 prevents Smo ubiquitination and elevates Smo accumulation, leading to increased Hh signaling activity. Mechanistically, we show that Hh promotes the interaction of USP8 with Smo aa625-753, which covers the three PKA and CK1 phosphorylation clusters. Finally, USP8 promotes the accumulation of Smo at the cell surface and prevents localization to the early endosomes, presumably by deubiquitinating Smo. Our studies identify USP8 as a positive regulator in Hh signaling by down-regulating Smo ubiquitination and thereby mediating Smo intracellular trafficking.

  19. Subcellular Localization and Ser-137 Phosphorylation Regulate Tumor-suppressive Activity of Profilin-1*

    PubMed Central

    Diamond, Marc I.; Cai, Shirong; Boudreau, Aaron; Carey, Clifton J.; Lyle, Nicholas; Pappu, Rohit V.; Swamidass, S. Joshua; Bissell, Mina; Piwnica-Worms, Helen; Shao, Jieya

    2015-01-01

    The actin-binding protein profilin-1 (Pfn1) inhibits tumor growth and yet is also required for cell proliferation and survival, an apparent paradox. We previously identified Ser-137 of Pfn1 as a phosphorylation site within the poly-l-proline (PLP) binding pocket. Here we confirm that Ser-137 phosphorylation disrupts Pfn1 binding to its PLP-containing ligands with little effect on actin binding. We find in mouse xenografts of breast cancer cells that mimicking Ser-137 phosphorylation abolishes cell cycle arrest and apoptotic sensitization by Pfn1 and confers a growth advantage to tumors. This indicates a previously unrecognized role of PLP binding in Pfn1 antitumor effects. Spatial restriction of Pfn1 to the nucleus or cytoplasm indicates that inhibition of tumor cell growth by Pfn1 requires its nuclear localization, and this activity is abolished by a phosphomimetic mutation on Ser-137. In contrast, cytoplasmic Pfn1 lacks inhibitory effects on tumor cell growth but rescues morphological and proliferative defects of PFN1 null mouse chondrocytes. These results help reconcile seemingly opposed cellular effects of Pfn1, provide new insights into the antitumor mechanism of Pfn1, and implicate Ser-137 phosphorylation as a potential therapeutic target for breast cancer. PMID:25681442

  20. Characterization and sub-cellular localization of GalNAc-binding proteins isolated from human hepatic stellate cells.

    PubMed

    Zhong, Yaogang; Zhang, Jing; Yu, Hanjie; Zhang, Jiaxu; Sun, Xiu-Xuan; Chen, Wentian; Bian, Huijie; Li, Zheng

    2015-12-25

    Although the expression levels of total GalNAc-binding proteins (GNBPs) were up-regulated significantly in human hepatic stellate cells (HSCs) activated with transforming growth factor-β1(TGF-β1), yet little is known about the precise types, distribution and sub-cellular localization of the GNBPs in HSCs. Here, 264 GNBPs from the activated HSCs and 257 GNBPs from the quiescent HSCs were identified and annotated. A total of 46 GNBPs were estimated to be significantly up-regulated and 40 GNBPs were estimated to be significantly down-regulated in the activated HSCs. For example, the GNBPs (i.e. BTF3, COX17, and ATP5A1) responsible for the regulation of protein binding were up-regulated, and those (i.e. FAM114A1, ENO3, and TKT) responsible for the regulation of protein binding were down-regulated in the activated HSCs. The motifs of the isolated GNBPs showed that Proline residue had the maximum preference in consensus sequences. The western blotting showed the expression levels of COX17, and PRMT1 were significantly up-regulated, while, the expression level of CLIC1(B5) was down-regulated in the activated HSCs and liver cirrhosis tissues. Moreover, the GNBPs were sub-localized in the Golgi apparatus of HSCs. In conclusion, the precision alteration of the GNBPs referred to pathological changes in liver fibrosis/cirrhosis may provide useful information to find new molecular mechanism of HSC activation and discover the biomarkers for diagnosis of liver fibrosis/cirrhosis as well as development of new anti-fibrotic strategies.

  1. Differential Subcellular Localization Renders HAI-2 a Matriptase Inhibitor in Breast Cancer Cells but Not in Mammary Epithelial Cells

    PubMed Central

    Chang, Hsiang-Hua D.; Xu, Yuan; Lai, Hongyu; Yang, Xiaoyu; Tseng, Chun-Che; Lai, Ying-Jung J.; Pan, Yu; Zhou, Emily; Johnson, Michael D.; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells. PMID:25786220

  2. Differential subcellular localization renders HAI-2 a matriptase inhibitor in breast cancer cells but not in mammary epithelial cells.

    PubMed

    Chang, Hsiang-Hua D; Xu, Yuan; Lai, Hongyu; Yang, Xiaoyu; Tseng, Chun-Che; Lai, Ying-Jung J; Pan, Yu; Zhou, Emily; Johnson, Michael D; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells.

  3. Identification, Biochemical Characterization, and Subcellular Localization of Allantoate Amidohydrolases from Arabidopsis and Soybean1[W

    PubMed Central

    Werner, Andrea K.; Sparkes, Imogen A.; Romeis, Tina; Witte, Claus-Peter

    2008-01-01

    Allantoate amidohydrolases (AAHs) hydrolize the ureide allantoate to ureidoglycolate, CO2, and two molecules of ammonium. Allantoate degradation is required to recycle purine-ring nitrogen in all plants. Tropical legumes additionally transport fixed nitrogen via allantoin and allantoate into the shoot, where it serves as a general nitrogen source. AAHs from Arabidopsis (Arabidopsis thaliana; AtAAH) and from soybean (Glycine max; GmAAH) were cloned, expressed in planta as StrepII-tagged variants, and highly purified from leaf extracts. Both proteins form homodimers and release 2 mol ammonium/mol allantoate. Therefore, they can truly be classified as AAHs. The kinetic constants determined and the half-maximal activation by 2 to 3 μm manganese are consistent with allantoate being the in vivo substrate of manganese-loaded AAHs. The enzymes were strongly inhibited by micromolar concentrations of fluoride as well as by borate, and by millimolar concentrations of l-asparagine and l-aspartate but not d-asparagine. l-Asparagine likely functions as competitive inhibitor. An Ataah T-DNA mutant, unable to grow on allantoin as sole nitrogen source, is rescued by the expression of StrepII-tagged variants of AtAAH and GmAAH, demonstrating that both proteins are functional in vivo. Similarly, an allantoinase (aln) mutant is rescued by a tagged AtAln variant. Fluorescent fusion proteins of allantoinase and both AAHs localize to the endoplasmic reticulum after transient expression and in transgenic plants. These findings demonstrate that after the generation of allantoin in the peroxisome, plant purine degradation continues in the endoplasmic reticulum. PMID:18065556

  4. Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils

    SciTech Connect

    Carmo, Lívia A.S.; Dias, Felipe F.; Malta, Kássia K.; Amaral, Kátia B.; Shamri, Revital; Weller, Peter F.; Melo, Rossana C.N.

    2015-10-01

    Background: SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Methods: Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. Results: STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. Conclusions: The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. - Highlights: • First demonstration of the Qa-SNARE syntaxin-17 (STX17) in human eosinophils. • High

  5. Subcellular localization and functional expression of the glycerol uptake protein 1 (GUP1) of Saccharomyces cerevisiae tagged with green fluorescent protein.

    PubMed

    Bleve, Gianluca; Zacheo, Giuseppe; Cappello, Maria Stella; Dellaglio, Franco; Grieco, Francesco

    2005-08-15

    GFP (green fluorescent protein) from Aequorea victoria was used as an in vivo reporter protein when fused to the N- and C-termini of the glycerol uptake protein 1 (Gup1p) of Saccharomyces cerevisiae. The subcellular localization and functional expression of biologically active Gup1-GFP chimaeras was monitored by confocal laser scanning and electron microscopy, thus supplying the first study of GUP1 dynamics in live yeast cells. The Gup1p tagged with GFP is a functional glycerol transporter localized at the plasma membrane and endoplasmic reticulum levels of induced cells. The factors involved in proper localization and turnover of Gup1p were revealed by expression of the Gup1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaerical protein was targeted to the plasma membrane through a Sec6-dependent process; on treatment with glucose, it was endocytosed through END3 and targeted for degradation in the vacuole. Gup1p belongs to the list of yeast proteins rapidly down-regulated by changing the carbon source in the culture medium, in agreement with the concept that post-translational modifications triggered by glucose affect proteins of peripheral functions. The immunoelectron microscopy assays of cells expressing either Gup1-GFP or GFP-Gup1 fusions suggested the Gup1p membrane topology: the N-terminus lies in the periplasmic space, whereas its C-terminal tail has an intracellular location. An extra cytosolic location of the N-terminal tail is not generally predicted or determined in yeast membrane transporters.

  6. EuLoc: a web-server for accurately predict protein subcellular localization in eukaryotes by incorporating various features of sequence segments into the general form of Chou's PseAAC

    NASA Astrophysics Data System (ADS)

    Chang, Tzu-Hao; Wu, Li-Ching; Lee, Tzong-Yi; Chen, Shu-Pin; Huang, Hsien-Da; Horng, Jorng-Tzong

    2013-01-01

    The function of a protein is generally related to its subcellular localization. Therefore, knowing its subcellular localization is helpful in understanding its potential functions and roles in biological processes. This work develops a hybrid method for computationally predicting the subcellular localization of eukaryotic protein. The method is called EuLoc and incorporates the Hidden Markov Model (HMM) method, homology search approach and the support vector machines (SVM) method by fusing several new features into Chou's pseudo-amino acid composition. The proposed SVM module overcomes the shortcoming of the homology search approach in predicting the subcellular localization of a protein which only finds low-homologous or non-homologous sequences in a protein subcellular localization annotated database. The proposed HMM modules overcome the shortcoming of SVM in predicting subcellular localizations using few data on protein sequences. Several features of a protein sequence are considered, including the sequence-based features, the biological features derived from PROSITE, NLSdb and Pfam, the post-transcriptional modification features and others. The overall accuracy and location accuracy of EuLoc are 90.5 and 91.2 %, respectively, revealing a better predictive performance than obtained elsewhere. Although the amounts of data of the various subcellular location groups in benchmark dataset differ markedly, the accuracies of 12 subcellular localizations of EuLoc range from 82.5 to 100 %, indicating that this tool is much more balanced than other tools. EuLoc offers a high, balanced predictive power for each subcellular localization. EuLoc is now available on the web at http://euloc.mbc.nctu.edu.tw/.

  7. EuLoc: a web-server for accurately predict protein subcellular localization in eukaryotes by incorporating various features of sequence segments into the general form of Chou's PseAAC.

    PubMed

    Chang, Tzu-Hao; Wu, Li-Ching; Lee, Tzong-Yi; Chen, Shu-Pin; Huang, Hsien-Da; Horng, Jorng-Tzong

    2013-01-01

    The function of a protein is generally related to its subcellular localization. Therefore, knowing its subcellular localization is helpful in understanding its potential functions and roles in biological processes. This work develops a hybrid method for computationally predicting the subcellular localization of eukaryotic protein. The method is called EuLoc and incorporates the Hidden Markov Model (HMM) method, homology search approach and the support vector machines (SVM) method by fusing several new features into Chou's pseudo-amino acid composition. The proposed SVM module overcomes the shortcoming of the homology search approach in predicting the subcellular localization of a protein which only finds low-homologous or non-homologous sequences in a protein subcellular localization annotated database. The proposed HMM modules overcome the shortcoming of SVM in predicting subcellular localizations using few data on protein sequences. Several features of a protein sequence are considered, including the sequence-based features, the biological features derived from PROSITE, NLSdb and Pfam, the post-transcriptional modification features and others. The overall accuracy and location accuracy of EuLoc are 90.5 and 91.2 %, respectively, revealing a better predictive performance than obtained elsewhere. Although the amounts of data of the various subcellular location groups in benchmark dataset differ markedly, the accuracies of 12 subcellular localizations of EuLoc range from 82.5 to 100 %, indicating that this tool is much more balanced than other tools. EuLoc offers a high, balanced predictive power for each subcellular localization. EuLoc is now available on the web at http://euloc.mbc.nctu.edu.tw/.

  8. SEPPA 2.0—more refined server to predict spatial epitope considering species of immune host and subcellular localization of protein antigen

    PubMed Central

    Qi, Tao; Qiu, Tianyi; Zhang, Qingchen; Tang, Kailin; Fan, Yangyang; Qiu, Jingxuan; Wu, Dingfeng; Zhang, Wei; Chen, Yanan; Gao, Jun; Zhu, Ruixin; Cao, Zhiwei

    2014-01-01

    Spatial Epitope Prediction server for Protein Antigens (SEPPA) has received lots of feedback since being published in 2009. In this improved version, relative ASA preference of unit patch and consolidated amino acid index were added as further classification parameters in addition to unit-triangle propensity and clustering coefficient which were previously reported. Then logistic regression model was adopted instead of the previous simple additive one. Most importantly, subcellular localization of protein antigen and species of immune host were fully taken account to improve prediction. The result shows that AUC of 0.745 (5-fold cross-validation) is almost the baseline performance with no differentiation like all the other tools. Specifying subcellular localization of protein antigen and species of immune host will generally push the AUC up. Secretory protein immunized to mouse can push AUC to 0.823. In this version, the false positive rate has been largely decreased as well. As the first method which has considered the subcellular localization of protein antigen and species of immune host, SEPPA 2.0 shows obvious advantages over the other popular servers like SEPPA, PEPITO, DiscoTope-2, B-pred, Bpredictor and Epitopia in supporting more specific biological needs. SEPPA 2.0 can be accessed at http://badd.tongji.edu.cn/seppa/. Batch query is also supported. PMID:24838566

  9. Differential Subcellular Localization of the Glucocorticoid Receptor in Distinct Neural Stem and Progenitor Populations of the Mouse Telencephalon In Vivo

    PubMed Central

    Tsiarli, Maria A.; Monaghan, A. Paula; DeFranco, Donald B.

    2013-01-01

    Glucocorticoids are given to pregnant women at risk for premature delivery to promote lung maturation. Despite reports of detrimental effects of glucocorticoids on telencephalic neural stem/progenitor cells (NSPCs), the regional and cellular expression of the glucocorticoid receptor (GR) in various NSPC populations in the intact brain has not been thoroughly assessed. Therefore in this study we performed a detailed analysis of GR protein expression in the developing mouse ventral and dorsal telencephalon in vivo. At embryonic day 11.5 (E11.5), the majority of Pax6-positive radial glial cells (RGCs) and Tbr2-positive intermediate progenitor cells (IPCs) expressed nuclear GR, while a small number of RGCs on the apical ventricular zone (aVZ), expressed cytoplasmic GR. However, on E13.5, the latter population of RGCs increased in size, whereas abventricular NSPCs and especially neurons of the cortical plate, expressed nuclear GR. In IPCs, GR was always nuclear. A similar expression profile was observed throughout the ventral telencephalon, hippocampus and olfactory bulb, with NSPCs of the aVZ primarily expressing cytoplasmic GR, while abventricular NSPCs and mature cells primarily expressed nuclear GR. Close to birth, nuclear GR accumulated within specific cortical areas such as layer V, the subplate and CA1 area of the hippocampus. In summary, our data show that GR protein is present in early NSPCs of the dorsal and ventral telencephalon at E11.5 and primarily occupies the nucleus. Moreover, our study suggests that the subcellular localization of the receptor may be subjected to region and neurodevelopmental stage-specific regulation. PMID:23751362

  10. Inflammation-induced abnormalities in the subcellular localization and trafficking of the neurokinin 1 receptor in the enteric nervous system

    PubMed Central

    Lieu, TinaMarie; Pelayo, Juan Carlos; Eriksson, Emily M.; Veldhuis, Nicholas A.; Bunnett, Nigel W.

    2015-01-01

    Activated G protein-coupled receptors traffic to endosomes and are sorted to recycling or degradative pathways. Endosomes are also a site of receptor signaling of sustained and pathophysiologically important processes, including inflammation. However, the mechanisms of endosomal sorting of receptors and the impact of disease on trafficking have not been fully defined. We examined the effects of inflammation on the subcellular distribution and trafficking of the substance P (SP) neurokinin 1 receptor (NK1R) in enteric neurons. We studied NK1R trafficking in enteric neurons of the mouse colon using immunofluorescence and confocal microscopy. The impact of inflammation was studied in IL10−/−-piroxicam and trinitrobenzenesulfonic acid colitis models. NK1R was localized to the plasma membrane of myenteric and submucosal neurons of the uninflamed colon. SP evoked NK1R endocytosis and recycling. Deletion of β-arrestin2, which associates with the activated NK1R, accelerated recycling. Inhibition of endothelin-converting enzyme-1 (ECE-1), which degrades endosomal SP, prevented recycling. Inflammation was associated with NK1R endocytosis in myenteric but not submucosal neurons. Whereas the NK1R in uninflamed neurons recycled within 60 min, NK1R recycling in inflamed neurons was delayed for >120 min, suggesting defective recycling machinery. Inflammation was associated with β-arrestin2 upregulation and ECE-1 downregulation, which may contribute to the defective NK1R recycling. We conclude that inflammation evokes redistribution of NK1R from the plasma membrane to endosomes of myenteric neurons through enhanced SP release and defective NK1R recycling. Defective recycling may be secondary to upregulation of β-arrestin2 and downregulation of ECE-1. Internalized NK1R may generate sustained proinflammatory signals that disrupt normal neuronal functions. PMID:26138465

  11. Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi.

    PubMed

    Gonzalez, Soledad Natalia; Valsecchi, Wanda Mariela; Maugeri, Dante; Delfino, José María; Cazzulo, Juan José

    2017-01-01

    The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these

  12. Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi

    PubMed Central

    Gonzalez, Soledad Natalia; Valsecchi, Wanda Mariela; Maugeri, Dante; Delfino, José María; Cazzulo, Juan José

    2017-01-01

    The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these

  13. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

    SciTech Connect

    Bhaskar,; Kumari, Neeti; Goyal, Neena

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer The study presents cloning and characterization of TCP1{gamma} gene from L. donovani. Black-Right-Pointing-Pointer TCP1{gamma} is a subunit of T-complex protein-1 (TCP1), a chaperonin class of protein. Black-Right-Pointing-Pointer LdTCP{gamma} exhibited differential expression in different stages of promastigotes. Black-Right-Pointing-Pointer LdTCP{gamma} co-localized with actin, a cytoskeleton protein. Black-Right-Pointing-Pointer The data suggests that this gene may have a role in differentiation/biogenesis. Black-Right-Pointing-Pointer First report on this chapronin in Leishmania. -- Abstract: T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1{gamma}), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1{gamma} of Leishmania donovani (LdTCP1{gamma}), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1{gamma} revealed the presence of all the characteristic features of TCP1{gamma}. However, leishmanial TCP1{gamma} represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1{gamma} exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1{gamma} as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1{gamma} was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1{gamma} with actin suggests

  14. Expression of cytochrome P450 CYP81A6 in rice: tissue specificity, protein subcellular localization, and response to herbicide application*

    PubMed Central

    Lu, Hai-ping; Edwards, Martin; Wang, Qi-zhao; Zhao, Hai-jun; Fu, Hao-wei; Huang, Jian-zhong; Gatehouse, Angharad; Shu, Qing-yao

    2015-01-01

    The cytochrome P450 gene CYP81A6 confers tolerance to bentazon and metsulfuron-methyl, two selective herbicides widely used for weed control in rice and wheat fields. Knockout mutants of CYP81A6 are highly susceptible to both herbicides. The present study aimed to characterize the CYP81A6 expression in rice. Quantitative real-time polymerase chain reaction (PCR) analyses demonstrated that foliar treatment of bentazon (500 mg/L) greatly induced expression of CYP81A6 in both wild-type (Jiazhe B) and its knockout mutant (Jiazhe mB): a 10-fold increase at 9 h before returning to basal levels at 24 h in Jiazhe B, while in the mutant the expression level rose to >20-fold at 12 h and maintained at such high level up to 24 h post exposure. In contrast, metsulfuron-methyl (500 mg/L) treatment did not affect the expression of CYP81A6 in Jiazhe B within 80 h; thereafter the expression peaked at 120 h and returned gradually to basal levels by Day 6. We suggest that a metabolite of metsulfuron-methyl, 1H-2,3-benzothiazin-4-(3H)-one-2,2-dioxide, is likely to be responsible for inducing CYP81A6 expression, rather than the metsulfuron-methyl itself. Use of a promoter-GUS reporter construct (CYP81A6Pro::GUS) demonstrated that CYP81A6 was constitutively expressed throughout the plant, with the highest expression in the upper surfaces of leaves. Subcellular localization studies in rice protoplasts showed that CYP81A6 was localized in the endoplasmic reticulum. These observations advance our understanding of CYP81A6 expression in rice, particularly its response to the two herbicides. PMID:25644466

  15. Subcellular localization of the K+ channel subunit Kv3.1b in selected rat CNS neurons.

    PubMed

    Sekirnjak, C; Martone, M E; Weiser, M; Deerinck, T; Bueno, E; Rudy, B; Ellisman, M

    1997-08-22

    Voltage-gated potassium channels constitute the largest group of heteromeric ion channels discovered to date. Over 20 genes have been isolated, encoding different channel subunit proteins which form functional tetrameric K+ channels. We have analyzed the subcellular localization of subunit Kv3.1b, a member of the Kv3 (Shaw-like) subfamily, in rat brain at the light and electron microscopic level, using immunocytochemical detection. Detailed localization was carried out in specific neurons of the neocortex, hippocampus and cerebellum. The identity of Kv3.1b-positive neurons was established using double labeling with markers for specific neuronal populations. In the neocortex, the Kv3.1b subunit was expressed in most parvalbumin-containing bipolar, basket or chandelier cells, and in some bipolar or double bouquet neurons containing calbindin. In the hippocampus, Kv3.1b was expressed in many parvalbumin-containing basket cells, as well as in calbindin-positive neurons in the stratum oriens, and in a small number of interneurons that did not stain for either parvalbumin or calbindin. Kv3.1b protein was not present in pyramidal cells in the neocortex and the hippocampus, but these cells were outlined by labeled presynaptic terminals from interneuron axons that surround the postsynaptic cell. In the cerebellar cortex, granule cells were the only population expressing the channel protein. Careful examination of individual granule cells revealed a non-uniform distribution of Kv3.1 staining on the somata: circular bands of labeling were present in the vicinity of the axon hillock. In cortical and hippocampal interneurons, as well as in cerebellar granule cells, the Kv3.1b subunit was present in somatic and unmyelinated axonal membranes and adjacent cytoplasm, as well as in the most proximal portion of dendritic processes, but not throughout most of the dendrite. Labeling was also seen in the terminals of labeled axons, but not at a higher concentration than in other parts

  16. Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation

    PubMed Central

    Rodríguez-Escudero, María; Cid, Víctor J.; Molina, María; Schulze-Luehrmann, Jan; Lührmann, Anja; Rodríguez-Escudero, Isabel

    2016-01-01

    Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors

  17. Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation.

    PubMed

    Rodríguez-Escudero, María; Cid, Víctor J; Molina, María; Schulze-Luehrmann, Jan; Lührmann, Anja; Rodríguez-Escudero, Isabel

    2016-01-01

    Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors.

  18. Effect of NGF on the subcellular localization of group IIA secretory phospholipase A(2) (GIIA) in PC12 cells: role in neuritogenesis.

    PubMed

    Ferrini, M; Nardicchi, V; Mannucci, R; Arcuri, C; Nicoletti, I; Donato, R; Goracci, G

    2010-12-01

    Phospholipases A(2) (PLA(2)s) are involved in neuritogenesis but the identity of the isoforms(s) contributing to this process is still not defined. Several reports have focused on secretory PLA(2)s (sPLA(2)) as the administration of exogenous sPLA(2)s to PC12 neuronal cells stimulates neurite outgrowth. The present study demonstrates that the endogenous group IIA sPLA(2) (GIIA), constitutively expressed in mammalian neural cells, changes its subcellular localization when PC12 cells are induced to differentiate by NGF treatment. Indeed, confocal analysis showed a time-dependent accumulation of GIIA in growth cones and neurite tips. Under identical conditions the subcellular distribution of another isoform (GV) was unaffected by NGF. Contrary to GX, another sPLA(2) isoform expressed by PC12 cells, the contribution of GIIA to neuritogenesis does not require its release in the extracellular medium.

  19. Subcellular localization analysis of the closely related Fps/Fes and Fer protein-tyrosine kinases suggests a distinct role for Fps/Fes in vesicular trafficking.

    PubMed

    Zirngibl, R; Schulze, D; Mirski, S E; Cole, S P; Greer, P A

    2001-05-15

    The subcellular localizations of the Fps/Fes and closely related Fer cytoplasmic tyrosine kinases were studied using green fluorescent protein (GFP) fusions and confocal fluorescence microscopy. In contrast to previous reports, neither kinase localized to the nucleus. Fer was diffusely cytoplasmic throughout the cell cycle. Fps/Fes also displayed a diffuse cytoplasmic localization, but in addition it showed distinct accumulations in cytoplasmic vesicles as well as in a perinuclear region consistent with the Golgi. This localization was very similar to that of TGN38, a known marker of the trans Golgi. The localization of Fps/Fes and TGN38 were both perturbed by brefeldin A, a fungal metabolite that disrupts the Golgi apparatus. Fps/Fes was also found to colocalize to various extents with several Rab proteins, which are members of the monomeric G-protein superfamily involved in vesicular transport between specific subcellular compartments. Using Rabs that are involved in endocytosis (Rab5B and Rab7) or exocytosis (Rab1A and Rab3A), we showed that Fps/Fes is localized in both pathways. These results suggest that Fps/Fes may play a general role in the regulation of vesicular trafficking.

  20. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

    PubMed

    Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C S; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah

    2016-01-01

    The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene

  1. Chronic alcohol exposure differentially affects activation of female locus coeruleus neurons and the subcellular distribution of corticotropin releasing factor receptors.

    PubMed

    Retson, T A; Reyes, B A; Van Bockstaele, E J

    2015-01-02

    Understanding the neurobiological bases for sex differences in alcohol dependence is needed to help guide the development of individualized therapies for alcohol abuse disorders. In the present study, alcohol-induced adaptations in (1) anxiety-like behavior, (2) patterns of c-Fos activation and (3) subcellular distribution of corticotropin releasing factor receptor in locus coeruleus (LC) neurons was investigated in male and female Sprague-Dawley rats that were chronically exposed to ethanol using a liquid diet. Results confirm and extend reports by others showing that chronic ethanol exposure produces an anxiogenic-like response in both male and female subjects. Ethanol-induced sex differences were observed with increased c-Fos expression in LC neurons of female ethanol-treated subjects compared to controls or male subjects. Results also reveal sex differences in the subcellular distribution of the CRFr in LC-noradrenergic neurons with female subjects exposed to ethanol exhibiting a higher frequency of plasmalemmal CRFrs. These adaptations have implications for LC neuronal activity and its neural targets across the sexes. Considering the important role of the LC in ethanol-induced activation of the hypothalamo-pituitary-adrenal (HPA) axis, the present results indicate important sex differences in feed-forward regulation of the HPA axis that may render alcohol dependent females more vulnerable to subsequent stress exposure.

  2. Temperature affects cadmium-induced phytotoxicity involved in subcellular cadmium distribution and oxidative stress in wheat roots.

    PubMed

    Li, Dandan; Zhou, Dongmei; Wang, Peng; Li, Lianzhen

    2011-10-01

    In this study, the effect of temperature on Cd toxicity to wheat roots was evaluated in terms of the relative root length and subcellular distribution of Cd as well as the antioxidant enzymatic activities after exposed to Cd for 72 h under different temperatures. The result showed that the EC(50)-values for the relative root length were 9.24, 4.91 and 3.62 μM Cd at 18, 25 and 30°C, respectively. The Cd concentrations in the cellular metal-sensitive fraction or the potentially toxic fraction (cell debris fraction-Cd) were well correlated with the toxicity of Cd. Interestingly, the superoxide dismutase (SOD) and catalase (CAT) in wheat roots without Cd exposure were increased at 18°C compared to those at 25°C, while decreased at 30°C. The CAT activities decreased with increasing Cd level at 25 and 18°C but did not show the same change at 30°C, which could be explained by the subcellular distribution of Cd.

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

    SciTech Connect

    Lee, Asaka; Asahina, Kota; Okamoto, Takumi; Kawaguchi, Kosuke; Kostsin, Dzmitry G.; Kashiwayama, Yoshinori; Takanashi, Kojiro; Yazaki, Kazufumi; Imanaka, Tsuneo; Morita, Masashi

    2014-10-24

    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 several 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.

  4. A recurrent KCNQ2 pore mutation causing early onset epileptic encephalopathy has a moderate effect on M current but alters subcellular localization of Kv7 channels.

    PubMed

    Abidi, Affef; Devaux, Jérôme J; Molinari, Florence; Alcaraz, Gisèle; Michon, François-Xavier; Sutera-Sardo, Julie; Becq, Hélène; Lacoste, Caroline; Altuzarra, Cécilia; Afenjar, Alexandra; Mignot, Cyril; Doummar, Diane; Isidor, Bertrand; Guyen, Sylvie N; Colin, Estelle; De La Vaissière, Sabine; Haye, Damien; Trauffler, Adeline; Badens, Catherine; Prieur, Fabienne; Lesca, Gaetan; Villard, Laurent; Milh, Mathieu; Aniksztejn, Laurent

    2015-08-01

    Mutations in the KCNQ2 gene encoding the voltage-dependent potassium M channel Kv7.2 subunit cause either benign epilepsy or early onset epileptic encephalopathy (EOEE). It has been proposed that the disease severity rests on the inhibitory impact of mutations on M current density. Here, we have analyzed the phenotype of 7 patients carrying the p.A294V mutation located on the S6 segment of the Kv7.2 pore domain (Kv7.2(A294V)). We investigated the functional and subcellular consequences of this mutation and compared it to another mutation (Kv7.2(A294G)) associated with a benign epilepsy and affecting the same residue. We report that all the patients carrying the p.A294V mutation presented the clinical and EEG characteristics of EOEE. In CHO cells, the total expression of Kv7.2(A294V) alone, assessed by western blotting, was only 20% compared to wild-type. No measurable current was recorded in CHO cells expressing Kv7.2(A294V) channel alone. Although the total Kv7.2(A294V) expression was rescued to wild-type levels in cells co-expressing the Kv7.3 subunit, the global current density was still reduced by 83% compared to wild-type heteromeric channel. In a configuration mimicking the patients' heterozygous genotype i.e., Kv7.2(A294V)/Kv7.2/Kv7.3, the global current density was reduced by 30%. In contrast to Kv7.2(A294V), the current density of homomeric Kv7.2(A294G) was not significantly changed compared to wild-type Kv7.2. However, the current density of Kv7.2(A294G)/Kv7.2/Kv7.3 and Kv7.2(A294G)/Kv7.3 channels were reduced by 30% and 50% respectively, compared to wild-type Kv7.2/Kv7.3. In neurons, the p.A294V mutation induced a mislocalization of heteromeric mutant channels to the somato-dendritic compartment, while the p.A294G mutation did not affect the localization of the heteromeric channels to the axon initial segment. We conclude that this position is a hotspot of mutation that can give rise to a severe or a benign epilepsy. The p.A294V mutation does not exert a

  5. Predict Gram-Positive and Gram-Negative Subcellular Localization via Incorporating Evolutionary Information and Physicochemical Features Into Chou's General PseAAC.

    PubMed

    Sharma, Ronesh; Dehzangi, Abdollah; Lyons, James; Paliwal, Kuldip; Tsunoda, Tatsuhiko; Sharma, Alok

    2015-12-01

    In this study, we used structural and evolutionary based features to represent the sequences of gram-positive and gram-negative subcellular localizations. To do this, we proposed a normalization method to construct a normalize Position Specific Scoring Matrix (PSSM) using the information from original PSSM. To investigate the effectiveness of the proposed method we compute feature vectors from normalize PSSM and by applying support vector machine (SVM) and naïve Bayes classifier, respectively, we compared achieved results with the previously reported results. We also computed features from original PSSM and normalized PSSM and compared their results. The archived results show enhancement in gram-positive and gram-negative subcellular localizations. Evaluating localization for each feature, our results indicate that employing SVM and concatenating features (amino acid composition feature, Dubchak feature (physicochemical-based features), normalized PSSM based auto-covariance feature and normalized PSSM based bigram feature) have higher accuracy while employing naïve Bayes classifier with normalized PSSM based auto-covariance feature proves to have high sensitivity for both benchmarks. Our reported results in terms of overall locative accuracy is 84.8% and overall absolute accuracy is 85.16% for gram-positive dataset; and, for gram-negative dataset, overall locative accuracy is 85.4% and overall absolute accuracy is 86.3%.

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

    PubMed Central

    2015-01-01

    Background 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. Results 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. Conclusions 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

  7. Bile acids alter the subcellular localization of CNT2 (concentrative nucleoside cotransporter) and increase CNT2-related transport activity in liver parenchymal cells

    PubMed Central

    Fernández-Veledo, Sonia; Huber-Ruano, Isabel; Aymerich, Ivette; Duflot, Sylvie; Casado, F. Javier; Pastor-Anglada, Marçal

    2006-01-01

    CNT2 (concentrative nucleoside cotransporter) is a plasma membrane high-affinity Na+-coupled adenosine transporter, also localized in intracellular structures. This transporter protein may play additional roles other than nucleoside salvage, since it has recently been shown to be under purinergic control via KATP channels, by a mechanism that does not seem to involve changes in its subcellular localization. In an attempt to identify the agents that promote CNT2 trafficking, bile acids were found to increase CNT2-related transport activity in a KATP channel-independent manner in both Fao hepatoma and rat liver parenchymal cells. A maximum effect was recorded after treatment with hydrophilic anions such as TCA (taurocholate). However, this effect did not involve changes in the amount of CNT2 protein, it was instead associated with a subcellular redistribution of CNT2, resulting in an accumulation of the transporter at the plasma membrane. This was deduced from subcellular fractionation studies, biotinylation of plasma membrane proteins and subsequent CNT2 detection in streptavidin precipitates and in vivo confocal microscopic analysis of the distribution of a YFP (yellow fluorescent protein)–CNT2 construct. The induction of CNT2 translocation, triggered by TCA, was inhibited by wortmannin, dibutyryl-AMPc, PD98059 and colchicine, thus suggesting the involvement of the PI3K/ERK (phosphoinositide 3-kinase/extracellular-signal related kinase) pathway in microtubule-dependent activation of recombinant CNT2. These are novel effects of bile-acid physiology and provide the first evidence for short-term regulation of CNT2 translocation into and from the plasma membrane. PMID:16390326

  8. Phosphorylation and subcellular localization of Na(+)/H(+) exchanger isoform 3 (NHE3) are associated with altered gallbladder absorptive function after formation of cholesterol gallstones.

    PubMed

    Chen, Yongsheng; Wu, Shuodong; Tian, Yu; Kong, Jing

    2017-02-01

    Na(+)/H(+) exchanger isoform 3 (NHE3) dysfunction is thought to contribute to the altered gallbladder absorption that occurs in cholesterol gallstone disease, but the mechanism is unknown. The current study was undertaken to examine the expression, phosphorylation, and subcellular localization of NHE3 in gallbladder epithelium cells (GBECs) of male C57BL/6 mice on a control or lithogenic diet. Thirty-six 8-week-old male C57BL/6 mice were randomly assigned to receive a high cholesterol diet or a regular diet for 8 weeks. Gallstone formation was recorded. Gallbladder bile cholesterol, phospholipid, and total bile acids were examined. RT-PCR was used to measure NHE3 mRNA expression. NHE3 protein expression and subcellular localization were examined by Western blotting and immunofluorescence microscopy, respectively. Gallstones were formed in all mice fed the lithogenic diet. Despite higher NHE3 mRNA expression in gallbladders of the mice on the lithogenic diet than in those on the control diet, there was no significant difference in expression of total NHE3 protein. However, a higher level of NHE3 phosphorylated at serine-552 (P-NHE3) was seen on the lithogenic diet. In immunofluorescence studies, NHE3 protein was expressed both on the apical membrane and in the cytoplasm of mouse GBEC. This pattern of subcellular distribution of NHE3 strongly corroborates an exchanger trafficking mechanism in NHE3 activity regulation in mouse GBEC. We conclude that increased phosphorylation of NHE3 following gallstone formation leads to turnover of the exchanger, resulting in decreased gallbladder concentrating function.

  9. Calpain-mediated Processing of p53-associated Parkin-like Cytoplasmic Protein (PARC) Affects Chemosensitivity of Human Ovarian Cancer Cells by Promoting p53 Subcellular Trafficking*

    PubMed Central

    Woo, Michael G.; Xue, Kai; Liu, Jiayin; McBride, Heidi; Tsang, Benjamin K.

    2012-01-01

    Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA), and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways, and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA are unknown. Here, we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation, and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibition of calpain but not of caspases or the 26 S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin-induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicate the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity. PMID:22117079

  10. Imaging of Caenorhabditis elegans samples and sub-cellular localization of new generation photosensitizers for photodynamic therapy, using non-linear microscopy

    NASA Astrophysics Data System (ADS)

    Filippidis, G.; Kouloumentas, C.; Kapsokalyvas, D.; Voglis, G.; Tavernarakis, N.; Papazoglou, T. G.

    2005-08-01

    Two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) are relatively new promising tools for the imaging and mapping of biological structures and processes at the microscopic level. The combination of the two image-contrast modes in a single instrument can provide unique and complementary information concerning the structure and the function of tissues and individual cells. The extended application of this novel, innovative technique by the biological community is limited due to the high price of commercial multiphoton microscopes. In this study, a compact, inexpensive and reliable setup utilizing femtosecond pulses for excitation was developed for the TPEF and SHG imaging of biological samples. Specific cell types of the nematode Caenorhabditis elegans were imaged. Detection of the endogenous structural proteins of the worm, which are responsible for observation of SHG signals, was achieved. Additionally, the binding of different photosensitizers in the HL-60 cell line was investigated, using non-linear microscopy. The sub-cellular localization of photosensitizers of a new generation, very promising for photodynamic therapy (PDT), (Hypericum perforatum L. extracts) was achieved. The sub-cellular localization of these novel photosensitizers was linked with their photodynamic action during PDT, and the possible mechanisms for cell killing have been elucidated.

  11. Genome-wide analysis of phylogeny, expression profile and sub-cellular localization of SKP1-Like genes in wild tomato.

    PubMed

    Zhang, YueQin; Wang, CuiPing; Lin, QingFang; Gao, FengHua; Ma, Yan; Zhang, Min; Lin, YueHui; Ma, QingHu; Hua, XueJun

    2015-09-01

    SKP1 is a core component of SCF complex, a major type of E3 ubiquitin ligase catalyzing the last step in ubiquitin-mediated protein degradation pathway. In present study, SKP1 gene family in Solanum pimpinellifolium (SSK), a wild species of tomato, was investigated. A total of 19 SSK genes were identified through homologous search. Their chromosomal locations, gene structures, phylogeny, expression profiles, sub-cellular localizations and protein-protein interaction patterns with putative F-box proteins were analyzed in detail. The high homology and similar expression patterns among clustered SSK genes in chromosome suggested that they may have evolved from duplication events and are functionally redundant. Sub-cellular localization indicated that most of the SSK proteins are distributed in both cytosol and nucleus, except for SSK8, which is detected in cytosol only. Tissue-specific expression patterns suggested that many SSK genes may be involved in tomato fruit development. Furthermore, several SSK genes were found to be responsive to heat stress and salicylic acid treatment. Based on phylogenetic analysis, expression profiles and protein interaction property, we proposed that tomato SSK1 and SSK2 might have similar function to ASK1 and ASK2 in Arabidopsis.

  12. Enhancing a Pathway-Genome Database (PGDB) to Capture Subcellular Localization of Metabolites and Enzymes: The Nucleotide-Sugar Biosynthetic Pathways of Populus trichocarpa

    SciTech Connect

    Nag, A.; Karpinets, T. V.; Chang, C. H.; Bar-Peled, M.

    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).

  13. Bioaccumulation, subcellular, and molecular localization and damage to physiology and ultrastructure in Nymphoides peltata (Gmel.) O. Kuntze exposed to yttrium.

    PubMed

    Fu, Yongyang; Li, Feifei; Xu, Ting; Cai, Sanjuan; Chu, Weiyue; Qiu, Han; Sha, Sha; Cheng, Guangyu; Xu, Qinsong

    2014-02-01

    Bioaccumulation, subcellular distribution, and acute toxicity of yttrium (Y) were evaluated in Nymphoides peltata. The effects of Y concentrations of 1-5 mg L(-1) applied for 4 days were assessed by measuring changes in photosynthetic pigments, nutrient contents, enzymatic and non-enzymatic antioxidants, and ultrastructure. The accumulation of Y in subcellular fractions decreased in the order of cell wall > organelle > soluble fraction. Much more Y was located in cellulose and pectin than in other biomacromolecules. The content of some mineral elements (Mg, Ca, Fe, Mn, and Mo) increased in N. peltata, but there was an opposite effect for P and K. Meanwhile, ascorbate, and catalase activity decreased significantly for all Y concentrations. In contrast, peroxidase activity was induced, while initial rises in superoxide dismutase activity and glutathione content were followed by subsequent declines. Morphological symptoms of senescence, such as chlorosis and damage to chloroplasts and mitochondria, were observed even at the lowest Y concentration. Pigment content decreased as the Y concentration rose and the calculated EC50 and MPC of Y for N. peltata were 2 and 0.2 mg L(-1) after 4 days of exposure, respectively. The results showed that exogenous Y was highly available in water and that its high concentration in water bodies might produce harmful effects on aquatic organisms. N. peltata is proposed as a biomonitor for the assessment of metal pollution in aquatic ecosystems.

  14. β-Adrenergic receptor gene expression in HIV-associated neurocognitive impairment and encephalitis: implications for MOR-1K subcellular localization.

    PubMed

    Dever, Seth M; Rodriguez, Myosotys; El-Hage, Nazira

    2016-12-01

    We previously reported that mRNA expression of the unique alternatively spliced OPRM1 isoform μ-opioid receptor-1K (MOR-1K), which exhibits excitatory cellular signaling, is elevated in HIV-infected individuals with combined neurocognitive impairment (NCI) and HIV encephalitis (HIVE). It has recently been shown that the β2-adrenergic receptor (β2-AR) chaperones MOR-1K, normally localized intracellularly, to the cell surface. Here, we found mRNA expression of the adrenoceptor beta 2 (ADRB2) gene is also elevated in NCI-HIVE individuals, as well as that β2-AR protein expression is elevated in HIV-1-infected primary human astrocytes treated with morphine, and discuss the implications for MOR-1K subcellular localization in this condition.

  15. Pregnancy Specific β-1 Glycoprotein 1 is Expressed in Pancreatic Ductal Adenocarcinoma and its Subcellular Localization Correlates with Overall Survival

    PubMed Central

    Shahinian, Jasmin H.; Fuellgraf, Hannah; Tholen, Stefan; Mastroianni, Justin; Knopf, Julia Daniela; Kuehs, Markus; Mayer, Bettina; Schlimpert, Manuel; Kulemann, Birte; Kuesters, Simon; Hoeppner, Jens; Wellner, Ulrich F.; Werner, Martin; Hopt, Ulrich T.; Zeiser, Robert; Bronsert, Peter; Schilling, Oliver

    2016-01-01

    Proteins of the pregnancy specific β-1 glycoprotein (PSG) family are renowned for their elevated expression during pregnancy. Only few reports have investigated their expression in adenocarcinomas. We studied the expression of PSG1 in pancreatic adenocarcinoma (PDAC). In a cohort of 104 patient samples, immunohistochemical analysis determined PSG1 expression in every specimen. PSG1 was found at apical and cytoplasmic localization or solely at cytoplasmic localization, with the latter case being correlated to shortened median survival (25 vs 11 months, logrank p-value < 0.001). At the same time, enzyme linked immunosorbent assay (ELISA) did not detect elevated PSG1 levels in the plasma of PDAC patients as opposed to the plasma of healthy, non-pregnant control individuals. We also probed the impact of PSG1 expression in a murine tumor model system, using subcutaneous injection of Colo-26 cells into immunocompetent BALB/c mice. Here, tumor growth was not affected by the expression of human PSG1. Our study reaffirms interest into the tumor-contextual biology of PSG proteins. PMID:27877217

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

    PubMed Central

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

    2015-01-01

    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. PMID:26359114

  17. Subcellular Localization, Stability, and trans-Cleavage Competence of the Hepatitis C Virus NS3-NS4A Complex Expressed in Tetracycline-Regulated Cell Lines

    PubMed Central

    Wölk, Benno; Sansonno, Domenico; Kräusslich, Hans-Georg; Dammacco, Franco; Rice, Charles M.; Blum, Hubert E.; Moradpour, Darius

    2000-01-01

    A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3′-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent trans-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic amino-terminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C. PMID:10666260

  18. Agents that Stabilize Mutated von Hippel Lindau Protein Result in Differential Post-Translational Modification and Subcellular Localization

    PubMed Central

    Ding, Zhiyong; German, Peter; Bai, Shanshan; Feng, Zhehui; Gao, Meng; Si, Wendy; Sobieski, Mary M.; Stephan, Clifford C.; Mills, Gordon B.; Jonasch, Eric

    2014-01-01

    Background von Hippel Lindau (VHL) disease is an autosomal dominant inherited disorder that results in multiple organ systems being affected. Treatment is mainly surgical, however, effective systemic therapies are needed. We developed and tested a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point mutated VHL. Methods The 786-0 cell line was infected with full-length W117A mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against the known proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of downstream functional readouts, including HIF and GLUT1 levels, were performed. Results Bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton and thioguanosine were found to reliably upregulate VHL-W117A-Venus in 786-0 cells. 8-azaguanine was found to downregulate HIF2α levels, and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate post-translational processing. In addition, nuclear-cytoplasmic localization of pVHL varied amongst the different compounds. Conclusion 786-0 cells containing VHL-W117A-Venus can be successfully used to identify compounds that upregulate VHL levels, and that have a differential effect on pVHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL. PMID:22357874

  19. Assessing the role of the ASP56/CAP homologue of Dictyostelium discoideum and the requirements for subcellular localization.

    PubMed

    Noegel, A A; Rivero, F; Albrecht, R; Janssen, K P; Köhler, J; Parent, C A; Schleicher, M

    1999-10-01

    The CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP(2)-regulation of the sequestering activity. Here we have constructed GFP-fusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination. They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect. Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria. In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild-type cells. This was paralleled by a reduction in fluid phase endocytosis. Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected.

  20. Two isoforms of Saccharomyces cerevisiae glutaredoxin 2 are expressed in vivo and localize to different subcellular compartments.

    PubMed Central

    Pedrajas, José R; Porras, Pablo; Martínez-Galisteo, Emilia; Padilla, C Alicia; Miranda-Vizuete, Antonio; Bárcena, J Antonio

    2002-01-01

    Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast. Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a 'classical' Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH. Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency. Western blot analysis of S. cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9 kDa respectively. The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one. From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction. On the other hand, only the long form is prominent in microsomes. Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99 bp upstream of the GRX2 start codon described thus far. These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria. The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product. PMID:11958675

  1. Cellular Distribution and Subcellular Localization of Molecular Components of Vesicular Transmitter Release in Horizontal Cells of Rabbit Retina

    PubMed Central

    HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN H.; BRECHA, NICHOLAS C.

    2010-01-01

    The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of γ-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca2+-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells. PMID:15912504

  2. Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes

    PubMed Central

    Melo, Rossana C N; Morgan, Ellen; Monahan-Earley, Rita; Dvorak, Ann M; Weller, Peter F

    2014-01-01

    Precise immunolocalization of proteins within a cell is central to understanding cell processes and functions such as intracellular trafficking and secretion of molecules during immune responses. Here we describe a protocol for ultrastructural detection of proteins in leukocytes. The method uses a pre-embedding approach (immunolabeling before standard processing for transmission electron microscopy (TEM)). This protocol combines several strategies for ultrastructure and antigen preservation, robust blocking of nonspecific binding sites, as well as superior antibody penetration for detecting molecules at subcellular compartments and membrane microdomains. A further advantage of this technique is that electron microscopy (EM) processing is quick. This method has been used to study leukocyte biology, and it has helped demonstrate how activated leukocytes deliver specific cargos. It may also potentially be applied to a variety of different cell types. Excluding the initial time required for sample preparation (15 h) and the final resin polymerization step (16 h), the protocol (immunolabeling and EM procedures) can be completed in 8 h. PMID:25211515

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

    PubMed

    Han, Jae-Yeong; Chung, Jinsoo; Kim, Jungkyu; Seo, Eun-Young; Kilcrease, James P; Bauchan, Gary R; Lim, Seungmo; Hammond, John; Lim, Hyoun-Sub

    2016-08-01

    In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution and variability of Turnip mosaic virus (TuMV). Brassica rapa ssp. pekinensis sap-inoculated with three isolates of TuMV from infected radish tissue showed different symptom severities, whereas symptoms in Raphanus sativus were similar for each isolate. The helper component-protease (HC-Pro) genes of each isolate were sequenced, and phylogenetic analysis showed that the three Korean isolates were clustered into the basal-BR group. The HC-Pro proteins of these isolates were tested for their RNA silencing suppressor (VSR) activity and subcellular localization in Nicotiana benthamiana. A VSR assay by co-agroinfiltration of HC-Pro with soluble-modified GFP (smGFP) showed that HC-Pro of isolate R007 and R041 showed stronger VSR activity than R065. The HC-Pros showed 98.25 % amino acid identity, and weak VSR isolate (R065) has a single variant residue in the C-terminal domain associated with protease activity and self-interaction compared to isolates with strong VSR activity. Formation of large subcellular aggregates of GFP:HC-Pro fusion proteins in N. benthamiana was only observed for HC-Pro from isolates with strong VSR activity, suggesting that R065 'weak' HC-Pro may have diminished self-association; substitution of the variant C-terminal residue largely reversed the HC-Pro aggregation and silencing suppressor characteristics. The lack of correlation between VSR efficiency and induction of systemic necrosis (SN) suggests that differences in viral accumulation due to HC-Pro are not responsible for SN.

  4. Mobility and subcellular localization of endogenous, gene-edited Tau differs from that of over-expressed human wild-type and P301L mutant Tau

    PubMed Central

    Di Xia; Gutmann, Julia M.; Götz, Jürgen

    2016-01-01

    Alzheimer’s disease (AD) and a subset of frontotemporal dementia termed FTLD-Tau are characterized by a massive, yet incompletely characterized and understood redistribution of Tau. To establish a framework for understanding this pathology, we used the genome-editing tool TALEN and generated Tau-mEOS2 knock-in mice to determine the mobility and subcellular localization of endogenous Tau in hippocampal cultures. We analysed Tau in axons, dendrites and spines at three stages of maturation using live-cell imaging, photo-conversion and FRAP assays. Tau-mEOS2 cultures were compared with those over-expressing EGFP-tagged forms of human wild-type (hWT-Tau) and P301L mutant Tau (hP301L-Tau), modelling Tau accumulation in AD and FTLD-Tau, respectively. In developing neurons, Tau-mEOS2 followed a proximo-distal gradient in axons and a subcellular distribution similar to that of endogenous Tau in neurons obtained from wild-type mice, which were abolished, when either hWT-Tau or hP301L-Tau was over-expressed. For the three conditions, FRAP analysis revealed a similar mobility in dendrites compared with axons; however, Tau-mEOS2 was less mobile than hWT-Tau and hP301L-Tau and the mobile fraction was smaller, possibly reflecting less efficient microtubule binding of Tau when over-expressed. Together, our study presents Tau-mEOS2 mice as a novel tool for the study of Tau in a physiological and a pathological context. PMID:27378256

  5. Cloning, characterization and subcellular localization of a gene encoding a human Ubiquitin-conjugating enzyme (E2) homologous to the Arabidopsis thaliana UBC-16 gene product.

    PubMed

    Yin, Gang; Ji, Chaoneng; Wu, Tong; Shen, Zhouliang; Xu, Xin; Xie, Yi; Mao, Yumin

    2006-05-01

    Ubiquitin charging and activation of class III E2 enzymes has been directly linked to their nuclear import. It has not been published whether other classes E2s also abide by this mechanism. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone that is 2252 base pair in length, encoding a putative 162 amino acid protein, which shares high homology to Arabidopsis thaliana ubiquitin-conjugating enzyme 16 (Accession number NP_565110, 51% identity and 71% similarity) at protein level. Bioinformatics analysis revealed that the gene is composed of 7 exons, located on human chromosome 8q13-8q21.1, and that the predicted protein of the gene is a class I E2, for only composed of a conserved approximately 150-amino acid catalytic core, ubiquitin-conjugating enzyme E2 domain (UBC domain). In the C-terminal of the UBC domain sequence, there are two nuclear localization signals (NLSs). RT-PCR showed that this gene is ubiquitously expressed in 16 kinds of normal human tissues, but expression level is very low, unless in human heart, brain, liver, and pancreas. The subcellular localizations of the new human Ubiquitin conjugating enzyme E2 and its mutation were also examined, which showed that the nuclear localization of hUBC16 depended on two conditions: It has NLS, and at the same time, has enzyme active site, too, at least in HEK293 cells.

  6. Diacylglycerol-dependent Binding Recruits PKCθ and RasGRP1 C1 Domains to Specific Subcellular Localizations in Living T LymphocytesV⃞

    PubMed Central

    Carrasco, Silvia; Merida, Isabel

    2004-01-01

    Diacylglycerol (DAG) signaling relies on the presence of conserved domain 1 (C1) in its target proteins. Phospholipase C–dependent generation of DAG after T cell receptor (TCR) triggering is essential for the correct immune response onset. Accordingly, two C1-containing proteins expressed in T lymphocytes, Ras guanyl nucleotide-releasing protein1 (RasGRP1) and protein kinase Cθ (PKCθ), were shown to be fundamental for T-cell activation and proliferation. Although containing the same regulatory domain, they are proposed to relocate to distinct subcellular locations in response to TCR triggering. Here we studied intracellular localization of RasGRP1 and PKCθ C1 domains in living Jurkat T cells. The results demonstrate that, in the absence of significant primary sequence differences, the C1 domains of these proteins show specific localization within the cell and distinct responses to pharmacological stimulation and TCR triggering. These differences help explain the divergent localization and distinct functional roles of the full-length proteins, which contains them. The properties of these DAG-binding modules allow their characterization as functional markers that discriminate between DAG pools. Finally, we show that by binding to different diacylglycerol forms, overexpression of distinct C1 modules can attenuate DAG-dependent signals originating from the plasma or internal membranes. This is shown by analyzing the contribution of these two lipid pools to PLC-dependent Ras activation in response to TCR triggering. PMID:15064353

  7. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production

    PubMed Central

    Ng, Ivan H. W.; Chan, Kitti W. K.; Zhao, Yongqian; Ooi, Eng Eong; Lescar, Julien; Jans, David A.; Forwood, Jade K.

    2016-01-01

    Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization. PMID:27622521

  8. Efficient Subcellular Targeting to the Cell Nucleus of Quantum Dots Densely Decorated with a Nuclear Localization Sequence Peptide.

    PubMed

    Maity, Amit Ranjan; Stepensky, David

    2016-01-27

    development of subcellularly targeted DDSs that will deliver specific drugs to the nuclei of the target cells and will enhance efficacy and reduce toxicity of these drugs.

  9. Expression level and subcellular localization of heme oxygenase-1 modulates its cytoprotective properties in response to lung injury: a mouse model.

    PubMed

    Namba, Fumihiko; Go, Hayato; Murphy, Jennifer A; La, Ping; Yang, Guang; Sengupta, Shaon; Fernando, Amal P; Yohannes, Mekdes; Biswas, Chhanda; Wehrli, Suzanne L; Dennery, Phyllis A

    2014-01-01

    Premature infants exposed to hyperoxia suffer acute and long-term pulmonary consequences. Nevertheless, neonates survive hyperoxia better than adults. The factors contributing to neonatal hyperoxic tolerance are not fully elucidated. In contrast to adults, heme oxygenase (HO)-1, an endoplasmic reticulum (ER)-anchored protein, is abundant in the neonatal lung but is not inducible in response to hyperoxia. The latter may be important, because very high levels of HO-1 overexpression are associated with significant oxygen cytotoxicity in vitro. Also, in contrast to adults, HO-1 localizes to the nucleus in neonatal mice exposed to hyperoxia. To understand the mechanisms by which HO-1 expression levels and subcellular localization contribute to hyperoxic tolerance in neonates, lung-specific transgenic mice expressing high or low levels of full-length HO-1 (cytoplasmic, HO-1-FL(H) or HO-1-FL(L)) or C-terminally truncated HO-1 (nuclear, Nuc-HO-1-TR) were generated. In HO-1-FL(L), the lungs had a normal alveolar appearance and lesser oxidative damage after hyperoxic exposure. In contrast, in HO-1-FL(H), alveolar wall thickness with type II cell hyperproliferation was observed as well worsened pulmonary function and evidence of abnormal lung cell hyperproliferation in recovery from hyperoxia. In Nuc-HO-1-TR, the lungs had increased DNA oxidative damage, increased poly (ADP-ribose) polymerase (PARP) protein expression, and reduced poly (ADP-ribose) (PAR) hydrolysis as well as reduced pulmonary function in recovery from hyperoxia. These data indicate that low cytoplasmic HO-1 levels protect against hyperoxia-induced lung injury by attenuating oxidative stress, whereas high cytoplasmic HO-1 levels worsen lung injury by increasing proliferation and decreasing apoptosis of alveolar type II cells. Enhanced lung nuclear HO-1 levels impaired recovery from hyperoxic lung injury by disabling PAR-dependent regulation of DNA repair. Lastly both high cytoplasmic and nuclear expression of

  10. Analysis of the subcellular localization of the proteins Rep, Rep' and Cap of porcine circovirus type 1

    SciTech Connect

    Finsterbusch, T. . E-mail: finsterbuscht@rki.de; Steinfeldt, T.; Caliskan, R.; Mankertz, A.

    2005-12-05

    Porcine circovirus type 1 (PCV1) encodes two major ORFs. The cap gene comprises the major structural protein of PCV, the rep gene specifies Rep and Rep', which are both essential for initiating the replication of the viral DNA. Rep corresponds to the full-length protein, whereas Rep' is a truncated splice product that is frame-shifted in its C-terminal sequence. In this study, the cellular localization of PCV1-encoded proteins was investigated by immune fluorescence techniques using antibodies against Rep, Rep' and Cap and by expression of viral proteins fused to green and red fluorescence proteins. Rep and Rep' protein co-localized in the nucleus of infected cells as well as in cells transfected with plasmids expressing Rep and Rep' fused to fluorescence proteins, but no signal was seen in the nucleoli. Rep and Rep' carry three potential nuclear localization signals in their identical N-termini, and the contribution of these motifs to nuclear import was experimentally dissected. In contrast to the rep gene products, the localization of the Cap protein varied. While the Cap protein was restricted to the nucleoli in plasmid-transfected cells and was also localized in the nucleoli at an early stage of PCV1 infection, it was seen in the nucleoplasm and the cytoplasm later in infection, suggesting that a shuttling between distinct cellular compartments occurs.

  11. CRM1-dependent nuclear export and dimerization with hMSH5 contribute to the regulation of hMSH4 subcellular localization

    SciTech Connect

    Neyton, Sophie; Lespinasse, Francoise; Lahaye, Francois; Staccini, Pascal; Paquis-Flucklinger, Veronique; Santucci-Darmanin, Sabine

    2007-10-15

    MSH4 and MSH5 are members of the MutS homolog family, a conserved group of proteins involved in DNA mismatch correction and homologous recombination. Although several studies have provided compelling evidences suggesting that MSH4 and MSH5 could act together in early and late stages of meiotic recombination, their precise roles are poorly understood and recent findings suggest that the human MSH4 protein may also exert a cytoplasmic function. Here we show that MSH4 is present in the cytoplasm and the nucleus of both testicular cells and transfected somatic cells. Confocal studies on transfected cells provide the first evidence that the subcellular localization of MSH4 is regulated, at least in part, by an active nuclear export pathway dependent on the exportin CRM1. We used deletion mapping and mutagenesis to define two functional nuclear export sequences within the C-terminal part of hMSH4 that mediate nuclear export through the CRM1 pathway. Our results suggest that CRM1 is also involved in MSH5 nuclear export. In addition, we demonstrate that dimerization of MSH4 and MSH5 facilitates their nuclear localization suggesting that dimerization may regulate the intracellular trafficking of these proteins. Our findings suggest that nucleocytoplasmic traffic may constitute a regulatory mechanism for MSH4 and MSH5 functions.

  12. Control of Glucocorticoid and Progesterone Receptor Subcellular Localization by the Ligand-binding Domain is Mediated by Distinct Interactions with Tetratricopeptide Repeat Proteins

    PubMed Central

    Banerjee, Ananya; Periyasamy, Sumudra; Wolf, Irene M.; Hinds, Terry; Yong, Weidong; Shou, Weinian; Sanchez, Edwin R.

    2011-01-01

    The TPR proteins FKBP52, FKBP51, Cyp40 and PP5 are found in steroid receptor (SR) complexes but their receptor-specific preferences and roles remain unresolved. We have undertaken a systematic approach to this problem by examining the contribution of all four TPRs to the localization properties of glucocorticoid (GR) and progesterone (PR) receptors. The GR of L929 cells was found in the cytoplasm in a complex containing PP5 and FKBP51, while GR of WCL2 cells was nuclear and contained PP5 and FKBP52. Cyp40 did not interact with GR in either cell line. To test whether FKBP interaction determined localization, we over-expressed Flag-tagged FKBP51 in WCL2 cells and Flag-FKBP52 in L929 cells. In WCL2 cells, GR showed a shift to greater cytoplasmic localization that correlated with recruitment of Flag-FKBP51. In contrast, Flag-FKBP52 was not recruited to GR of L929 cells and no change in localization was observed, suggesting that both cell-type specific mechanisms and TPR abundance contribute to the SR/TPR interaction. As a further test, GR-GFP and PR-GFP constructs were expressed in COS cells. GR-GFP localized to the cytoplasm, while PR-GFP was predominantly nuclear. Similar to L929 cells, GR in COS interacted with PP5 and FKBP51, while PR interacted with FKBP52. Analysis of GR/PR chimeric constructs revealed that the ligand-binding domain of each receptor determines both TPR specificity and localization. Lastly, we analyzed GR and PR localization in cells completely lacking TPR. PR in FKBP52 KO cells showed a complete shift to the cytoplasm, while GR in FKBP51 KO and PP5 KO cells showed a moderate shift to the nucleus, indicating that both TPRs contribute to GR localization. Our results demonstrate that SRs have distinct preferences for TPR proteins – a property that resides in the LBD and which can now explain long-standing differences in receptor subcellular localization. PMID:18771283

  13. Effects of prostaglandin E{sub 2} on the subcellular localization of Epac-1 and Rap1 proteins during Fc{gamma}-receptor-mediated phagocytosis in alveolar macrophages

    SciTech Connect

    Brock, Thomas G.; Serezani, Carlos H.; Carstens, Jennifer K.; Peters-Golden, Marc; Aronoff, David M.

    2008-01-15

    Recent studies have demonstrated a central role for the exchange protein activated by cAMP (Epac) in the inhibition of Fc{gamma}-receptor-mediated phagocytosis and bacterial killing by prostaglandin E{sub 2} (PGE{sub 2}) in macrophages. However, the subcellular localization of Epac, and its primary target Rap1, has yet to be determined in primary macrophages. Therefore, we used immunofluorescent techniques and phagosome isolation to localize Epac-1 and Rap1 in alveolar macrophages. Epac-1 was predominantly expressed on punctate and tubular membranes throughout the cell body; on the plasma membrane; and co-localized with microtubule organizing centers (MTOCs). Rap1 was abundant on punctate membranes, less abundant on plasma membrane, and also found on MTOCs. Following PGE{sub 2} treatment, Epac-1, but not Rap1, accumulated on the nuclear envelope and disappeared from MTOCs. By immunofluorescent microscopy, both Epac-1 and Rap1 were seen to associate with phagosomes containing IgG-opsonized beads, but this association appeared weak, as we failed to observe such interactions in phagosomes isolated from cells at various time points after bead ingestion. Strikingly, however, Epac-1, but not Rap1, appeared to accumulate on maturing phagosomes, but only after PGE{sub 2} treatment (or treatment with a selective Epac-1 agonist). This association was confirmed in isolated phagosome preparations. The changes in Epac-1 localization were too slow to account for the inhibitory effects of PGE{sub 2} on phagocytosis. However, the appearance of Epac-1 on late phagosomes following PGE{sub 2} treatment might be important for suppressing H{sub 2}O{sub 2} production and inhibiting the killing of intraphagosomal pathogens. The absence of Rap1 on late phagosomes suggests that the effect of Epac-1 might not require Rap1.

  14. Subcellular localizations of Arabidopsis myotubularins MTM1 and MTM2 suggest possible functions in vesicular trafficking between ER and cis-Golgi.

    PubMed

    Nagpal, Akanksha; Ndamukong, Ivan; Hassan, Ammar; Avramova, Zoya; Baluška, František

    2016-08-01

    The two Arabidopsis genes AtMTM1 and AtMTM2 encode highly similar phosphoinositide 3-phosphatases from the myotubularin family. Despite the high-level conservation of structure and biochemical activities, their physiological roles have significantly diverged. The nature of a membrane and the concentrations of their membrane-anchored substrates (PtdIns3P or PtdIns3,5P2) and/or products (PtdIns5P and PtdIns) are considered critical for determining the functional specificity of myotubularins. We have performed comprehensive analyses of the subcellular localization of AtMTM1 and AtMTM2 using a variety of specific constructs transiently expressed in Nicotiana benthamiana leaf epidermal cells under the control of 35S promoter. AtMTM1 co-localized preferentially with cis-Golgi membranes, while AtMTM2 associated predominantly with ER membranes. In a stark contrast with animal/human MTMs, neither AtMTM1 nor AtMTM2 co-localizes with early or late endosomes or with TGN/EE compartments, making them unlikely participants in the endosomal trafficking system. Localization of the AtMTM2 is sensitive to cold and osmotic stress challenges. In contrast to animal myotubularins, Arabidopsis myotubularins do not associate with endosomes. Our results suggest that Arabidopsis myotubularins play a role in the vesicular trafficking between ER exit sites and cis-Golgi elements. The significance of these results is discussed also in the context of stress biology and plant autophagy.

  15. Population imaging at subcellular resolution supports specific and local inhibition by granule cells in the olfactory bulb

    PubMed Central

    Wienisch, Martin; Murthy, Venkatesh N.

    2016-01-01

    Information processing in early sensory regions is modulated by a diverse range of inhibitory interneurons. We sought to elucidate the role of olfactory bulb interneurons called granule cells (GCs) in odor processing by imaging the activity of hundreds of these cells simultaneously in mice. Odor responses in GCs were temporally diverse and spatially disperse, with some degree of non-random, modular organization. The overall sparseness of activation of GCs was highly correlated with the extent of glomerular activation by odor stimuli. Increasing concentrations of single odorants led to proportionately larger population activity, but some individual GCs had non-monotonic relations to concentration due to local inhibitory interactions. Individual dendritic segments could sometimes respond independently to odors, revealing their capacity for compartmentalized signaling in vivo. Collectively, the response properties of GCs point to their role in specific and local processing, rather than global operations such as response normalization proposed for other interneurons. PMID:27388949

  16. Phosphorylation and subcellular localization of p27Kip1 regulated by hydrogen peroxide modulation in cancer cells.

    PubMed

    Ibañez, Irene L; Bracalente, Candelaria; Notcovich, Cintia; Tropper, Ivanna; Molinari, Beatriz L; Policastro, Lucía L; Durán, Hebe

    2012-01-01

    The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2)O(2)) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2)O(2) removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2)O(2) (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2)O(2) scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27

  17. Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells

    PubMed Central

    Ibañez, Irene L.; Bracalente, Candelaria; Notcovich, Cintia; Tropper, Ivanna; Molinari, Beatriz L.; Policastro, Lucía L.; Durán, Hebe

    2012-01-01

    The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H2O2) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H2O2 (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H2O2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1. PMID

  18. Subcellular localization and clues for the function of the HetN factor influencing heterocyst distribution in Anabaena sp. strain PCC 7120.

    PubMed

    Corrales-Guerrero, Laura; Mariscal, Vicente; Nürnberg, Dennis J; Elhai, Jeff; Mullineaux, Conrad W; Flores, Enrique; Herrero, Antonia

    2014-10-01

    In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, heterocysts are formed in the absence of combined nitrogen, following a specific distribution pattern along the filament. The PatS and HetN factors contribute to the heterocyst pattern by inhibiting the formation of consecutive heterocysts. Thus, inactivation of any of these factors produces the multiple contiguous heterocyst (Mch) phenotype. Upon N stepdown, a HetN protein with its C terminus fused to a superfolder version of green fluorescent protein (sf-GFP) or to GFP-mut2 was observed, localized first throughout the whole area of differentiating cells and later specifically on the peripheries and in the polar regions of mature heterocysts, coinciding with the location of the thylakoids. Polar localization required an N-terminal stretch comprising residues 2 to 27 that may represent an unconventional signal peptide. Anabaena strains expressing a version of HetN lacking this fragment from a mutant gene placed at the native hetN locus exhibited a mild Mch phenotype. In agreement with previous results, deletion of an internal ERGSGR sequence, which is identical to the C-terminal sequence of PatS, also led to the Mch phenotype. The subcellular localization in heterocysts of fluorescence resulting from the fusion of GFP to the C terminus of HetN suggests that a full HetN protein is present in these cells. Furthermore, the full HetN protein is more conserved among cyanobacteria than the internal ERGSGR sequence. These observations suggest that HetN anchored to thylakoid membranes in heterocysts may serve a function besides that of generating a regulatory (ERGSGR) peptide.

  19. Subcellular localization of caspase-3 activation correlates with changes in apoptotic morphology in MOLT-4 leukemia cells exposed to X-ray irradiation.

    PubMed

    Feng, Yongdong; Hu, Junbo; Xie, Daxin; Qin, Jichao; Zhong, Yisheng; Li, Xiaolan; Xiao, Wei; Wu, Jianhong; Tao, Deding; Zhang, Manchao; Zhu, Yunfeng; Song, Yuping; Reed, Eddie; Li, Qingdi Q; Gong, Jianping

    2005-09-01

    Caspase-3 is a critical effector caspase for apoptosis, which cleaves proteins, including cytoskeletal and associated proteins, kinases, and members of the Bcl-2 family of apoptosis-related proteins. This leads to changes in apoptotic morphology, such as membrane externalization and cytoplasm and nuclear condensation. It has been reported that pro-caspase-3 is activated in the cytosol. However, it remains obscure how caspase-3 activation correlates to serial changes in cell morphology during apoptosis. The current study was therefore undertaken to assess the relationship between caspase-3 activation and its subcellular localization and alterations in apoptotic morphology in MOLT-4 human leukemia cells exposed to X-ray irradiation. Fluorescence labeled inhibitor of caspases (FLICA) was used to detect caspase-3 activity in apoptotic cells in this project; cell morphology and caspase-3 sub-localization were determined by confocal microscopy. Our data showed that MOLT-4 cells presented typical morphological changes in apoptosis, such as membrane reversion, DNA fragmentation, and formation of apoptotic cell bodies following 10 Gray (Gy) of X-ray irradiation. Caspase-3 was activated 2 h after X-ray irradiation, and its activity increased markedly after 4-6-h exposure. Membrane reversion in MOLT-4 leukemia cells was detected by Annexin V assay at 4 h following X-ray irradiation, 2 h after the elevated caspase-3 activity was measured. Cytologically, activation of caspase-3 was first observed close to the inside surface of the cellular membrane, then transferred to the cytoplasm, and finally translocated to the nuclear region. We conclude that caspase-3 is activated in MOLT-4 cells following exposure to X-rays, and that the enhanced caspase-3 activity and its sub-localization shifting is correlated to changes in apoptotic morphology. The spatial shift of activated caspase-3 in X-ray-induced apoptotic MOLT-4 leukemia cells is a process of crucial importance for apoptosis.

  20. Subcellular localization of proline-rich tyrosine kinase 2 during oocyte fertilization and early-embryo development in mice

    PubMed Central

    MENG, Xiao-qian; DAI, Yuan-yuan; JING, Lai-dong; BAI, Jing; LIU, Shu-zhen; ZHENG, Ke-gang; PAN, Jie

    2016-01-01

    Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase, is a member of the focal adhesion kinase family and is highly expressed in oocytes. Using a combination of confocal microscopy and RNAi, we localized and studied the function of both Pyk2 and tyrosine-phosphorylated Pyk2 (p-Pyk2) during mouse oocyte fertilization and early embryo development. At the onset of fertilization, Pyk2 and p-Pyk2 were detected predominantly in sperm heads and the oocyte cytoplasm. Upon formation of male and female pronuclei, Pyk2 and its activated form leave the cytoplasm and accumulate in the two pronuclei. We detected Pyk2 in blastomere nuclei and found both Pyk2 and p-Pyk2 in the pre-blastula cytoplasm. Pyk2 and its activated form then disappeared from the blastula nuclei and localized to the perinuclear regions, where blastula cells come into contact with each other. Pyk2 knockdown via microinjection of siRNA into the zygote did not inhibit early embryo development. Our results suggest that Pyk2 plays multiple functional roles in mouse oocyte fertilization as well as throughout early embryo development. PMID:27086609

  1. Subcellular localization of proteins in the anaerobic sulfate reducer Desulfovibrio vulgaris via SNAP-tag labeling and photoconversion

    SciTech Connect

    Gorur, A.; Leung, C. M.; Jorgens, D.; Tauscher, A.; Remis, J. P.; Ball, D. A.; Chhabra, S.; Fok, V.; Geller, J. T.; Singer, M.; Hazen, T. C.; Juba, T.; Elias, D.; Wall, J.; Biggin, M.; Downing, K. H.; Auer, M.

    2010-06-01

    Systems Biology studies the temporal and spatial 3D distribution of macromolecular complexes with the aim that such knowledge will allow more accurate modeling of biological function and will allow mathematical prediction of cellular behavior. However, in order to accomplish accurate modeling precise knowledge of spatial 3D organization and distribution inside cells is necessary. And while a number of macromolecular complexes may be identified by its 3D structure and molecular characteristics alone, the overwhelming number of proteins will need to be localized using a reporter tag. GFP and its derivatives (XFPs) have been traditionally employed for subcelllar localization using photoconversion approaches, but this approach cannot be taken for obligate anaerobic bacteria, where the intolerance towards oxygen prevents XFP approaches. As part of the GTL-funded PCAP project (now ENIGMA) genetic tools have been developed for the anaerobe sulfate reducer Desulfovibrio vulgaris that allow the high-throughput generation of tagged-protein mutant strains, with a focus on the commercially available SNAP-tag cell system (New England Biolabs, Ipswich, MA), which is based on a modified O6-alkylguanine-DNA alkyltransferase (AGT) tag, that has a dead-end reaction with a modified O6-benzylguanine (BG) derivative and has been shown to function under anaerobic conditions. After initial challenges with respect to variability, robustness and specificity of the labeling signal we have optimized the labeling. Over the last year, as a result of the optimized labeling protocol, we now obtain robust labeling of 20 out of 31 SNAP strains. Labeling for 13 strains were confirmed at least five times. We have also successfully performed photoconversion on 5 of these 13 strains, with distinct labeling patterns for different strains. For example, DsrC robustly localizes to the periplasmic portion of the inner membrane, where as a DNA-binding protein localizes to the center of the cell, where the

  2. Establishing the subcellular localization of photodynamically-induced ROS using 3,3'-diaminobenzidine: A methodological proposal, with a proof-of-concept demonstration.

    PubMed

    Stockert, Juan C; Blázquez-Castro, Alfonso

    2016-10-15

    The critical involvement of reactive oxygen species (ROS) in both physiological and pathological processes in cell biology makes their detection and assessment a fundamental topic in biomedical research. Established methodologies to study ROS in cell biology take advantage of oxidation reactions between the ROS and a reduced probe. After reacting the probe reveals the presence of ROS either by the appearance of colour (chromogenic reaction) or fluorescence (fluorogenic reaction). However current methodologies rarely allow for a site-specific detection of ROS production. Here we propose a colorimetric reaction driven by the oxidation of 3,3'-diaminobenzidine (DAB) by photodynamically-produced ROS that allows for fine detection of the ROS production site. The introduced methodology is fast, easy to implement and permits cellular resolution at the submicrometric level. Although the basic protocol is proved in a photodynamic model of ROS generation, the principle is applicable to many different scenarios of intracellular ROS production. As a consequence this proposed methodology should greatly complement other techniques aiming at establishing a precise subcellular localization of ROS generation.

  3. Investigation of subcellular localization and dynamics of membrane proteins in living bacteria by combining optical micromanipulation and high-resolution microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Barroso Peña, Álvaro; Nieves, Marcos; Teper, Konrad; Wedlich-Soldner, Roland; Denz, Cornelia

    2016-09-01

    The plasma membrane serves as protective interface between cells and their environment. It also constitutes a hub for selective nutrient uptake and signal transduction. Increasing evidence over the last years indicates that, similar to eukaryotic cells, lateral membrane organization plays an important role in the regulation of prokaryotic signaling pathways. However, the mechanisms underlying this phenomenon are still poorly understood. Spatiotemporal characterization of bacterial signal transduction demands very sensitive high-resolution microscopy techniques due to the low expression levels of most signaling proteins and the small size of bacterial cells. In addition, direct study of subcellular confinement and dynamics of bacterial signaling proteins during the different stages of the signal transduction also requires immobilization in order to avoid cell displacement caused by Brownian motion, local fluid flows and bacterial self-propulsion. In this work we present a novel approach based on the combination of high resolution imaging and optical manipulation that enables the investigation of the distribution and dynamics of proteins at the bacterial plasma membrane. For this purpose, we combine the versatility of holographic optical tweezers (HOT) with the sensitivity and resolution of total internal reflection fluorescence (TIRF) microscopy. Furthermore, we discuss the implementation of microfluidic devices in our integrated HOT+TIRF system for the control of growth conditions of bacterial cells. The capabilities of our workstation provides thus new valuable insights into the fundamental cellular and physical mechanisms underlying the regulation of bacterial signal transduction.

  4. Static magnetic fields inhibit proliferation and disperse subcellular localization of gamma complex protein3 in cultured C2C12 myoblast cells.

    PubMed

    Kim, SeungChan; Im, Wooseok

    2010-05-01

    Magnetic fields may delay the rate of cell cycle progression, and there are reports that magnetic fields induce neurite outgrowth in cultured neuronal cells. To demonstrate whether magnetic field also effects on myoblast cells in cell growth, C2C12 cell lines were cultured and 2000G static magnetic field was applied. After 48 h of incubation, both the WST-1 assay (0.01 < P < 0.025, t-test) and direct cell counting (P < 0.0005, t-test) showed that static magnetic fields inhibit the proliferation of cultured C2C12 cells. Immunocytochemistry for alpha and tubulin gamma complex protein (TUBA and GCP3) was made and applying a static magnetic field-dispersed tubulin GCP3 formation, a intracellular apparatus for tubulin structuring in cell division. This protein expression was not altered by western blot. This study indicates that applying a static magnetic field alters the subcellular localizing of GCP3, and may delay the cell growth in cultured C2C12 myoblast cells.

  5. Subcellular localization of the human papillomavirus 16 E7 oncoprotein in CaSki cells and its detection in cervical adenocarcinoma and adenocarcinoma in situ.

    PubMed

    Dreier, Kerstin; Scheiden, René; Lener, Barbara; Ehehalt, Daniela; Pircher, Haymo; Müller-Holzner, Elisabeth; Rostek, Ursula; Kaiser, Andreas; Fiedler, Marc; Ressler, Sigrun; Lechner, Stefan; Widschwendter, Andreas; Even, Jos; Capesius, Catherine; Jansen-Dürr, Pidder; Zwerschke, Werner

    2011-01-05

    E7 is the major oncoprotein of high-risk human papillomaviruses (HPV) which causes cervical cancer. To date E7 oncoproteins have not been investigated in cervical adenocarcinoma. In this study we generated a rabbit monoclonal anti-HPV-16 E7 antibody, RabMab42-3, which recognizes a conformational epitope in the E7 carboxy-terminal zinc-finger resulting in a strong increase in the sensitivity for the detection of cell-associated HPV-16 E7 protein relative to conventional polyclonal anti-HPV-16 E7 antibodies. Using RabMab42-3, we show that the subcellular localization of endogenous HPV-16 E7 oncoprotein varies during the cell cycle in cervical cancer cells. Moreover, we demonstrate for the first time that the HPV-16 E7 oncoprotein is abundantly expressed in cervical adenocarcinoma in situ and adenocarcinoma, suggesting an important role of HPV-16 E7 for the development of these tumors. Our findings suggest that the HPV-16 E7 oncoprotein could be a useful marker for the detection of cervical adenocarcinoma and their precursors.

  6. Subcellular localization of the inositol 1,4,5-trisphosphate receptor, P400, in the vestibular complex and dorsal cochlear nucleus of the rat.

    PubMed

    Rodrigo, J; Uttenthal, O; Bentura, M L; Maeda, N; Mikoshiba, K; Martinez-Murillo, R; Polak, J M

    1994-01-21

    The subcellular localization of the inositol 1,4,5-trisphosphate receptor protein, P400, was studied in the vestibular complex, an area to which Purkinje cells project, as well as in neurons of the dorsal cochlear nucleus and in ectopic Purkinje cells of adult rat brain. The receptor was demonstrated by electron microscopical immunocytochemistry using the avidin-biotin peroxidase complex procedure, with the monoclonal antibody 4C11 raised against mouse cerebellar inositol 1,4,5-trisphosphate receptor protein. Immunoreactivity was found in preterminal fibres and terminal boutons in the nuclei of the vestibular complex, generally associated with the subsurface systems and stacks or fragments of smooth endoplasmic reticulum. Ectopic Purkinje cells and cartwheel cells of the dorsal cochlear nucleus also displayed immunoreactivity, but this was much less intense in the latter. The results of the present study suggest that this receptor protein, involved in the release of Ca2+, is located in sites that enable it to influence the synthesis, transport and release of neurotransmitters.

  7. cDNA cloning and characterization of Npap60: a novel rat nuclear pore-associated protein with an unusual subcellular localization during male germ cell differentiation.

    PubMed

    Fan, F; Liu, C P; Korobova, O; Heyting, C; Offenberg, H H; Trump, G; Arnheim, N

    1997-03-15

    We have cloned and characterized a cDNA, Npap60, encoding a rat nuclear pore-associated protein. The 3-kb cDNA was obtained by antibody screening of a rat testis expression library. The predicted NPAP60 contains 381 amino acids with a composition of 25.6% charged residues and is highly hydrophilic. The Npap60 gene appears to be conserved in mouse, rat, and human. Immunofluorescence studies with anti-NPAP60 fusion protein antibody show that the NPAP60 protein colocalizes with nuclear pore complexes in RAT1A cells. The expression of Npap60 is about 10-20 times higher in rat testis than in somatic tissues. The subcellular localization of NPAP60 protein changes dramatically during male germ cell differentiation, from nuclear pore complex-like staining in spermatocytes to whole nucleus staining in spermatids and finally to a nuclear surface staining in mature spermatozoa. These changes are temporally and spatially related to nuclear reorganization during male germ cell differentiation.

  8. Subcellular localization of neptunium-237 in lung and kidney after intratracheal administration in the rat: an ultrastructural and microanalytical study.

    PubMed

    Boulahdour, H; Poncy, J L; Berry, J P; Galle, P

    1996-12-01

    Chronic intratracheal administration of 237Np to rate was performed during 6 weeks. The total dose administered was 45.8 kBq. Two methods, electron microscopy and electron probe X-ray microanalysis, were used to determine the intracellular sites of localization of 237Np. Clusters of dense granules were observed in nuclei of pneumocytes and proximal tubular cells of the kidneys. These clusters have been shown to contain neptunium associated with phosphorus, sulfur and calcium. Alterations of nuclei and ultrastructural cytoplasmic lesions were observed. The absorbed doses in lungs and kidneys were very low. These results suggest that the chemical toxicity of 237Np is more important than its radiological toxicity.

  9. Cloning, Functional Expression, and Subcellular Localization of Multiple NADPH-Cytochrome P450 Reductases from Hybrid Poplar1

    PubMed Central

    Ro, Dae-Kyun; Ehlting, Jürgen; Douglas, Carl J.

    2002-01-01

    NADPH:cytochrome P450 reductase (CPR) provides reducing equivalents to diverse cytochrome P450 monooxygenases. We isolated cDNAs for three CPR genes (CPR1, CPR2, and CPR3) from hybrid poplar (Populus trichocarpa × Populus deltoides). Deduced CPR2 and CPR3 amino acid sequences were 91% identical, but encoded isoforms divergent from CPR1 (72% identity). CPR1 and CPR2 were co-expressed together with the P450 enzyme cinnamate-4-hydroxylase (C4H) in yeast (Saccharomyces cerevisiae). Microsomes isolated from strains expressing CPR1/C4H or CPR2/C4H enhanced C4H activities approximately 10-fold relative to the C4H-only control strain, and catalyzed NADPH-dependent cytochrome c reduction. The divergent CPR isoforms (CPR1 and CPR2/3) contained entirely different N-terminal sequences, which are conserved in other plant CPRs and are diagnostic for two distinct classes of CPRs within the angiosperms. C-terminal green fluorescent protein fusions to CPR1 and CPR2 were constructed and expressed in both yeast and Arabidopsis. The fusion proteins expressed in yeast retained the ability to support C4H activity and, thus, were catalytically active. Both CPR::green fluorescent protein fusion proteins were strictly localized to the endoplasmic reticulum in transgenic Arabidopsis. The lack of localization of either isoform to chloroplasts, where P450s are known to be present, suggests that an alternative P450 reduction system may be operative in this organelle. Transcripts for the three poplar CPR genes were present ubiquitously in all tissues examined, but CPR2 showed highest expression in young leaf tissue. PMID:12481067

  10. Molecular cloning, expression analysis and subcellular localization of a Transparent Testa 12 ortholog in brown cotton (Gossypium hirsutum L.).

    PubMed

    Gao, Jun-Shan; Wu, Nan; Shen, Zhi-Lin; Lv, Kai; Qian, Sen-He; Guo, Ning; Sun, Xu; Cai, Yong-Ping; Lin, Yi

    2016-02-01

    Transparent Testa 12 (TT12) is a kind of transmembrane transporter of proanthocyanidins (PAs), which belongs to a membrane-localized multidrug and toxin efflux (MATE) family, but the molecular basis of PAs transport is still poorly understood. Here, we cloned a full-length TT12 cDNA from the fiber of brown cotton (Gossypium hirsutum), named GhTT12 (GenBank accession No. KF240564), which comprised 1733 bp with an open reading frame (ORF) of 1503 bp and encoded a putative protein containing 500 amino acid residues with a typical MATE conserved domain. The GhTT12 gene had 96.8% similarity to AA genome in Gossypium arboretum. Quantitative RT-PCR analysis denoted that the relative expression of GhTT12 in brown cotton was 1-5 folds higher than that in white cotton. The mRNA level was the highest at 5 days post anthesis (DPA) and reduced gradually during the fiber development. Expressing GhTT12-fused green fluorescent protein (GFP) in Nicotiana tabacum showed that GhTT12-GFP was localized in the vacuole membrane. The content of PAs increased firstly and decreased afterwards, and reached the maximum at 15 DPA in brown cotton. But for white cotton, the content of PAs remained at a low level during the fiber development. We speculate that GhTT12 may participate in the transportation of PAs from the cytoplasmic matrix to the vacuole. Taken together, our data revealed that GhTT12 was functional as a PAs transmembrane transporter.

  11. Cellular and Subcellular Localization of the RGS7/Gβ5/R7BP Complex in the Cerebellar Cortex

    PubMed Central

    Aguado, Carolina; Orlandi, Cesare; Fajardo-Serrano, Ana; Gil-Minguez, Mercedes; Martemyanov, Kirill A.; Luján, Rafael

    2016-01-01

    A member of regulator of G-protein signaling family, RGS7, is an essential modulator of signaling through GABAB receptors. RGS7 functions as a macromolecular complex with type 5 G protein β (Gβ5) and R7 binding protein (R7BP) to control the localization and function of the resultant heterotrimeric complexes. Here, we used co-immunoprecipitation, in situ hybridization, histoblot and immunohistochemical techniques at the light and electron microscopic level to advance understanding of RGS7-Gβ5-R7BP complexes in the central nervous system, focusing on distinct neuronal populations in the cerebellar cortex. Histoblot analysis showed that RGS7, Gβ5 and R7BP proteins were widely expressed in the brain, with mostly an overlapping pattern and showing a high expression level in the molecular layer of the cerebellar cortex. Co-immunoprecipitation experiments established that the RGS7/Gβ5 forms complexes with R7BP in the cerebellum. At the cellular level, RGS7 and R7BP mRNAs were expressed at the highest level in Purkinje cells (PCs) and Golgi cells, and at low levels in granule cells. Immunohistochemistry confirmed that labeling for RGS7, Gβ5 and R7BP were present in the three neuronal populations and concentrated in dendrites and spines. At the electron microscopic level, immunolabeling for RGS7, Gβ5 and R7BP proteins was found both at postsynaptic and presynaptic sites and showed similar distribution patterns. Immunoreactivity for the three proteins was mostly localized along the extrasynaptic plasma membrane of dendritic shafts and spines of PCs and to a lesser extent, in axon terminals (AT) establishing excitatory synapses. Quantitative analysis of immunogold particles for RGS7, Gβ5 and R7BP revealed that they are non-uniformly distributed along the surface of PCs, and show enrichment around excitatory synapses on dendritic spines. We further report that deletion of R7BP in mice reduced the targeting of both RGS7 and Gβ5 to the plasma membrane. Altogether, these

  12. Identification, subcellular localization, biochemical properties, and high-resolution crystal structure of Trypanosoma brucei UDP-glucose pyrophosphorylase

    PubMed Central

    Mariño, Karina; Güther, Maria Lucia Sampaio; Wernimont, Amy K; Amani, Mernhaz; Hui, Raymond; Ferguson, Michael AJ

    2010-01-01

    The protozoan parasite Trypanosoma brucei is the causative agent of the cattle disease Nagana and human African sleeping sickness. Glycoproteins play key roles in the parasite’s survival and infectivity, and the de novo biosyntheses of the sugar nucleotides UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine, and GDP-fucose have been shown to be essential for their growth. The only route to UDP-Gal in T. brucei is through the epimerization of UDP-glucose (UDP-Glc) by UDP-Glc 4′-epimerase. UDP-Glc is also the glucosyl donor for the unfolded glycoprotein glucosyltransferase (UGGT) involved in glycoprotein quality control in the endoplasmic reticulum and is the presumed donor for the synthesis of base J (β-d-glucosylhydroxymethyluracil), a rare deoxynucleotide found in telomere-proximal DNA in the bloodstream form of T. brucei. Considering that UDP-Glc plays such a central role in carbohydrate metabolism, we decided to characterize UDP-Glc biosynthesis in T. brucei. We identified and characterized the parasite UDP-glucose pyrophosphorylase (TbUGP), responsible for the formation of UDP-Glc from glucose-1-phosphate and UTP, and localized the enzyme to the peroxisome-like glycosome organelles of the parasite. Recombinant TbUGP was shown to be enzymatically active and specific for glucose-1-phosphate. The high-resolution crystal structure was also solved, providing a framework for the design of potential inhibitors against the parasite enzyme. PMID:20724435

  13. Spastin subcellular localization is regulated through usage of different translation start sites and active export from the nucleus

    SciTech Connect

    Claudiani, Pamela; Riano, Elena; Errico, Alessia; Andolfi, Gennaro; Rugarli, Elena I. . E-mail: rugarli@tigem.it

    2005-10-01

    Most cases of autosomal-dominant hereditary spastic paraplegia are linked to mutations in SPG4 encoding spastin, a protein involved in microtubule dynamics and membrane trafficking. In pyramidal neurons of the motor cortex and in immortalized motor neurons, spastin is localized to the synaptic terminals and growth cones. However, in other neurons and in proliferating cells spastin is prevalently nuclear. The mechanisms that determine targeting of spastin to the nucleus or the cytoplasm are unknown. We show here that the SPG4 mRNA is able to direct synthesis of two spastin isoforms, 68 and 60 kDa, respectively, through usage of two different translational start sites. Both isoforms are imported into the nucleus, but the 68-kDa isoform contains two nuclear export signals that efficiently drive export to the cytoplasm. Nuclear export is leptomycin-B sensitive. The cytoplasmic 68-kDa spastin isoform is more abundant in the brain and the spinal cord than in other tissues. Our data indicate that spastin function is modulated through usage of alternative translational start sites and active nuclear import and export, and open new perspectives for the pathogenesis of hereditary spastic paraplegia.

  14. Localization of polyamine enhancement of protein synthesis to subcellular components of Escherichia coli and Pseudomonas sp. strain Kim.

    PubMed Central

    Rosano, C L; Bunce, S C; Hurwitz, C

    1983-01-01

    At 5 mM Mg2+, spermidine stimulation of polyphenylalanine synthesis by cell-free extracts of Escherichia coli was found to be about 30 times greater than that by extracts of Pseudomonas sp. strain Kim, a unique organism which lacks detectable levels of spermidine. By means of reconstitution experiments, the target of spermidine stimulation was localized to the protein fraction of the highspeed supernatant component (S-100) of E. coli and was absent from, or deficient in, the S-100 fraction of Pseudomonas sp. strain Kim. The spermidine stimulation did not appear to be due to the presence in the E. coli S-100 fraction of ribosomal protein S1, elongation factors, or E. coli aminoacyl-tRNA synthetases. The failure to observe spermidine stimulation by the Pseudomonas sp. strain Kim S-100 fraction was also not due to a spermidine-enhanced polyuridylic acid degradation. The synthesis of polyphenylalanine by Pseudomonas sp. strain Kim extracts was stimulated by putrescine and by S-(+)-2-hydroxyputrescine to a greater degree than was synthesis by E. coli extracts. The enhancement by putrescine and by S-(+)-2-hydroxyputrescine with Pseudomonas sp. strain Kim extracts was found to be due to effects on its ribosomes. PMID:6336736

  15. Aspergillus nidulans VeA subcellular localization is dependent on the importin alpha carrier and on light.

    PubMed

    Stinnett, Suzanne M; Espeso, Eduardo A; Cobeño, Laura; Araújo-Bazán, Lidia; Calvo, Ana M

    2007-01-01

    The veA gene is a light-dependent regulator governing development and secondary metabolism in Aspergillus nidulans. We have identified a putative bipartite nuclear localization signal (NLS) motif in the A. nidulans VeA amino acid sequence and demonstrated its functionality when expressed in yeast. Furthermore, migration of VeA to the nucleus was dependent on the importin alpha. This bipartite NLS is also functional when VeA is expressed in A. nidulans. Interestingly, we found that VeA migration to the nucleus is light-dependent. While in the dark VeA is located mainly in the nuclei, under light VeA is found abundantly in the cytoplasm. The VeA1 mutant protein (lacking the first 36 amino acids at the N-terminus) was found predominantly in the cytoplasm independent of illumination. This indicates that the truncated bipartite NLS in VeA1 is not functional and fails to respond to light. These results might explain the lack of the morphological light-dependent response in strains carrying the veA1 allele. We also evaluated the effect of light on production of the mycotoxin sterigmatocystin in a veA wild-type and the veA1 mutant strains and found that the highest amount of toxin was produced by the veA+ strain growing in the dark, condition favouring accumulation of VeA in the nucleus.

  16. Subcellular localization, interactions and dynamics of the phage-shock protein-like Lia response in Bacillus subtilis.

    PubMed

    Domínguez-Escobar, Julia; Wolf, Diana; Fritz, Georg; Höfler, Carolin; Wedlich-Söldner, Roland; Mascher, Thorsten

    2014-05-01

    The liaIH operon of Bacillus subtilis is the main target of the envelope stress-inducible two-component system LiaRS. Here, we studied the localization, interaction and cellular dynamics of Lia proteins to gain insights into the physiological role of the Lia response. We demonstrate that LiaI serves as the membrane anchor for the phage-shock protein A homologue LiaH. Under non-inducing conditions, LiaI locates in highly motile membrane-associated foci, while LiaH is dispersed throughout the cytoplasm. Under stress conditions, both proteins are strongly induced and colocalize in numerous distinct static spots at the cytoplasmic membrane. This behaviour is independent of MreB and does also not correlate with the stalling of the cell wall biosynthesis machinery upon antibiotic inhibition. It can be induced by antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis, while compounds that inhibit the cytoplasmic or extracytoplasmic steps do not trigger this response. Taken together, our data are consistent with a model in which the Lia system scans the cytoplasmic membrane for envelope perturbations. Upon their detection, LiaS activates the cognate response regulator LiaR, which in turn strongly induces the liaIH operon. Simultaneously, LiaI recruits LiaH to the membrane, presumably to protect the envelope and counteract the antibiotic-induced damage.

  17. Protein kinase C gamma interneurons in the rat medullary dorsal horn: distribution and synaptic inputs to these neurons, and subcellular localization of the enzyme.

    PubMed

    Peirs, Cédric; Patil, Sudarshan; Bouali-Benazzouz, Rabia; Artola, Alain; Landry, Marc; Dallel, Radhouane

    2014-02-01

    The γ isoform of protein kinase C (PKCγ), which is concentrated in interneurons in the inner part of lamina II (IIi ) of the dorsal horn, has been implicated in the expression of tactile allodynia. Lamina IIi PKCγ interneurons were shown to be activated by tactile inputs and to participate in local circuits through which these inputs can reach lamina I, nociceptive output neurons. That such local circuits are gated by glycinergic inhibition and that A- and C-fibers low threshold mechanoreceptors (LTMRs) terminate in lamina IIi raise the general issue of synaptic inputs to lamina IIi PKCγ interneurons. Combining light and electron microscopic immunochemistry in the rat spinal trigeminal nucleus, we show that PKCγ-immunoreactivity is mostly restricted to interneurons in lamina IIi of the medullary dorsal horn, where they constitute 1/3 of total neurons. The majority of synapses on PKCγ-immunoreactive interneurons are asymmetric (likely excitatory). PKCγ-immunoreactive interneurons appear to receive exclusively myelinated primary afferents in type II synaptic glomeruli. Neither large dense core vesicle terminals nor type I synaptic glomeruli, assumed to be the endings of unmyelinated nociceptive terminals, were found on these interneurons. Moreover, there is no vesicular glutamate transporter 3-immunoreactive bouton, specific to C-LTMRs, on PKCγ-immunoreactive interneurons. PKCγ-immunoreactive interneurons contain GABAA ergic and glycinergic receptors. At the subcellular level, PKCγ-immunoreactivity is mostly concentrated on plasma membranes, close to, but not within, postsynaptic densities. That only myelinated primary afferents were found to contact PKCγ-immunoreactive interneurons suggests that myelinated, but not unmyelinated, LTMRs play a critical role in the expression of mechanical allodynia.

  18. Expression, subcellular localization, and cis-regulatory structure of duplicated phytoene synthase genes in melon (Cucumis melo L.).

    PubMed

    Qin, Xiaoqiong; Coku, Ardian; Inoue, Kentaro; Tian, Li

    2011-10-01

    Carotenoids perform many critical functions in plants, animals, and humans. It is therefore important to understand carotenoid biosynthesis and its regulation in plants. Phytoene synthase (PSY) catalyzes the first committed and rate-limiting step in carotenoid biosynthesis. While PSY is present as a single copy gene in Arabidopsis, duplicated PSY genes have been identified in many economically important monocot and dicot crops. CmPSY1 was previously identified from melon (Cucumis melo L.), but was not functionally characterized. We isolated a second PSY gene, CmPSY2, from melon in this work. CmPSY2 possesses a unique intron/exon structure that has not been observed in other plant PSYs. Both CmPSY1 and CmPSY2 are functional in vitro, but exhibit distinct expression patterns in different melon tissues and during fruit development, suggesting differential regulation of the duplicated melon PSY genes. In vitro chloroplast import assays verified the plastidic localization of CmPSY1 and CmPSY2 despite the lack of an obvious plastid target peptide in CmPSY2. Promoter motif analysis of the duplicated melon and tomato PSY genes and the Arabidopsis PSY revealed distinctive cis-regulatory structures of melon PSYs and identified gibberellin-responsive motifs in all PSYs except for SlPSY1, which has not been reported previously. Overall, these data provide new insights into the evolutionary history of plant PSY genes and the regulation of PSY expression by developmental and environmental signals that may involve different regulatory networks.

  19. Characterization of bovine immunodeficiency virus rev cDNAs and identification and subcellular localization of the Rev protein.

    PubMed Central

    Oberste, M S; Williamson, J C; Greenwood, J D; Nagashima, K; Copeland, T D; Gonda, M A

    1993-01-01

    One of the six putative accessory genes of bovine immunodeficiency virus (BIV) is similar to those identified as rev in the human immunodeficiency virus and visna virus genomes. To further analyze the BIV rev gene locus, protein, and function, rev cDNAs were cloned and characterized. BIV rev mRNA is derived from the full-length transcript by multiple splicing events and consists of three exons, including the untranslated leader sequence and two coding exons. BIV rev cDNA was expressed in bacteria and in a mammalian in vitro translation expression system. A 23-kDa Rev protein (p23rev) was immunologically detected in lysates from both systems by using an antiserum made to a synthetic Rev peptide. Recombinant p23rev made in bacteria was purified and used to make a polyvalent antiserum. Antisera to Rev peptide and recombinant p23rev immunoprecipitated p23rev from BIV-infected mammalian cells but not from virions. A mammalian expression vector using the BIV rev cDNA was constructed; p23rev was immunoprecipitated with anti-Rev serum from 32P-labeled lysates of monkey cells transfected with this plasmid, demonstrating that BIV Rev is phosphorylated. Immunofluorescence and immunoelectron microscopy with anti-BIV Rev antisera localized Rev in the nucleus and, particularly, in the nucleoli of BIV-infected cells. In functional studies, the expression of BIV Rev was shown to positively regulate the appearance both of Gag protein, which is translated from the unspliced primary viral transcript, and of singly spliced env mRNA but not that of the multiply spliced tat mRNA. These results demonstrate that BIV Rev activity correlates with the known function of lentivirus Rev proteins. Images PMID:8411341

  20. Establishment of a human cell line stably overexpressing mouse Nip45 and characterization of Nip45 subcellular localization

    SciTech Connect

    Hashiguchi, Kohtaro; Ozaki, Masumi; Kuraoka, Isao; Saitoh, Hisato

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer A human cell line expressing a mouse Nip45 has facilitated Nip45 analysis. Black-Right-Pointing-Pointer Nip45 does not effectively inhibit polySUMOylation in vivo. Black-Right-Pointing-Pointer Nip45 interacts directly with SUMO and SUMO chains. Black-Right-Pointing-Pointer Nip45 accumulates at PML bodies in response to proteasome inhibition. -- Abstract: The nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2 interacting protein, Nfatc2ip (Nip45), has been implicated as a crucial coordinator of the immune response and of cellular differentiation in humans and mice, and contains SUMO-like domains in its C-terminal region. However, the significance of its N-terminal region and its correlation to the SUMO modification pathway remain largely uncharacterized. In this study, a human cultured cell line was established, in which FLAG-tagged mouse Nip45 (FLAG-mNip45) was stably overexpressed. Under standard, non-stressful conditions, we detected FLAG-mNip45 diffusely distributed in the nucleus. Intriguingly, proteasome inhibition by MG132 caused FLAG-mNip45, together with SUMOylated proteins, to localize in nuclear domains associated with promyelocytic leukemia protein. Finally, using an in vitro binding assay, we showed interaction of the N-terminal region of mNip45 with both free SUMO-3 and SUMO-3 chains, indicating that Nip45 may, in part, exert its function via interaction with SUMO/SUMOylated proteins. Taken together, our study provides novel information on a poorly characterized mammalian protein and suggests that our newly established cell line will be useful for elucidating the physiological role of Nip45.

  1. p53 Degradation Activity, Expression, and Subcellular Localization of E6 Proteins from 29 Human Papillomavirus Genotypes

    PubMed Central

    Mesplède, Thibault; Gagnon, David; Bergeron-Labrecque, Fanny; Azar, Ibrahim; Sénéchal, Hélène; Coutlée, François

    2012-01-01

    Human papillomaviruses (HPVs) are the etiological agents of cervical cancer and other human malignancies. HPVs are classified into high- and low-risk genotypes according to their association with cancer. Host cell transformation by high-risk HPVs relies in part on the ability of the viral E6 protein to induce the degradation of p53. We report the development of a cellular assay that accurately quantifies the p53 degradation activity of E6 in vivo, based on the fusion of p53 to Renilla luciferase (RLuc-p53). This assay was used to measure the p53 degradation activities of E6 proteins from 29 prevalent HPV types and variants of HPV type 16 (HPV16) and HPV33 by determining the amount of E6 expression vector required to reduce by half the levels of RLuc-p53 (50% effective concentration [EC50]). These studies revealed an unexpected variability in the p53 degradation activities of different E6 proteins, even among active types whose EC50s span more than 2 log units. Differences in activity were greater between types than between variants and did not correlate with differences in the intracellular localization of E6, with most being predominantly nuclear. Protein and mRNA expression of the 29 E6 proteins was also examined. For 16 high-risk types, spliced transcripts that encode shorter E6*I proteins of variable sizes and abundances were detected. Mutation of the splice donor site in five different E6 proteins increased their p53 degradation activity, suggesting that mRNA splicing can limit the activity of some high-risk E6 types. The quantification of p53 degradation in vivo represents a novel tool to systematically compare the oncogenic potentials of E6 proteins from different HPV types and variants. PMID:22013048

  2. Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).

    PubMed

    Hugo, Martín; Martínez, Alejandra; Trujillo, Madia; Estrada, Damián; Mastrogiovanni, Mauricio; Linares, Edlaine; Augusto, Ohara; Issoglio, Federico; Zeida, Ari; Estrín, Darío A; Heijnen, Harry F G; Piacenza, Lucía; Radi, Rafael

    2017-02-21

    The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 10(7) M(-1)⋅s(-1)) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 10(5) versus 3.5 × 10(4) M(-1)⋅s(-1), respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp(233•+)). Mutation of Trp(233) to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp(233•+), a Cys(222)-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp(233) and Cys(222) is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.

  3. Subcellular Nutrient Element Localization and Enrichment in Ecto- and Arbuscular Mycorrhizas of Field-Grown Beech and Ash Trees Indicate Functional Differences

    PubMed Central

    Seven, Jasmin; Polle, Andrea

    2014-01-01

    Mycorrhizas are the chief organ for plant mineral nutrient acquisition. In temperate, mixed forests, ash roots (Fraxinus excelsior) are colonized by arbuscular mycorrhizal fungi (AM) and beech roots (Fagus sylvatica) by ectomycorrhizal fungi (EcM). Knowledge on the functions of different mycorrhizal species that coexist in the same environment is scarce. The concentrations of nutrient elements in plant and fungal cells can inform on nutrient accessibility and interspecific differences of mycorrhizal life forms. Here, we hypothesized that mycorrhizal fungal species exhibit interspecific differences in mineral nutrient concentrations and that the differences correlate with the mineral nutrient concentrations of their associated root cells. Abundant mycorrhizal fungal species of mature beech and ash trees in a long-term undisturbed forest ecosystem were the EcM Lactarius subdulcis, Clavulina cristata and Cenococcum geophilum and the AM Glomus sp. Mineral nutrient subcellular localization and quantities of the mycorrhizas were analysed after non-aqueous sample preparation by electron dispersive X-ray transmission electron microscopy. Cenococcum geophilum contained the highest sulphur, Clavulina cristata the highest calcium levels, and Glomus, in which cations and P were generally high, exhibited the highest potassium levels. Lactarius subdulcis-associated root cells contained the highest phosphorus levels. The root cell concentrations of K, Mg and P were unrelated to those of the associated fungal structures, whereas S and Ca showed significant correlations between fungal and plant concentrations of those elements. Our results support profound interspecific differences for mineral nutrient acquisition among mycorrhizas formed by different fungal taxa. The lack of correlation between some plant and fungal nutrient element concentrations may reflect different retention of mineral nutrients in the fungal part of the symbiosis. High mineral concentrations, especially of

  4. Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in Its Subcellular Localization and Shutoff Activity

    PubMed Central

    Hayashi, Tsuyoshi; Chaimayo, Chutikarn; McGuinness, James

    2016-01-01

    ABSTRACT PA-X is a recently identified influenza virus protein that is composed of the PA N-terminal 191 amino acids and unique C-terminal 41 or 61 residues. We and others showed that PA-X has a strong ability to suppress host protein synthesis via host mRNA decay, which is mediated by endonuclease activity in its N-terminal domain (B. W. Jagger, H. M. Wise, J. C. Kash, K. A. Walters, N. M. Wills, Y. L. Xiao, R. L. Dunfee, L. M. Schwartzman, A. Ozinsky, G. L. Bell, R. M. Dalton, A. Lo, S. Efstathiou, J. F. Atkins, A. E. Firth, J. K. Taubenberger, and P. Digard, 2012, Science 337:199–204, http://dx.doi.org/10.1126/science.1222213, and E. A. Desmet, K. A. Bussey, R. Stone, and T. Takimoto, 2013, J Virol 87:3108–3118, http://dx.doi.org/10.1128/JVI.02826-12). However, the mechanism of host mRNA degradation, especially where and how PA-X targets mRNAs, has not been analyzed. In this study, we determined the localization of PA-X and the role of the C-terminal unique region in shutoff activity. Quantitative subcellular localization analysis revealed that PA-X was located equally in both cytoplasm and nucleus. By characterizing a series of PA-X C-terminal deletion mutants, we found that the first 9 amino acids were sufficient for nuclear localization, but an additional 6 residues were required to induce the maximum shutoff activity observed with intact PA-X. Importantly, forced nuclear localization of the PA-X C-terminal deletion mutant enhanced shutoff activity, highlighting the ability of nuclear PA-X to degrade host mRNAs more efficiently. However, PA-X also inhibited luciferase expression from transfected mRNAs synthesized in vitro, suggesting that PA-X also degrades mRNAs in the cytoplasm. Among the basic amino acids in the PA-X C-terminal region, 3 residues, 195K, 198K, and 199R, were identified as key residues for inducing host shutoff and nuclear localization. Overall, our data indicate a critical role for the 15 residues in the PA-X C-terminal domain in

  5. Subcellular localization of phospholipase Cζ in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation

    PubMed Central

    Escoffier, Jessica; Yassine, Sandra; Lee, Hoi Chang; Martinez, Guillaume; Delaroche, Julie; Coutton, Charles; Karaouzène, Thomas; Zouari, Raoudha; Metzler-Guillemain, Catherine; Pernet-Gallay, Karin; Hennebicq, Sylviane; Ray, Pierre F.; Fissore, Rafael; Arnoult, Christophe

    2015-01-01

    We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospholipase C, zeta (PLCζ), the sperm factor thought to induce the Ca2+ oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLCζ in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLCζ and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLCζ is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca2+ oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLCζ in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equatorial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLCζ in globozoospermic sperm is thus consistent and reinforces the role of PLCζ as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2

  6. The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons

    PubMed Central

    Miyagi, Tatsuhiro; Tanaka, Shigeru; Hide, Izumi; Shirafuji, Toshihiko; Sakai, Norio

    2016-01-01

    G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation. PMID:26800526

  7. Carcass Type Affects Local Scavenger Guilds More than Habitat Connectivity

    PubMed Central

    Olson, Zachary H.; Beasley, James C.; Rhodes, Olin E.

    2016-01-01

    Scavengers and decomposers provide an important ecosystem service by removing carrion from the environment. Scavenging and decomposition are known to be temperature-dependent, but less is known about other factors that might affect carrion removal. We conducted an experiment in which we manipulated combinations of patch connectivity and carcass type, and measured responses by local scavenger guilds along with aspects of carcass depletion. We conducted twelve, 1-month trials in which five raccoon (Procyon lotor), Virginia opossum (Didelphis virginiana), and domestic rabbit (Oryctolagus spp.) carcasses (180 trials total) were monitored using remote cameras in 21 forest patches in north-central Indiana, USA. Of 143 trials with complete data, we identified fifteen species of vertebrate scavengers divided evenly among mammalian (N = 8) and avian species (N = 7). Fourteen carcasses (9.8%) were completely consumed by invertebrates, vertebrates exhibited scavenging behavior at 125 carcasses (87.4%), and four carcasses (2.8%) remained unexploited. Among vertebrates, mammals scavenged 106 carcasses, birds scavenged 88 carcasses, and mammals and birds scavenged 69 carcasses. Contrary to our expectations, carcass type affected the assemblage of local scavenger guilds more than patch connectivity. However, neither carcass type nor connectivity explained variation in temporal measures of carcass removal. Interestingly, increasing richness of local vertebrate scavenger guilds contributed moderately to rates of carrion removal (≈6% per species increase in richness). We conclude that scavenger-specific differences in carrion utilization exist among carcass types and that reliable delivery of carrion removal as an ecosystem service may depend on robust vertebrate and invertebrate communities acting synergistically. PMID:26886299

  8. ADP1 Affects Plant Architecture by Regulating Local Auxin Biosynthesis

    PubMed Central

    Li, Shibai; Qin, Genji; Novák, Ondřej; Pěnčík, Aleš; Ljung, Karin; Aoyama, Takashi; Liu, Jingjing; Murphy, Angus; Gu, Hongya; Tsuge, Tomohiko; Qu, Li-Jia

    2014-01-01

    Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs. PMID:24391508

  9. ADP1 affects plant architecture by regulating local auxin biosynthesis.

    PubMed

    Li, Ruixi; Li, Jieru; Li, Shibai; Qin, Genji; Novák, Ondřej; Pěnčík, Aleš; Ljung, Karin; Aoyama, Takashi; Liu, Jingjing; Murphy, Angus; Gu, Hongya; Tsuge, Tomohiko; Qu, Li-Jia

    2014-01-01

    Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs.

  10. Subcellular localization of (latent) transforming growth factor beta and the latent TGF-beta binding protein in rat hepatocytes and hepatic stellate cells.

    PubMed

    Roth-Eichhorn, S; Kühl, K; Gressner, A M

    1998-12-01

    Recently, the existence of the large latent transforming growth factor beta (TGF-beta) complex, consisting of TGF-beta, the N-terminal part of its precursor (latency-associated peptide [LAP]), and the latent TGF-beta binding protein (LTBP), was demonstrated in rat liver parenchymal cells (PC) and stellate cells (HSC). However, in contrast to HSC, in freshly isolated PC, no message of these proteins is detectable. This study was performed to investigate the subcellular distribution of the proteins forming the latent TGF-beta complex in PC and HSC from rat liver to obtain more information about their origin and potential intracellular functions. PC and HSC were isolated from rat liver by protease reperfusion and investigated for TGF-beta1,-2,-3, beta1-LAP, and LTBP-1 after cultivation using double-immunofluorescent staining, followed by high-resolution confocal microscopic analysis. Subcellular fractions obtained by standard differential centrifugation of rat liver homogenate were analyzed using a TGF-beta1 enzyme-linked immunosorbent assay (ELISA) and Western blotting for beta1-LAP and LTBP-1. By confocal microscopy, a diffuse distribution of TGF-beta and LAP in the cytoplasm of PC is noticed, whereas the LTBP immunostaining predominates at plasma membranes. In PC, distinct intracellular granules were superimposed with TGF-beta, LAP, and LTBP stainings identified as lysosomal compartments and mitochondria by ELISA and immunoblotting of subcellular fractions. In HSC, stainings of colocalized TGF-beta, LAP, and LTBP are strongest in the perinuclear area, indicating synthesis and secretion via endoplasmic reticulum and Golgi, respectively. Partially, the proteins were also found in HSC nuclei. During the transformation of HSC to myofibroblasts, LAP and LTBP become strongly colocalized with other components of the cytoskeletal network like smooth muscle--actin, desmin, and talin. The results confirm biochemical data about the existence and expression of the large latent

  11. Novel and recurrent CIB2 variants, associated with nonsyndromic deafness, do not affect calcium buffering and localization in hair cells.

    PubMed

    Seco, Celia Zazo; Giese, Arnaud P; Shafique, Sobia; Schraders, Margit; Oonk, Anne M M; Grossheim, Mike; Oostrik, Jaap; Strom, Tim; Hegde, Rashmi; van Wijk, Erwin; Frolenkov, Gregory I; Azam, Maleeha; Yntema, Helger G; Free, Rolien H; Riazuddin, Saima; Verheij, Joke B G M; Admiraal, Ronald J; Qamar, Raheel; Ahmed, Zubair M; Kremer, Hannie

    2016-04-01

    Variants in CIB2 can underlie either Usher syndrome type I (USH1J) or nonsyndromic hearing impairment (NSHI) (DFNB48). Here, a novel homozygous missense variant c.196C>T and compound heterozygous variants, c.[97C>T];[196C>T], were found, respectively, in two unrelated families of Dutch origin. Besides, the previously reported c.272 T>C functional missense variant in CIB2 was identified in two families of Pakistani origin. The missense variants are demonstrated not to affect subcellular localization of CIB2 in vestibular hair cells in ex vivo expression experiments. Furthermore, these variants do not affect the ATP-induced calcium responses in COS-7 cells. However, based on the residues affected, the variants are suggested to alter αIIβ integrin binding. HI was nonsyndromic in all four families. However, deafness segregating with the c.272T>C variant in one Pakistani family is remarkably less severe than that in all other families with this mutation. Our results contribute to the insight in genotype-phenotype correlations of CIB2 mutations.

  12. Prediction of protein subcellular localization by incorporating multiobjective PSO-based feature subset selection into the general form of Chou's PseAAC.

    PubMed

    Mandal, Monalisa; Mukhopadhyay, Anirban; Maulik, Ujjwal

    2015-04-01

    In this article, the possible subcellular location of a protein is predicted using multiobjective particle swarm optimization-based feature selection technique. In general form of pseudo-amino acid composition, the protein sequences are used for constructing protein features. Here, the different amino acids compositions are used to construct the feature sets. Therefore, the data are presented as sample of protein versus amino acid compositions as features. The proposed algorithm tries to maximize the feature relevance and minimize the feature redundancy simultaneously. After proposed algorithm is executed on the multiclass dataset, some features are selected. On this resultant feature subset, tenfold cross-validation is applied and corresponding accuracy, F score, entropy, representation entropy and average correlation are calculated. The performance of the proposed method is compared with that of its single objective versions, sequential forward search, sequential backward search, minimum redundancy maximum relevance with two schemes, CFS, CBFS, [Formula: see text], Fisher discriminant and a Cluster-based technique.

  13. 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

  14. The B56γ3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1

    PubMed Central

    Lai, Tai-Yu; Yang, Yu-San; Hong, Wei-Fu; Chiang, Chi-Wu

    2016-01-01

    The B56γ-containing protein phosphatase 2A (PP2A-B56γ) has been postulated to have tumor suppressive functions. Here, we report regulation of p27KIP1 subcellular localization by PP2A-B56γ3. B56γ3 overexpression enhanced nuclear localization of p27KIP1, whereas knockdown of B56γ3 decreased p27KIP1 nuclear localization. B56γ3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157), whose phosphorylation promotes cytoplasmic localization of p27KIP1, whereas B56γ3 knockdown significantly increased the level of phospho-Thr157. In vitro, PP2A-B56γ3 catalyzed dephosphorylation of phospho-Thr157 in a dose-dependent and okadaic acid-sensitive manner. B56γ3 did not increase p27KIP1 nuclear localization by down-regulating the upstream kinase Akt activity and outcompeted a myristoylated constitutively active Akt (Aktca) in regulating Thr157 phosphorylation and subcellular localization of p27KIP1. In addition, results of interaction domain mapping revealed that both the N-terminal and C-terminal domains of p27 and a domain at the C-terminus of B56γ3 are required for interaction between p27 and B56γ3. Furthermore, we demonstrated that p27KIP1 levels are positively correlated with B56γ levels in both non-tumor and tumor parts of a set of human colon tissue specimens. However, positive correlation between nuclear p27KIP1 levels and B56γ levels was found only in the non-tumor parts, but not in tumor parts of these tissues, implicating a dysregulation in PP2A-B56γ3-regulated p27KIP1 nuclear localization in these tumor tissues. Altogether, this study provides a new mechanism by which the PP2A-B56γ3 holoenzyme plays its tumor suppressor role. PMID:26684356

  15. Specific Soybean Lipoxygenases Localize to Discrete Subcellular Compartments and Their mRNAs Are Differentially Regulated by Source-Sink Status1

    PubMed Central

    Stephenson, Lowry C.; Bunker, Thomas W.; Dubbs, Wesley E.; Grimes, Howard D.

    1998-01-01

    Members of the lipoxygenase multigene family, found widely in eukaryotes, have been proposed to function in nitrogen partitioning and storage in plants. Lipoxygenase gene responses to source-sink manipulations in mature soybean (Glycine max [L.] Merr.) leaves were examined using gene-specific riboprobes to the five vegetative lipoxygenases (vlxA–vlxE). Steady-state levels of all vlx mRNAs responded strongly to sink limitation, but specific transcripts exhibited differential patterns of response as well. During reproductive sink limitation, vlxA and vlxB messages accumulated to high levels, whereas vlxC and vlxD transcript levels were modest. Immunolocalization using peptide-specific antibodies demonstrated that under control conditions, VLXB was present in the cytosol of the paraveinal mesophyll and with pod removal accumulated additionally in the bundle-sheath and adjacent cells. With sink limitation VLXD accumulated to apparent high levels in the vacuoles of the same cells. Segregation of gene products at the cellular and subcellular levels may thus permit complex patterns of differential regulation within the same cell type. Specific lipoxygenase isoforms may have a role in short-term nitrogen storage (VLXC/D), whereas others may simultaneously function in assimilate partitioning as active enzymes (VLXA/B). PMID:9501125

  16. Subcellular localization of Clostridium thermocellum ORF3p, a protein carrying a receptor for the docking sequence borne by the catalytic components of the cellulosome.

    PubMed Central

    Salamitou, S; Lemaire, M; Fujino, T; Ohayon, H; Gounon, P; Béguin, P; Aubert, J P

    1994-01-01

    The ORF3 gene of Clostridium thermocellum encodes a polypeptide (ORF3p) which contains a receptor domain for the docking sequence borne by the catalytic subunits of the cellulosome and a triplicated domain related to some bacterial cell surface proteins. It was thus surmised that ORF3p is a surface protein. In this study, this hypothesis was confirmed. Subcellular fractionation, Western blotting (immunoblotting), and electron microscopy of immunocytochemically labeled cells indicated that ORF3p produced by C. thermocellum was located in the outer surface layer of the bacterium. This layer appeared to consist of a soft matrix shedding off particulate fragments. Nonsedimenting ORF3p derived from sonicated cells was associated with high-molecular-mass fractions (> 20 MDa), probably corresponding to fragments of the outer cell layer. The same high-molecular-mass fractions also contained the cellulosomal marker CipA. Contrary to CipA, however, ORF3p was not associated with 2- to 4-MDa fractions corresponding to individual cellulosomes, and a significant fraction of ORF3p failed to bind to cellulose. It is proposed that ORF3 and ORF3p be renamed olpA and OlpA, respectively (for outer layer protein). Images PMID:8188584

  17. Multitask learning for protein subcellular location prediction.

    PubMed

    Xu, Qian; Pan, Sinno Jialin; Xue, Hannah Hong; Yang, Qiang

    2011-01-01

    Protein subcellular localization is concerned with predicting the location of a protein within a cell using computational methods. The location information can indicate key functionalities of proteins. Thus, accurate prediction of subcellular localizations of proteins can help the prediction of protein functions and genome annotations, as well as the identification of drug targets. Machine learning methods such as Support Vector Machines (SVMs) have been used in the past for the problem of protein subcellular localization, but have been shown to suffer from a lack of annotated training data in each species under study. To overcome this data sparsity problem, we observe that because some of the organisms may be related to each other, there may be some commonalities across different organisms that can be discovered and used to help boost the data in each localization task. In this paper, we formulate protein subcellular localization problem as one of multitask learning across different organisms. We adapt and compare two specializations of the multitask learning algorithms on 20 different organisms. Our experimental results show that multitask learning performs much better than the traditional single-task methods. Among the different multitask learning methods, we found that the multitask kernels and supertype kernels under multitask learning that share parameters perform slightly better than multitask learning by sharing latent features. The most significant improvement in terms of localization accuracy is about 25 percent. We find that if the organisms are very different or are remotely related from a biological point of view, then jointly training the multiple models cannot lead to significant improvement. However, if they are closely related biologically, the multitask learning can do much better than individual learning.

  18. LC8 dynein light chain (DYNLL1) binds to the C-terminal domain of ATM-interacting protein (ATMIN/ASCIZ) and regulates its subcellular localization

    SciTech Connect

    Rapali, Peter; Garcia-Mayoral, Maria Flor; Martinez-Moreno, Monica; Tarnok, Krisztian; Schlett, Katalin; Albar, Juan Pablo; Bruix, Marta; Nyitray, Laszlo; Rodriguez-Crespo, Ignacio

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer We have screened a human library with dynein light chain DYNLL1 (DLC8) as bait. Black-Right-Pointing-Pointer Dynein light chain DYNLL1 binds to ATM-kinase interacting protein (ATMIN). Black-Right-Pointing-Pointer ATMIN has 17 SQ/TQ motifs, a motif frequently found in DYNLL1-binding partners. Black-Right-Pointing-Pointer The two proteins interact in vitro, with ATMIN displaying at least five binding sites. Black-Right-Pointing-Pointer The interaction of ATMIN and DYNNL1 in transfected cells can also be observed. -- Abstract: LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1

  19. Effects of chronic cerebral hypoperfusion and low-dose progesterone treatment on apoptotic processes, expression and subcellular localization of key elements within Akt and Erk signaling pathways in rat hippocampus.

    PubMed

    Stanojlović, M; Guševac, I; Grković, I; Zlatković, J; Mitrović, N; Zarić, M; Horvat, A; Drakulić, D

    2015-12-17

    The present study attempted to investigate how chronic cerebral hypoperfusion (CCH) and repeated low-dose progesterone (P) treatment affect gene and protein expression, subcellular distribution of key apoptotic elements within protein kinase B (Akt) and extracellular signal-regulated kinases (Erk) signal transduction pathways, as well as neurodegenerative processes and behavior. The results revealed the absence of Erk activation in CCH in cytosolic and synaptosomal fractions, indicating a lower threshold of Akt activation in brain ischemia, while P increased their levels above control values. CCH induced an increase in caspase 3 (Casp 3) and poly (ADP-ribose) polymerase (PARP) gene and protein expression. However, P restored expression of examined molecules in all observed fractions, except for the levels of Casp 3 in synapses which highlighted its possible non-apoptotic or even protective function. Our study showed the absence of nuclear factor kappa-light-chain-enhancer of activated b cells (NF-κB) response to this type of ischemic condition and its strong activation under the influence of P. Further, the initial increase in the number of apoptotic cells and amount of DNA fragmentation induced by CCH was significantly reduced by P. Finally, P reversed the CCH-induced reduction in locomotor activity, while promoting a substantial decrease in anxiety-related behavior. Our findings support the concept that repeated low-dose post-ischemic P treatment reduces CCH-induced neurodegeneration in the hippocampus. Neuroprotection is initiated through the activation of investigated kinases and regulation of their downstream molecules in subcellular specific manner, indicating that this treatment may be a promising therapy for alleviation of CCH-induced pathologies.

  20. Socioeconomic factors affecting local support for black bear recovery strategies.

    PubMed

    Morzillo, Anita T; Mertig, Angela G; Hollister, Jeffrey W; Garner, Nathan; Liu, Jianguo

    2010-06-01

    There is global interest in recovering locally extirpated carnivore species. Successful efforts to recover Louisiana black bear in Louisiana have prompted interest in recovery throughout the species' historical range. We evaluated support for three potential black bear recovery strategies prior to public release of a black bear conservation and management plan for eastern Texas, United States. Data were collected from 1,006 residents living in proximity to potential recovery locations, particularly Big Thicket National Preserve. In addition to traditional logistic regression analysis, we used conditional probability analysis to statistically and visually evaluate probabilities of public support for potential black bear recovery strategies based on socioeconomic characteristics. Allowing black bears to repopulate the region on their own (i.e., without active reintroduction) was the recovery strategy with the greatest probability of acceptance. Recovery strategy acceptance was influenced by many socioeconomic factors. Older and long-time local residents were most likely to want to exclude black bears from the area. Concern about the problems that black bears may cause was the only variable significantly related to support or non-support across all strategies. Lack of personal knowledge about black bears was the most frequent reason for uncertainty about preferred strategy. In order to reduce local uncertainty about possible recovery strategies, we suggest that wildlife managers focus outreach efforts on providing local residents with general information about black bears, as well as information pertinent to minimizing the potential for human-black bear conflict.

  1. Socioeconomic Factors Affecting Local Support for Black Bear Recovery Strategies

    NASA Astrophysics Data System (ADS)

    Morzillo, Anita T.; Mertig, Angela G.; Hollister, Jeffrey W.; Garner, Nathan; Liu, Jianguo

    2010-06-01

    There is global interest in recovering locally extirpated carnivore species. Successful efforts to recover Louisiana black bear in Louisiana have prompted interest in recovery throughout the species’ historical range. We evaluated support for three potential black bear recovery strategies prior to public release of a black bear conservation and management plan for eastern Texas, United States. Data were collected from 1,006 residents living in proximity to potential recovery locations, particularly Big Thicket National Preserve. In addition to traditional logistic regression analysis, we used conditional probability analysis to statistically and visually evaluate probabilities of public support for potential black bear recovery strategies based on socioeconomic characteristics. Allowing black bears to repopulate the region on their own (i.e., without active reintroduction) was the recovery strategy with the greatest probability of acceptance. Recovery strategy acceptance was influenced by many socioeconomic factors. Older and long-time local residents were most likely to want to exclude black bears from the area. Concern about the problems that black bears may cause was the only variable significantly related to support or non-support across all strategies. Lack of personal knowledge about black bears was the most frequent reason for uncertainty about preferred strategy. In order to reduce local uncertainty about possible recovery strategies, we suggest that wildlife managers focus outreach efforts on providing local residents with general information about black bears, as well as information pertinent to minimizing the potential for human-black bear conflict.

  2. Liver X receptor ligand cytotoxicity in colon cancer cells and not in normal colon epithelial cells depends on LXRβ subcellular localization.

    PubMed

    Courtaut, Flavie; Derangère, Valentin; Chevriaux, Angélique; Ladoire, Sylvain; Cotte, Alexia K; Arnould, Laurent; Boidot, Romain; Rialland, Mickaël; Ghiringhelli, François; Rébé, Cédric

    2015-09-29

    Increasing evidence indicates that Liver X Receptors (LXRs) have some anticancer properties. We recently demonstrated that LXR ligands induce colon cancer cell pyroptosis through an LXRβ-dependent pathway. In the present study, we showed that human colon cancer cell lines presented differential cytoplasmic localizations of LXRβ. This localization correlated with caspase-1 activation and cell death induction under treatment with LXR ligand. The association of LXRβ with the truncated form of RXRα (t-RXRα) was responsible for the sequestration of LXRβ in the cytoplasm in colon cancer cells. Moreover t-RXRα was not expressed in normal colon epithelial cells. These cells presented a predominantly nuclear localization of LXRβ and were resistant to LXR ligand cytotoxicity. Our results showed that predominant cytoplasmic localization of LXRβ, which occurs in colon cancer cells but not in normal colon epithelial cells, allowed LXR ligand-induced pyroptosis. This study strengthens the hypothesis that LXRβ could be a promising target in cancer therapy.

  3. Subcellular proteomics in neuroscience.

    PubMed

    Li, Ka Wan; Smit, August B

    2008-05-01

    The brain is the most complex and dynamically organized organ of the human body, with a high degree of computation capability enabling the execution of a wide spectrum of physiological processes and behaviors. In the past decades a large number of genomics studies have been undertaken to investigate brain function and brain disorders, but despite these efforts many of the underlying molecular mechanisms still remain largely unknown. The implementation of mass spectrometry based quantitative proteomics in recent years enabled to tap into condition-specific protein trafficking and protein interaction that are the key to organelle proteome (dys)function. The technology for neuroproteomics is still evolving; currently there are no standardized protocols. In this review we describe the most commonly used methods to prepare brain subcellular fractions suitable for proteomics analysis, and highlight the various approaches for quantitative neuroproteomics.

  4. Different subcellular localizations for the related interferon-induced GTPases, MuGBP-1 and MuGBP-2: implications for different functions?

    PubMed

    Vestal, D J; Gorbacheva, V Y; Sen, G C

    2000-11-01

    The guanylate-binding proteins (GBPs) are a family of 65-67-kDa proteins induced by both type I and type II interferons (IFN). Members of the GBP family of GTPases are among the most abundant IFN-gamma-induced proteins. GBPs contain an unusual GTP binding site, which is consistent with GBP hydrolysis of GTP to both GDP and GMP. In addition, six of the eight known GBPs have a carboxy-terminal CaaX motif for the addition of isoprenyl lipids. Despite their abundance, however, little is known about the biologic function or cellular location of GBPs. We report here on studies to localize both a newly identified murine GBP (MuGBP-2) and its closely related family member, MuGBP-1. In both IFN-treated macrophages and fibroblasts, MuGBP-2 is found in both a granular distribution throughout the cytoplasm and localized to vesicle populations of heterogeneous sizes. The localization of MuGBP-2 to vesicles is dependent on its isoprenylation. Despite a high degree of sequence identity and the presence of an identical CaaX sequence, MuGBP-1 has a very homogeneous cytoplasmic distribution and fails to localize to intracellular vesicles. The different intracellular distribution of these two closely related family members suggests differential function(s).

  5. Subcellular targeting and cytoskeletal attachment of SAP97 to the epithelial lateral membrane.

    PubMed

    Wu, H; Reuver, S M; Kuhlendahl, S; Chung, W J; Garner, C C

    1998-08-01

    The synapse-associated protein SAP97 is a member of a novel family of cortical cytoskeletal proteins involved in the localization of ion channels at such membrane specializations as synaptic junctions. These multidomain proteins have binding sites for protein 4.1, GKAPs/SAPAPs, voltage- and ligand-gated ion channels and cell-adhesion molecules containing C-terminal T/SXV motifs. In this study, we evaluated the contribution of individual domains in SAP97 to its selective recruitment and attachment to the cortical cytoskeleton in epithelial cells. We find that the PDZ, SH3 and GK domains, as well as the I3 insert in SAP97, are not essential for subcellular targeting, though both PDZ1-2 domains and the I3 insert affect the efficiency of localization. Instead, we show that the first 65 amino acid residues in SAP97, which are absent from SAP90/PSD-95 and SAP102, direct the selective subcellular localization and can mediate at least one point of attachment of SAP97 to the cytoskeleton assembled at sites of cell-cell contact. Our data demonstrate that it is the sequences unique to SAP97 that direct its subcellular targeting to the epithelial lateral membrane.

  6. Local bacteria affect the efficacy of chemotherapeutic drugs

    PubMed Central

    Lehouritis, Panos; Cummins, Joanne; Stanton, Michael; Murphy, Carola T.; McCarthy, Florence O.; Reid, Gregor; Urbaniak, Camilla; Byrne, William L.; Tangney, Mark

    2015-01-01

    In this study, the potential effects of bacteria on the efficacy of frequently used chemotherapies was examined. Bacteria and cancer cell lines were examined in vitro and in vivo for changes in the efficacy of cancer cell killing mediated by chemotherapeutic agents. Of 30 drugs examined in vitro, the efficacy of 10 was found to be significantly inhibited by certain bacteria, while the same bacteria improved the efficacy of six others. HPLC and mass spectrometry analyses of sample drugs (gemcitabine, fludarabine, cladribine, CB1954) demonstrated modification of drug chemical structure. The chemoresistance or increased cytotoxicity observed in vitro with sample drugs (gemcitabine and CB1954) was replicated in in vivo murine subcutaneous tumour models. These findings suggest that bacterial presence in the body due to systemic or local infection may influence tumour responses or off-target toxicity during chemotherapy. PMID:26416623

  7. Trapped energetic ion dynamics affected by localized electric field perturbations

    NASA Astrophysics Data System (ADS)

    Nishimura, Seiya

    2016-01-01

    Trapped energetic ion orbits in helical systems are numerically simulated using the Lorentz model. Simulation results of precession drift frequencies of trapped energetic ions are benchmarked by those of analytic solutions. The effects of the electric field perturbation localized at the rational surface on trapped energetic ions are examined, where the perturbation has an arbitrary rotation frequency and an amplitude fixed in time. It is found that the trapped energetic ions resonantly interact with the perturbation, when the rotation frequency of the perturbation is comparable to the precession drift frequencies of trapped energetic ions. The simulation results are suggestive to a mechanism of the energetic-ion-induced interchange mode, which might be associated with the fishbone mode observed in helical systems.

  8. Subcellular localization of the magnetosome protein MamC in the marine magnetotactic bacterium Magnetococcus marinus strain MC-1 using immunoelectron microscopy.

    PubMed

    Valverde-Tercedor, C; Abadía-Molina, F; Martinez-Bueno, M; Pineda-Molina, Estela; Chen, Lijun; Oestreicher, Zachery; Lower, Brian H; Lower, Steven K; Bazylinski, Dennis A; Jimenez-Lopez, C

    2014-07-01

    Magnetotactic bacteria are a diverse group of prokaryotes that biomineralize intracellular magnetosomes, composed of magnetic (Fe3O4) crystals each enveloped by a lipid bilayer membrane that contains proteins not found in other parts of the cell. Although partial roles of some of these magnetosome proteins have been determined, the roles of most have not been completely elucidated, particularly in how they regulate the biomineralization process. While studies on the localization of these proteins have been focused solely on Magnetospirillum species, the goal of the present study was to determine, for the first time, the localization of the most abundant putative magnetosome membrane protein, MamC, in Magnetococcus marinus strain MC-1. MamC was expressed in Escherichia coli and purified. Monoclonal antibodies were produced against MamC and immunogold labeling TEM was used to localize MamC in thin sections of cells of M. marinus. Results show that MamC is located only in the magnetosome membrane of Mc. marinus. Based on our findings and the abundance of this protein, it seems likely that it is important in magnetosome biomineralization and might be used in controlling the characteristics of synthetic nanomagnetite.

  9. Subcellular localization of the magnetosome protein MamC in the marine magnetotactic bacterium Magnetococcus marinus strain MC-1 using immunoelectron microscopy

    SciTech Connect

    Valverde-Tercedor, C; Abada-Molina, F; Martinez-Bueno, M; Pineda-Molina, Estela; Chen, Lijun; Oestreicher, Zachery; Lower, Brian H; Lower, Steven K; Bazylinski, Dennis A; Jimenez-Lopez, C

    2014-04-24

    Magnetotactic bacteria are a diverse group of prokaryotes that biomineralize intracellular magnetosomes, composed of magnetic (Fe3O4) crystals each enveloped by a lipid bilayer membrane that contains proteins not found in other parts of the cell. Although partial roles of some of these magnetosome proteins have been determined, the roles of most have not been completely elucidated, particularly in how they regulate the biomineralization process. While studies on the localization of these proteins have been focused solely on Magnetospirillum species, the goal of the present study was to determine, for the first time, the localization of the most abundant putative magnetosome membrane protein, MamC, in Magnetococcus marinus strain MC-1. MamC was expressed in Escherichia coli and purified. Monoclonal antibodies were produced against MamC and immunogold labeling TEM was used to localize MamC in thin sections of cells of M. marinus. Results show that MamC is located only in the magnetosome membrane of Mc. marinus. Based on our findings and the abundance of this protein, it seems likely that it is important in magnetosome biomineralization and might be used in controlling the characteristics of synthetic nanomagnetite.

  10. Structural and functional plasticity of subcellular tethering, targeting and processing of RPGRIP1 by RPGR isoforms

    PubMed Central

    Patil, Hemangi; Guruju, Mallikarjuna R.; Cho, Kyoung-in; Yi, Haiqing; Orry, Andrew; Kim, Hyesung; Ferreira, Paulo A.

    2012-01-01

    Summary Mutations affecting the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) interactome cause syndromic retinal dystrophies. RPGRIP1 interacts with the retinitis pigmentosa GTPase regulator (RPGR) through a domain homologous to RCC1 (RHD), a nucleotide exchange factor of Ran GTPase. However, functional relationships between RPGR and RPGRIP1 and their subcellular roles are lacking. We show by molecular modeling and analyses of RPGR disease-mutations that the RPGR-interacting domain (RID) of RPGRIP1 embraces multivalently the shared RHD of RPGR1–19 and RPGRORF15 isoforms and the mutations are non-overlapping with the interface found between RCC1 and Ran GTPase. RPGR disease-mutations grouped into six classes based on their structural locations and differential impairment with RPGRIP1 interaction. RPGRIP1α1 expression alone causes its profuse self-aggregation, an effect suppressed by co-expression of either RPGR isoform before and after RPGRIP1α1 self-aggregation ensue. RPGR1–19 localizes to the endoplasmic reticulum, whereas RPGRORF15 presents cytosolic distribution and they determine uniquely the subcellular co-localization of RPGRIP1α1. Disease mutations in RPGR1–19, RPGRORF15, or RID of RPGRIP1α1, singly or in combination, exert distinct effects on the subcellular targeting, co-localization or tethering of RPGRIP1α1 with RPGR1–19 or RPGRORF15 in kidney, photoreceptor and hepatocyte cell lines. Additionally, RPGRORF15, but not RPGR1–19, protects the RID of RPGRIP1α1 from limited proteolysis. These studies define RPGR- and cell-type-dependent targeting pathways with structural and functional plasticity modulating the expression of mutations in RPGR and RPGRIP1. Further, RPGR isoforms distinctively determine the subcellular targeting of RPGRIP1α1, with deficits in RPGRORF15-dependent intracellular localization of RPGRIP1α1 contributing to pathomechanisms shared by etiologically distinct syndromic retinal dystrophies. PMID

  11. Cloning and recombinant expression of active full-length xylosyltransferase I (XT-I) and characterization of subcellular localization of XT-I and XT-II.

    PubMed

    Schön, Sylvia; Prante, Christian; Bahr, Claudia; Kuhn, Joachim; Kleesiek, Knut; Götting, Christian

    2006-05-19

    Xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to serine residues in proteoglycan core proteins. This is the first and apparently rate-limiting step in the biosynthesis of the tetrasaccharide linkage region in glycosaminoglycan-containing proteoglycans. The XYLT-II gene codes for a highly homologous protein, but its physiological function is not yet known. Here we present for the first time the construction of a vector encoding the full-length GFP-tagged human XT-I and the recombinant expression of the active enzyme in mammalian cells. We expressed XT-I-GFP and various GFP-tagged XT-I and XT-II mutants with C-terminal truncations and deletions in HEK-293 and SaOS-2 cells in order to investigate the intracellular localization of XT-I and XT-II. Immunofluorescence analysis showed a distinct perinuclear pattern of XT-I-GFP and XT-II-GFP similar to that of alpha-mannosidase II, which is a known enzyme of the Golgi cisternae. Furthermore, a co-localization of native human XT-I and alpha-mannosidase II could also be demonstrated in untransfected cells. Using brefeldin A, we could also show that both xylosyltransferases are resident in the early cisternae of the Golgi apparatus. For its complete Golgi retention, XT-I requires the N-terminal 214 amino acids. Unlike XT-I, for XT-II, the first 45 amino acids are sufficient to target and retain the GFP reporter in the Golgi compartment. Here we show evidence that the stem regions were indispensable for Golgi localization of XT-I and XT-II.

  12. Subcellular localization of SREBP1 depends on its interaction with the C-terminal region of wild-type and disease related A-type lamins

    SciTech Connect

    Duband-Goulet, Isabelle; Woerner, Stephanie; Gasparini, Sylvaine; Attanda, Wikayatou; Konde, Emilie; Tellier-Lebegue, Carine; Craescu, Constantin T.; Roussel, Pascal; Vadrot, Nathalie; Vicart, Patrick; Oestlund, Cecilia; Worman, Howard J.; and others

    2011-12-10

    Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome ( Increment 607-656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.

  13. Subcellular localization of SREBP1 depends on its interaction with the C-terminal region of wild-type and disease related A-type lamins

    PubMed Central

    Duband-Goulet, Isabelle; Woerner, Stephanie; Gasparini, Sylvaine; Attanda, Wikayatou; Kondé, Emilie; Tellier-Lebègue, Carine; Craescu, Constantin T.; Gombault, Aurélie; Roussel, Pascal; Vadrot, Nathalie; Vicart, Patrick; Östlund, Cecilia; Worman, Howard J.; Zinn-Justin, Sophie; Buendia, Brigitte

    2011-01-01

    Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome (Δ607–656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function. PMID:21993218

  14. Cloning of Arabidopsis serotonin N-acetyltransferase and its role with caffeic acid O-methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localizations.

    PubMed

    Lee, Hyoung Yool; Byeon, Yeong; Lee, Kyungjin; Lee, Hye-Jung; Back, Kyoungwhan

    2014-11-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis. We cloned SNAT from Arabidopsis thaliana (AtSNAT) and functionally characterized this enzyme for the first time from dicotyledonous plants. Similar to rice SNAT, AtSNAT was found to localize to chloroplasts with peak enzyme activity at 45 °C (Km , 309 μm; Vmax , 1400 pmol/min/mg protein). AtSNAT also catalyzed 5-methoxytryptamine (5-MT) into melatonin with high catalytic activity (Km , 51 μm; Vmax , 5300 pmol/min/mg protein). In contrast, Arabidopsis caffeic acid O-methyltransferase (AtCOMT) localized to the cytoplasm. Interestingly, AtCOMT can methylate serotonin into 5-MT with low catalytic activity (Km , 3.396 mm; Vmax , 528 pmol/min/mg protein). These data suggest that serotonin can be converted into either N-acetylserotonin by SNAT or into 5-MT by COMT, after which it is metabolized into melatonin by COMT or SNAT, respectively. To support this hypothesis, serotonin was incubated in the presence of both AtSNAT and AtCOMT enzymes. In addition to melatonin production, the production of major intermediates depended on incubation temperatures; N-acetylserotonin was predominantly produced at high temperatures (45 °C), while low temperatures (37 °C) favored the production of 5-MT. Our results provide biochemical evidence for the presence of a serotonin O-methylation pathway in plant melatonin biosynthesis.

  15. Trimethyltin retinopathy: relationship of subcellular response to neuronal subspecialization

    SciTech Connect

    Bouldin, T.W.; Goines, N.D.; Krigman, M.R.

    1984-03-01

    Retinal neurons from rats acutely intoxicated with trimethyltin (TMT) were examined by light and electron microscopy to determine if there is a relationship between the subcellular response of a neuron to TMT and its morphologic subspecialization. Subcellular pathologic alterations were present in neurons from all three cellular layers of the sensory retina. However, the type and degree of subcellular response varied among the highly subspecialized neurons of the different retinal layers. Clusters of dense-cored vesicles and tubules were mainly limited to neurons of the ganglion-cell layer, large accumulations of dense bodies were mainly limited to neurons of the inner nuclear layer, and neuronal necrosis was mainly limited to the photoreceptor cells. The inner segment of the photoreceptor cell shared with the perikaryon of more conventional neurons a special vulnerability to TMT cytotoxicity. Our results suggest that the subspecialization of neurons affects the type and the degree of subcellular response to TMT.

  16. Tissue distribution and subcellular localization of trace metals in the pond snail Lymnaea stagnalis with special reference to the role of lysosomal granules in metal sequestration.

    PubMed

    Desouky, Mahmoud M A

    2006-05-01

    The present study was undertaken to elucidate the cellular mechanisms, which govern metal sequestration and detoxification in gastropods. For this purpose the pond snail, Lymnaea stagnalis was exposed to environmentally relevant concentrations of three species of metals (Al, Zn and Cd) for 30 days and the localization and fate of these metals were followed in different tissues of the snails. The measurement of relative distribution of metals between tissues revealed that the digestive gland and kidney account for most of the accumulated metals. Al and Cd (non-essential metals) were redistributed to the digestive gland, possibly because of the presence of specific binding entities in the digestive glands of the herein species. This study focuses on the role of intracellular metal-containing granules on metal sequestration. Three main types of granules were identified in the digestive gland cells namely small, green and yellow granules. The morphological examination and the progressive accumulation of elements within these granules revealed that they are developmental stages with the yellow granule being the mature one. The total number of these granules was found to be significantly increased upon exposure of the snails to Al only. This increase may be a response to the large amount of Al that is accumulated through feeding route of this grazing snail. X-ray microanalysis (XRMA) revealed that metals were localized in all three types of digestive gland granules. The increased amount of ligands (P and S) in the granules may give evidence for their role in metal sequestration. Levels of Al and P were positively correlated in the digestive gland granules. It is possible that aluminium is bound to phosphorus to render it insoluble and so to both immobilize it within the lysosome and to be excreted in a highly insoluble form. On the other hand, both Zn and Cd induced marked upregulation of S in mature (yellow) granules by 26- and 11-folds, respectively. The lysosomal

  17. Sub-Cellular Localization and Complex Formation by Aminoacyl-tRNA Synthetases in Cyanobacteria: Evidence for Interaction of Membrane-Anchored ValRS with ATP Synthase.

    PubMed

    Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A G; Olmedo-Verd, Elvira; Bru-Martínez, Roque; Luque, Ignacio

    2016-01-01

    tRNAs are charged with cognate amino acids by aminoacyl-tRNA synthetases (aaRSs) and subsequently delivered to the ribosome to be used as substrates for gene translation. Whether aminoacyl-tRNAs are channeled to the ribosome by transit within translational complexes that avoid their diffusion in the cytoplasm is a matter of intense investigation in organisms of the three domains of life. In the cyanobacterium Anabaena sp. PCC 7120, the valyl-tRNA synthetase (ValRS) is anchored to thylakoid membranes by means of the CAAD domain. We have investigated whether in this organism ValRS could act as a hub for the nucleation of a translational complex by attracting other aaRSs to the membranes. Out of the 20 aaRSs, only ValRS was found to localize in thylakoid membranes whereas the other enzymes occupied the soluble portion of the cytoplasm. To investigate the basis for this asymmetric distribution of aaRSs, a global search for proteins interacting with the 20 aaRSs was conducted. The interaction between ValRS and the FoF1 ATP synthase complex here reported is of utmost interest and suggests a functional link between elements of the gene translation and energy production machineries.

  18. Sub-Cellular Localization and Complex Formation by Aminoacyl-tRNA Synthetases in Cyanobacteria: Evidence for Interaction of Membrane-Anchored ValRS with ATP Synthase

    PubMed Central

    Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A. G.; Olmedo-Verd, Elvira; Bru-Martínez, Roque; Luque, Ignacio

    2016-01-01

    tRNAs are charged with cognate amino acids by aminoacyl-tRNA synthetases (aaRSs) and subsequently delivered to the ribosome to be used as substrates for gene translation. Whether aminoacyl-tRNAs are channeled to the ribosome by transit within translational complexes that avoid their diffusion in the cytoplasm is a matter of intense investigation in organisms of the three domains of life. In the cyanobacterium Anabaena sp. PCC 7120, the valyl-tRNA synthetase (ValRS) is anchored to thylakoid membranes by means of the CAAD domain. We have investigated whether in this organism ValRS could act as a hub for the nucleation of a translational complex by attracting other aaRSs to the membranes. Out of the 20 aaRSs, only ValRS was found to localize in thylakoid membranes whereas the other enzymes occupied the soluble portion of the cytoplasm. To investigate the basis for this asymmetric distribution of aaRSs, a global search for proteins interacting with the 20 aaRSs was conducted. The interaction between ValRS and the FoF1 ATP synthase complex here reported is of utmost interest and suggests a functional link between elements of the gene translation and energy production machineries. PMID:27375579

  19. ABC Transporter Subfamily D: Distinct Differences in Behavior between ABCD1–3 and ABCD4 in Subcellular Localization, Function, and Human Disease

    PubMed Central

    2016-01-01

    ATP-binding cassette (ABC) transporters are one of the largest families of membrane-bound proteins and transport a wide variety of substrates across both extra- and intracellular membranes. They play a critical role in maintaining cellular homeostasis. To date, four ABC transporters belonging to subfamily D have been identified. ABCD1–3 and ABCD4 are localized to peroxisomes and lysosomes, respectively. ABCD1 and ABCD2 are involved in the transport of long and very long chain fatty acids (VLCFA) or their CoA-derivatives into peroxisomes with different substrate specificities, while ABCD3 is involved in the transport of branched chain acyl-CoA into peroxisomes. On the other hand, ABCD4 is deduced to take part in the transport of vitamin B12 from lysosomes into the cytosol. It is well known that the dysfunction of ABCD1 results in X-linked adrenoleukodystrophy, a severe neurodegenerative disease. Recently, it is reported that ABCD3 and ABCD4 are responsible for hepatosplenomegaly and vitamin B12 deficiency, respectively. In this review, the targeting mechanism and physiological functions of the ABCD transporters are summarized along with the related disease. PMID:27766264

  20. Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16.

    PubMed

    Pfeiffer, Daniel; Wahl, Andreas; Jendrossek, Dieter

    2011-11-01

    A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1.

  1. A new immunoreactive recombinant protein designated as rBoSA2 from Babesia ovis: Its molecular characterization, subcellular localization and antibody recognition by infected sheep.

    PubMed

    Sevinc, Ferda; Cao, Shinuo; Zhou, Mo; Sevinc, Mutlu; Ceylan, Onur; Xuan, Xuenan

    2015-11-30

    Ovine babesiosis, caused by the intra-erythrocytic protozoan parasite Babesia ovis, is an infectious and economically important tick-borne disease of sheep. Diagnostic testing is an essential tool used for the control of the disease. In order to identify and characterize the immunoreactive proteins which are useful in serological diagnosis of the disease, a complementary DNA (cDNA) expression library was constructed from B. ovis merozoite mRNA. A cDNA clone designated as BoSA2 was identified by immunoscreening of a cDNA library using immune sheep serum. The sequence of the BoSA2 cDNA had a partial open reading frame of 1156 nucleotides encoding a polypeptide of 384 amino acid residues. Theoretical molecular mass for the mature protein was 43.5 kDa. The sequence of the BoSA2 was inserted into the expression vector pGEX-4T-1 and then expressed in Escherichia coli DH5α cells as a glutathione S-transferase (GST)-tagged fusion protein. This recombinant fusion protein (rBoSA2) was purified by GST-affinity chromatography. Immunoreactivity of the rBoSA2 was evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using the sera from the animals naturally and experimentally infected with B. ovis. ELISA results demonstrated that this antigen was useful for the diagnosis of ovine babesiosis. The localization of the BoSA2 protein was shown in and on the parasite and in the cytoplasm of the infected erythrocyte by confocal laser microscope. To our knowledge, rBoSA2 is the second immunoreactive recombinant protein of B. ovis until the present.

  2. Autographa californica nuclear polyhedrosis virus: subcellular localization and protein trafficking of BV/ODV-E26 to intranuclear membranes and viral envelopes.

    PubMed

    Beniya, H; Braunagel, S C; Summers, M D

    1998-01-05

    The Autographa californica nuclear polyhedrosis virus da26 gene codes for an envelope protein of both budded virus (BV) and occlusion derived virus (ODV). Western blot and temporal analysis of infected cell extracts detected a protein of 26 kDa by 4 h postinfection (p.i.). The amount of protein increased by 16 h p.i. and remained at high levels throughout infection. By 36 h p.i. several additional immunoreactive proteins were detected which migrated at approximately 18 kDa and remained through 96 h p.i. Western blot analysis of purified virus envelope and nucleocapsid preparations revealed that both the 26- and 18-kDa proteins are structural proteins of the envelope of BV and ODV. Immunoelectron microscopy performed at a time when only the 26-kDa species of the protein was present confirmed that the protein located to ODV envelope. The protein was named BV/ODV-E26 to designate incorporation into viral progeny, envelope location, and apparent molecular weight. Studies designed to follow localization of BV/ODV-E26 demonstrated that early in infection, the protein was incorporated into cytoplasmic vesicles and by 16 h p.i., BV/ODV-E26 was detected in the nucleus associated with virus-induced intranuclear microvesicles and ODV envelope. Coimmunoprecipitation and yeast two-hybrid assays showed that BV/ODV-E26 and FP25K were capable of interacting with each other to form a complex and coimmunoprecipitation assays indicated that cellular actin was a third component of this complex. Together, these data suggest that FP25K and cellular actin may participate in the regulation, or movement through the cell, of baculovirus proteins and/or virus nucleocapsids.

  3. Differential subcellular distribution of glucose transporters GLUT1-6 and GLUT9 in human cancer: ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues.

    PubMed

    Godoy, Alejandro; Ulloa, Viviana; Rodríguez, Federico; Reinicke, Karin; Yañez, Alejandro J; García, María de los Angeles; Medina, Rodolfo A; Carrasco, Mónica; Barberis, Sofía; Castro, Tamara; Martínez, Fernando; Koch, Ximena; Vera, Juan Carlos; Poblete, María Teresa; Figueroa, Carlos D; Peruzzo, Bruno; Pérez, Fernando; Nualart, Francisco

    2006-06-01

    It has been proposed that the enhanced metabolic activity of tumor cells is accompanied by an increased expression of facilitative hexose transporters (GLUTs). However, a previous immunohistochemical analysis of GLUT1 expression in 154 malignant human neoplasms failed to detect the GLUT1 isoform in 87 tumors. We used 146 normal human tissues and 215 tumor samples to reassess GLUT1 expression. A similar number of samples were used to compare the expression of GLUT2-6 and 9. The classical expression of GLUT1-5 in different normal human tissues was confirmed, however, we were unable to detect GLUT2 in human pancreatic islet cells. GLUT6 was principally detected in testis germinal cells and GLUT9 was localized in kidney, liver, heart, and adrenal. In tumor samples, GLUT1, 2, and 5 were the main transporters detected. GLUT1 was the most widely expressed transporter, however, 42% of the samples had very low-to-negative expression levels. GLUT2 was detected in 31% of the samples, being mainly expressed in breast, colon, and liver carcinoma. GLUT5 was detected in 27% of breast and colon adenocarcinoma, liver carcinoma, lymphomas, and testis seminoma samples. In situ RT-PCR and ultrastructural immunohistochemistry confirmed GLUT5 expression in breast cancer. GLUT6 and 9 are not clearly over-expressed in human cancer. The extensive expression of GLUT2 and 5 (glucose/fructose and fructose transporters, respectively) in malignant human tissues indicates that fructose may be a good energy substrate in tumor cells. Our functional data obtained in vitro in different tumor cells support this hypothesis. Additionally, these results suggest that fructose uptake could be used for positron emission tomography imaging and, may possibly represent a novel target for the development of therapeutic agents in different human cancers.

  4. Characterization of Xenopus egg membrane microdomains containing uroplakin Ib/III complex: roles of their molecular interactions for subcellular localization and signal transduction.

    PubMed

    Mahbub Hasan, A K M; Ou, Zhize; Sakakibara, Keiichi; Hirahara, Shino; Iwasaki, Tetsushi; Sato, Ken-ichi; Fukami, Yasuo

    2007-02-01

    A single-transmembrane protein uroplakin III (UPIII) and its tetraspanin binding-partner uroplakin Ib (UPIb) are members of the UP proteins that were originally identified in mammalian urothelium. In Xenopus laevis eggs, these proteins: xUPIII and xUPIb, are components of the cholesterol-enriched membrane microdomains or "rafts" and involved in the sperm-egg membrane interaction and subsequent egg activation signaling via Src tyrosine kinase at fertilization. Here, we investigate whether the xUPIII-xUPIb complex is in close proximity to CD9, a tetraspanin that has been implicated in the sperm-egg fusion in the mouse and GM1, a ganglioside typically enriched in egg rafts. Preparation of the egg membrane microdomains using different non-ionic detergents (Brij 98 and Triton X-100), chemical cross-linking, co-immunoprecipitation, in vitro kinase assay and in vitro fertilization experiments demonstrated that GM1, but not CD9, is in association with the xUPIII-xUPIb complex and contributes to the sperm-dependent egg activation. Transfection experiments using HEK293 cells demonstrated that xUPIII and xUPIb localized efficiently to the cholesterol-dependent membrane microdomains when they were co-expressed, whereas co-expression of xUPIII and CD9, instead of xUPIb, did not show this effect. Furthermore, xUPIII and xUPIb were shown to suppress kinase activity of the wild type, but not a constitutively active form of, Xenopus Src protein co-expressed in HEK293 cells. These results provide novel insight into the molecular architecture of the egg membrane microdomains containing xUPIII, xUPIb and Src, which may contribute to the understanding of sperm-egg interaction and signaling during Xenopus fertilization.

  5. Subcellular localization of selectively permeable aquaporins in the male germ line of a marine teleost reveals spatial redistribution in activated spermatozoa.

    PubMed

    Chauvigné, François; Boj, Mónica; Vilella, Sebastiano; Finn, Roderick Nigel; Cerdà, Joan

    2013-08-01

    In oviparous vertebrates such as the marine teleost gilthead seabream, water and fluid homeostasis associated with testicular physiology and the external activation of spermatozoa is potentially mediated by multiple aquaporins. To test this hypothesis, we isolated five novel members of the aquaporin superfamily from gilthead seabream and developed paralog-specific antibodies to localize the cellular sites of protein expression in the male reproductive tract. Together with phylogenetic classification, functional characterization of four of the newly isolated paralogs, Aqp0a, -7, -8b, and -9b, demonstrated that they were water permeable, while Aqp8b was also permeable to urea, and Aqp7 and -9b were permeable to glycerol and urea. Immunolocalization experiments indicated that up to seven paralogous aquaporins are differentially expressed in the seabream testis: Aqp0a and -9b in Sertoli and Leydig cells, respectively; Aqp1ab, -7, and -10b from spermatogonia to spermatozoa; and Aqp1aa and -8b in spermatids and sperm. In the efferent duct, only Aqp10b was found in the luminal epithelium. Ejaculated spermatozoa showed a segregated spatial distribution of five aquaporins: Aqp1aa and -7 in the entire flagellum or the head, respectively, and Aqp1ab, -8b, and -10b both in the head and the anterior tail. The combination of immunofluorescence microscopy and biochemical fractionation of spermatozoa indicated that Aqp10b and phosphorylated Aqp1ab are rapidly translocated to the head plasma membrane upon activation, whereas Aqp8b accumulates in the mitochondrion of the spermatozoa. In contrast, Aqp1aa and -7 remained unchanged. These data reveal that aquaporin expression in the teleost testis shares conserved features of the mammalian system, and they suggest that the piscine channels may play different roles in water and solute transport during spermatogenesis, sperm maturation and nutrition, and the initiation and maintenance of sperm motility.

  6. Global analysis of biogenesis, stability and sub-cellular localization of lncRNAs mapping to intragenic regions of the human genome

    PubMed Central

    Ayupe, Ana C; Tahira, Ana C; Camargo, Lauren; Beckedorff, Felipe C; Verjovski-Almeida, Sergio; Reis, Eduardo M

    2015-01-01

    Long noncoding RNAs (lncRNAs) that map to intragenic regions of the human genome with the same (intronic lncRNAs) or opposite orientation (antisense lncRNAs) relative to protein-coding mRNAs have been largely dismissed from biochemical and functional characterization due to the belief that they are mRNA precursors, byproducts of RNA splicing or simply transcriptional noise. In this work, we used a custom microarray to investigate aspects of the biogenesis, processing, stability, evolutionary conservation, and cellular localization of ∼6,000 intronic lncRNAs and ∼10,000 antisense lncRNAs. Most intronic (2,903 of 3,427, 85%) and antisense lncRNAs (4,945 of 5,214, 95%) expressed in HeLa cells showed evidence of 5′ cap modification, compatible with their transcription by RNAP II. Antisense lncRNAs (median t1/2 = 3.9 h) were significantly (p < 0.0001) more stable than mRNAs (median t1/2 = 3.2 h), whereas intronic lncRNAs (median t1/2 = 2.1 h) comprised a more heterogeneous class that included both stable (t1/2 > 3 h) and unstable (t1/2 < 1 h) transcripts. Intragenic lncRNAs display evidence of evolutionary conservation, have little/no coding potential and were ubiquitously detected in the cytoplasm. Notably, a fraction of the intronic and antisense lncRNAs (13 and 15%, respectively) were expressed from loci at which the corresponding host mRNA was not detected. The abundances of a subset of intronic/antisense lncRNAs were correlated (r ≥ |0.8|) with those of genes encoding proteins involved in cell division and DNA replication. Taken together, the findings of this study contribute novel biochemical and genomic information regarding intronic and antisense lncRNAs, supporting the notion that these classes include independently transcribed RNAs with potentials for exerting regulatory functions in the cell. PMID:26151857

  7. Differential characteristics and subcellular localization of two starch-branching enzyme isoforms encoded by a single gene in Phaseolus vulgaris L.

    PubMed

    Hamada, Shigeki; Ito, Hiroyuki; Hiraga, Susumu; Inagaki, Keisuke; Nozaki, Kouichi; Isono, Naoto; Yoshimoto, Yasushi; Takeda, Yasuhito; Matsui, Hirokazu

    2002-05-10

    Starch-branching enzymes (SBE) have a dominant role for amylopectin structure as they define chain length and frequency of branch points. We have previously shown that one of the SBE isoforms of kidney bean (Phaseolus vulgaris L.), designated PvSBE2, has a molecular mass (82 kDa) significantly smaller than those reported for isologous SBEs from pea (SBEI), maize (BEIIb), and rice (RBE3). Additionally, in contrast to the dual location of the pea SBEI in both the soluble and starch granule fractions, PvSBE2 was found only in the soluble fraction during seed development. Analysis of a pvsbe2 cDNA suggested that PvSBE2 is generated from a larger precursor with a putative plastid targeting sequence of 156 residues. Here we describe the occurrence of a larger 100-kDa form (LF-PvSBE2) of PvSBE2 found both in the soluble and starch granule fractions of the developing seeds. The determined N-terminal sequence, VKSSHDSD, of LF-PvSBE2 corresponded to a peptide sequence located 111 amino acids upstream from the N terminus of purified PvSBE2, suggesting that LF-PvSBE2 and PvSBE2 are products of the same gene. Analysis of the products by 5'-RACE (rapid amplification of cDNA ends) and reverse transcription PCR indicated that the two transcripts for pre-LF-PvSBE2 and pre-PvSBE2 are generated by alternative splicing. Recombinant LF-PvSBE2 (rLF-PvSBE2) was purified from Escherichia coli and the kinetic properties were compared with those of recombinant PvSBE2 (rPvSBE2). rLF-PvSBE2 had much higher affinity for amylopectin (K(m) = 4.4 mg/ml) than rPvSBE2 (18.4 mg/ml), whereas the V(max) of rLF-PvSBE2 (135 units/mg) for this substrate was much lower than that of rPvSBE2 (561 units/mg). These results suggest that the N-terminal extension of LF-PvSBE2 plays a critical role for localization in starch granules by altering its enzymatic properties.

  8. Subcellular localization of FOXO3a as a potential biomarker of response to combined treatment with inhibitors of PI3K and autophagy in PIK3CA-mutant cancer cells

    PubMed Central

    Young, Chan Kim; Hwan, Yun Kim; Ju, Woong; Cheol, Seung Kim

    2017-01-01

    Autophagy is the process of lysosome-mediated degradation and recycling that functions as an adaptive survival mechanism during anti-cancer therapy. Aberrant activation of the phosphoinositide-3-kinase (PI3K) pathway frequently occurs in solid tumors, including cervical cancer. However, single-agent PI3K inhibitors show modest anti-tumor efficacy in clinics. To see whether autophagy inhibition improves the efficacy of PI3K inhibitor in PIK3CA-mutant cancer cells, cells were treated with BKM120, a pan-PI3K inhibitor, and the autophagy inhibitor hydroxychloroquine (HCQ). Autophagy inhibition augmented the efficacy of BKM120 depending on PIK3CA-mutant cancer cell type. BKM120 treatment led to the nuclear accumulation of forkhead box O3 (FOXO3a) in Caski and T47D cells, which showed a synergistic effect of BKM120 and HCQ and the strong induction of autophagy. However, most FOXO3a remained in cytoplasm in C33A and ME180 cells, which did not exhibit synergy. These data suggest that BKM120-induced nuclear translocation of FOXO3a might elicit autophagy and be a critical factor determining the synergistic activity of BKM120 and HCQ in PIK3CA-mutant cancer cells. The release of FOXO3a from 14-3-3 by BV02 or 14-3-3 knockdown induced autophagy by BKM120 in C33A cells and sensitized the cells to the combined BKM120 and HCQ treatment, suggesting that cytoplasmic retention of FOXO3a by 14-3-3 even in the presence of BKM120 inhibit autophagy induction and synergistic effect of BKM120 and HCQ combination. Taken together, our study shows that subcellular localization of FOXO3a might be a potential biomarker for predicting response to the combination treatment with PI3K and autophagy inhibitors in PIK3CA-mutant cervical cancer patients. PMID:28036259

  9. N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate that trans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice

    PubMed Central

    Kaneko, Kentaro; Takamatsu, Takeshi; Inomata, Takuya; Oikawa, Kazusato; Itoh, Kimiko; Hirose, Kazuko; Amano, Maho; Nishimura, Shin-Ichiro; Toyooka, Kiminori; Matsuoka, Ken; Pozueta-Romero, Javier; Mitsui, Toshiaki

    2016-01-01

    Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1–NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)–Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2–GFP and NPP6–GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER–Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs. PMID:27335351

  10. Knock-Down of a Tonoplast Localized Low-Affinity Nitrate Transporter OsNPF7.2 Affects Rice Growth under High Nitrate Supply

    PubMed Central

    Hu, Rui; Qiu, Diyang; Chen, Yi; Miller, Anthony J.; Fan, Xiaorong; Pan, Xiaoping; Zhang, Mingyong

    2016-01-01

    The large nitrate transporter 1/peptide transporter family (NPF) has been shown to transport diverse substrates, including nitrate, amino acids, peptides, phytohormones, and glucosinolates. However, the rice (Oryza sativa) root-specific family member OsNPF7.2 has not been functionally characterized. Here, our data show that OsNPF7.2 is a tonoplast localized low-affinity nitrate transporter, that affects rice growth under high nitrate supply. Expression analysis showed that OsNPF7.2 was mainly expressed in the elongation and maturation zones of roots, especially in the root sclerenchyma, cortex and stele. It was also induced by high concentrations of nitrate. Subcellular localization analysis showed that OsNPF7.2 was localized on the tonoplast of large and small vacuoles. Heterologous expression in Xenopus laevis oocytes suggested that OsNPF7.2 was a low-affinity nitrate transporter. Knock-down of OsNPF7.2 retarded rice growth under high concentrations of nitrate. Therefore, we deduce that OsNPF7.2 plays a role in intracellular allocation of nitrate in roots, and thus influences rice growth under high nitrate supply. PMID:27826301

  11. Phenosafranin inhibits nuclear localization of transglutaminase 2 without affecting its transamidase activity.

    PubMed

    Furutani, Yutaka; Toguchi, Mariko; Shrestha, Rajan; Kojima, Soichi

    2017-03-01

    Transglutaminase 2 (TG2) localizes to the nucleus and induces apoptosis through a crosslinking inactivation of Sp1 in JHH-7 cells treated with acyclic retinoid. We screened an inhibitor suppressing transamidase activity in the nucleus without affecting transamidase activity itself. Phenosafranin was found to inhibit nuclear localization of EGFP-tagged TG2 and dose-dependently reduce nuclear transamidase activity without affecting the activity in a tube. We concluded that phenosafranin was a novel TG2 inhibitor capable of suppressing its nuclear localization.

  12. Analysis of curated and predicted plastid subproteomes of Arabidopsis. Subcellular compartmentalization leads to distinctive proteome properties.

    PubMed

    Sun, Qi; Emanuelsson, Olof; van Wijk, Klaas J

    2004-06-01

    Carefully curated proteomes of the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen of chloroplasts from Arabidopsis were assembled based on published, well-documented localizations. These curated proteomes were evaluated for distribution of physical-chemical parameters, with the goal of extracting parameters for improved subcellular prediction and subsequent identification of additional (low abundant) components of each membrane system. The assembly of rigorously curated subcellular proteomes is in itself also important as a parts list for plant and systems biology. Transmembrane and subcellular prediction strategies were evaluated using the curated data sets. The three curated proteomes differ strongly in average isoelectric point and protein size, as well as transmembrane distribution. Removal of the cleavable, N-terminal transit peptide sequences greatly affected isoelectric point and size distribution. Unexpectedly, the Cys content was much lower for the thylakoid proteomes than for the inner envelope. This likely relates to the role of the thylakoid membrane in light-driven electron transport and helps to avoid unwanted oxidation-reduction reactions. A rule of thumb for discriminating between the predicted integral inner envelope membrane and integral thylakoid membrane proteins is suggested. Using a combination of predictors and experimentally derived parameters, four plastid subproteomes were predicted from the fully annotated Arabidopsis genome. These predicted subproteomes were analyzed for their properties and compared to the curated proteomes. The sensitivity and accuracy of the prediction strategies are discussed. Data can be extracted from the new plastid proteome database (http://ppdb.tc.cornell.edu).

  13. Local network parameters can affect inter-network phase lags in central pattern generators.

    PubMed

    Jones, S R; Kopell, N

    2006-01-01

    Weakly coupled phase oscillators and strongly coupled relaxation oscillators have different mechanisms for creating stable phase lags. Many oscillations in central pattern generators combine features of each type of coupling: local networks composed of strongly coupled relaxation oscillators are weakly coupled to similar local networks. This paper analyzes the phase lags produced by this combination of mechanisms and shows how the parameters of a local network, such as the decay time of inhibition, can affect the phase lags between the local networks. The analysis is motivated by the crayfish central pattern generator used for swimming, and uses techniques from geometrical singular perturbation theory.

  14. Subcellular compartmentation of glutathione in dicotyledonous plants

    PubMed Central

    Müller, Maria

    2010-01-01

    This study describes the subcellular distribution of glutathione in roots and leaves of different plant species (Arabidopsis, Cucurbita, and Nicotiana). Glutathione is an important antioxidant and redox buffer which is involved in many metabolic processes including plant defense. Thus information on the subcellular distribution in these model plants especially during stress situations provides a deeper insight into compartment specific defense reactions and reflects the occurrence of compartment specific oxidative stress. With immunogold cytochemistry and computer-supported transmission electron microscopy glutathione could be localized in highest contents in mitochondria, followed by nuclei, peroxisomes, the cytosol, and plastids. Within chloroplasts and mitochondria, glutathione was restricted to the stroma and matrix, respectively, and did not occur in the lumen of cristae and thylakoids. Glutathione was also found at the membrane and in the lumen of the endoplasmic reticulum. It was also associated with the trans and cis side of dictyosomes. None or only very little glutathione was detected in vacuoles and the apoplast of mesophyll and root cells. Additionally, glutathione was found in all cell compartments of phloem vessels, vascular parenchyma cells (including vacuoles) but was absent in xylem vessels. The specificity of this method was supported by the reduction of glutathione labeling in all cell compartments (up to 98%) of the glutathione-deficient Arabidopsis thaliana rml1 mutant. Additionally, we found a similar distribution of glutathione in samples after conventional fixation and rapid microwave-supported fixation. Thus, indicating that a redistribution of glutathione does not occur during sample preparation. Summing up, this study gives a detailed insight into the subcellular distribution of glutathione in plants and presents solid evidence for the accuracy and specificity of the applied method. PMID:20186447

  15. Subcellular localization of the voltage-gated potassium channels Kv3.1b and Kv3.3 in the cerebellar dentate nucleus of glutamic acid decarboxylase 67-green fluorescent protein transgenic mice.

    PubMed

    Alonso-Espinaco, V; Elezgarai, I; Díez-García, J; Puente, N; Knöpfel, T; Grandes, P

    2008-09-09

    Deep cerebellar dentate nuclei are in a key position to control motor planning as a result of an integration of cerebropontine inputs and hemispheric Purkinje neurons signals, and their influence through synaptic outputs onto extracerebellar hubs. GABAergic dentate neurons exhibit broader action potentials and slower afterhyperpolarization than non-GABAergic (presumably glutamatergic) neurons. Specific potassium channels may be involved in these distinct firing profiles, particularly, Kv3.1 and Kv3.3 subunits which rapidly activate at relatively positive potentials to support the generation of fast action potentials. To investigate the subcellular localization of Kv3.1b and Kv3.3 in GAD- and GAD+ dentate neurons of glutamic acid decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice a preembedding immunocytochemical method for electron microscopy was used. Kv3.1b and Kv3.3 were in membranes of cell somata, dendrites, axons and synaptic terminals of both GAD- and GAD+ dentate neurons. The vast majority of GAD- somatodendritic membrane segments domains labeled for Kv3.1b and Kv3.3 (96.1% and 84.7%, respectively) whereas 56.2% and 69.8% of GAD- axonal membrane segments were immunopositive for these subunits. Furthermore, density of Kv3.1b immunoparticles was much higher in GAD- somatodendritic than axonal domains. As to GAD+ neurons, only 70.6% and 50% of somatodendritic membrane segments, and 53.3% and 59.5% of axonal membranes exhibited Kv3.1b and Kv3.3 labeling, respectively. In contrast to GAD- cells, GAD+ cells exhibited a higher density labeling for both Kv3 subunits at their axonal than at their somatodendritic membranes. Taken together, Kv3.1b and Kv3.3 potassium subunits are expressed in both GAD- and GAD+ cells, albeit at different densities and distribution. They likely contribute to the distinct biophysical properties of both GAD- and GAD+ neurons in the dentate nucleus.

  16. National, State, and Local Trends: Environmental Scan of Trends and Key Issues Affecting Planning.

    ERIC Educational Resources Information Center

    Kangas, Jon; Budros, Kathleen; Yoshioka, Joyce

    This document is a collection of one-page summaries, or "Trends Newsletters" (Numbers 50, 54-7, 59-60, 67-71, and 73-91), that analyze the national, state, and local trends affecting planning for the San Jose/Evergreen Community College District in California. The document is divided into two sections: External Scan and Internal Scan.…

  17. How the Relativistic Motion Affect Quantum Fisher Information and Bell Non-locality for Multipartite state

    NASA Astrophysics Data System (ADS)

    Huang, Chun Yu; Ma, Wenchao; Wang, Dong; Ye, Liu

    2017-02-01

    In this work, the quantum fisher information (QFI) and Bell non-locality of a multipartite fermionic system are investigated. Unlike the currently existing research of QFI, we focus our attention on the differences between quantum fisher information and Bell non-locality under the relativistic framework. The results show that although the relativistic motion affects the strength of the non-locality, it does not change the physical structure of non-locality. However, unlike the case of non-locality, the relativistic motion not only influence the precision of the QFI Fϕ but also broke the symmetry of the function Fϕ. The results also show that for a special multipartite system, , the number of particles of a initial state do not affect the Fθ. Furthermore, we also find that Fθ is completely unaffected in non-inertial frame if there are inertial observers. Finally, in view of the decay behavior of QFI and non-locality under the non-inertial frame, we proposed a effective scheme to battle against Unruh effect.

  18. How the Relativistic Motion Affect Quantum Fisher Information and Bell Non-locality for Multipartite state

    PubMed Central

    Huang, Chun Yu; Ma, Wenchao; Wang, Dong; Ye, Liu

    2017-01-01

    In this work, the quantum fisher information (QFI) and Bell non-locality of a multipartite fermionic system are investigated. Unlike the currently existing research of QFI, we focus our attention on the differences between quantum fisher information and Bell non-locality under the relativistic framework. The results show that although the relativistic motion affects the strength of the non-locality, it does not change the physical structure of non-locality. However, unlike the case of non-locality, the relativistic motion not only influence the precision of the QFI Fϕ but also broke the symmetry of the function Fϕ. The results also show that for a special multipartite system, , the number of particles of a initial state do not affect the Fθ. Furthermore, we also find that Fθ is completely unaffected in non-inertial frame if there are inertial observers. Finally, in view of the decay behavior of QFI and non-locality under the non-inertial frame, we proposed a effective scheme to battle against Unruh effect. PMID:28145437

  19. Temporal dynamics of a local fish community are strongly affected by immigration from the surrounding metacommunity.

    PubMed

    Stoffels, Rick J; Clarke, Kenneth Robert; Linklater, Danielle S

    2015-01-01

    A 5-year time series of annual censuses was collected from a large floodplain lake to determine how dynamics of the local fish community were affected by changes in hydrological connectivity with the surrounding metacommunity. The lake was disconnected from the metacommunity for 1 year prior to our study and remained disconnected until 3 months before our third annual census, when a flood reconnected the lake to the metacommunity. We determined how changes in connectivity affected temporal dynamics of (1) local community composition and (2) the population composition, condition, and growth of catfish, to shed light on how immigration of other species might affect local population dynamics. Before reconnection, the community was likely shaped by interactions between the local environment and species traits. The reconnection caused significant immigration and change in community composition and correlated with a significant and abrupt decline in catfish condition, growth, and abundance; effects likely due to the immigration of a competitor with a similar trophic niche: carp. The community was slow to return to its preconnection state, which may be due to dispersal traits of the fishes, and a time-lag in the recovery of the local catfish population following transient intensification of species interactions. The dynamics observed were concordant with the species sorting and mass-effects perspectives of metacommunity theory. Floods cause episodic dispersal in floodplain fish metacommunities, and so, flood frequency determines the relative importance of regional and local processes. Local processes may be particularly important to certain species, but these species may need sufficient time between floods for population increase, before the next flood-induced dispersal episode brings competitors and predators that might cause population decline. Accordingly, species coexistence in these metacommunities may be facilitated by spatiotemporal storage effects, which may in

  20. Temporal dynamics of a local fish community are strongly affected by immigration from the surrounding metacommunity

    PubMed Central

    Stoffels, Rick J; Clarke, Kenneth Robert; Linklater, Danielle S

    2015-01-01

    A 5-year time series of annual censuses was collected from a large floodplain lake to determine how dynamics of the local fish community were affected by changes in hydrological connectivity with the surrounding metacommunity. The lake was disconnected from the metacommunity for 1 year prior to our study and remained disconnected until 3 months before our third annual census, when a flood reconnected the lake to the metacommunity. We determined how changes in connectivity affected temporal dynamics of (1) local community composition and (2) the population composition, condition, and growth of catfish, to shed light on how immigration of other species might affect local population dynamics. Before reconnection, the community was likely shaped by interactions between the local environment and species traits. The reconnection caused significant immigration and change in community composition and correlated with a significant and abrupt decline in catfish condition, growth, and abundance; effects likely due to the immigration of a competitor with a similar trophic niche: carp. The community was slow to return to its preconnection state, which may be due to dispersal traits of the fishes, and a time-lag in the recovery of the local catfish population following transient intensification of species interactions. The dynamics observed were concordant with the species sorting and mass-effects perspectives of metacommunity theory. Floods cause episodic dispersal in floodplain fish metacommunities, and so, flood frequency determines the relative importance of regional and local processes. Local processes may be particularly important to certain species, but these species may need sufficient time between floods for population increase, before the next flood-induced dispersal episode brings competitors and predators that might cause population decline. Accordingly, species coexistence in these metacommunities may be facilitated by spatiotemporal storage effects, which may in

  1. Regulating Subcellular Metal Homeostasis: The Key to Crop Improvement

    PubMed Central

    Bashir, Khurram; Rasheed, Sultana; Kobayashi, Takanori; Seki, Motoaki; Nishizawa, Naoko K.

    2016-01-01

    Iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) are essential micronutrient mineral elements for living organisms, as they regulate essential cellular processes, such as chlorophyll synthesis and photosynthesis (Fe, Cu, and Mn), respiration (Fe and Cu), and transcription (Zn). The storage and distribution of these minerals in various cellular organelles is strictly regulated to ensure optimal metabolic rates. Alteration of the balance in uptake, distribution, and/or storage of these minerals severely impairs cellular metabolism and significantly affects plant growth and development. Thus, any change in the metal profile of a cellular compartment significantly affects metabolism. Different subcellular compartments are suggested to be linked through complex retrograde signaling networks to regulate cellular metal homeostasis. Various genes regulating cellular and subcellular metal distribution have been identified and characterized. Understanding the role of these transporters is extremely important to elaborate the signaling between various subcellular compartments. Moreover, modulation of the proteins involved in cellular metal homeostasis may help in the regulation of metabolism, adaptability to a diverse range of environmental conditions, and biofortification. Here, we review progress in the understanding of different subcellular metal transport components in plants and discuss the prospects of regulating cellular metabolism and strategies to develop biofortified crop plants. PMID:27547212

  2. Removal of the local geomagnetic field affects reproductive growth in Arabidopsis.

    PubMed

    Xu, Chunxiao; Wei, Shufeng; Lu, Yan; Zhang, Yuxia; Chen, Chuanfang; Song, Tao

    2013-09-01

    The influence of the geomagnetic field-removed environment on Arabidopsis growth was investigated by cultivation of the plants in a near-null magnetic field and local geomagnetic field (45 µT) for the whole growth period under laboratory conditions. The biomass accumulation of plants in the near-null magnetic field was significantly suppressed at the time when plants were switching from vegetative growth to reproductive growth compared with that of plants grown in the local geomagnetic field, which was caused by a delay in the flowering of plants in the near-null magnetic field. At the early or later growth stage, no significant difference was shown in the biomass accumulation between the plants in the near-null magnetic field and local geomagnetic field. The average number of siliques and the production of seeds per plant in the near-null magnetic field was significantly lower by about 22% and 19%, respectively, than those of control plants. These resulted in a significant reduction of about 20% in the harvest index of plants in the near-null magnetic field compared with that of the controls. These results suggest that the removal of the local geomagnetic field negatively affects the reproductive growth of Arabidopsis, which thus affects the yield and harvest index.

  3. Plasma effects on subcellular structures

    SciTech Connect

    Gweon, Bomi; Kim, Dan Bee; Jung, Heesoo; Choe, Wonho; Kim, Daeyeon; Shin, Jennifer H.

    2010-03-08

    Atmospheric pressure helium plasma treated human hepatocytes exhibit distinctive zones of necrotic and live cells separated by a void. We propose that plasma induced necrosis is attributed to plasma species such as oxygen radicals, charged particles, metastables and/or severe disruption of charged cytoskeletal proteins. Interestingly, uncharged cytoskeletal intermediate filaments are only minimally disturbed by plasma, elucidating the possibility of plasma induced electrostatic effects selectively destroying charged proteins. These bona fide plasma effects, which inflict alterations in specific subcellular structures leading to necrosis and cellular detachment, were not observed by application of helium flow or electric field alone.

  4. Understanding the Local Socio-political Processes Affecting Conservation Management Outcomes in Corbett Tiger Reserve, India

    NASA Astrophysics Data System (ADS)

    Rastogi, Archi; Hickey, Gordon M.; Badola, Ruchi; Hussain, Syed Ainul

    2014-05-01

    Several measures have been recommended to guarantee a sustainable population of tigers: sufficient inviolate spaces for a viable population, sufficient prey populations, trained and skilled manpower to guard against poaching and intrusion, banning trade in tiger products to reduce poaching, and importantly, the political will to precipitate these recommendations into implementation. Of these, the creation of sufficient inviolate spaces (generally in the form of protected areas) has created the most issues with local resource-dependent communities, often resulting in significant challenges for tiger conservation policy and management. Very little empirical research has, however, been done to understand and contextualize the local-level socio-political interactions that may influence the efficacy of tiger conservation in India. In this paper, we present the results of exploratory research into the ways in which local-stakeholder groups affect the management of Corbett Tiger Reserve (CTR). Using a combined grounded theory-case study research design, and the Institutional Analysis and Development framework for analysis, we identify the socio-political processes through which local-stakeholder groups are able to articulate their issues and elicit desirable actions from the management of CTR. Increasing our awareness of these processes can help inform the design and implementation of more effective tiger conservation management and policy strategies that have the potential to create more supportive coalitions of tiger conservation stakeholders at the local level.

  5. Understanding the local socio-political processes affecting conservation management outcomes in Corbett Tiger Reserve, India.

    PubMed

    Rastogi, Archi; Hickey, Gordon M; Badola, Ruchi; Hussain, Syed Ainul

    2014-05-01

    Several measures have been recommended to guarantee a sustainable population of tigers: sufficient inviolate spaces for a viable population, sufficient prey populations, trained and skilled manpower to guard against poaching and intrusion, banning trade in tiger products to reduce poaching, and importantly, the political will to precipitate these recommendations into implementation. Of these, the creation of sufficient inviolate spaces (generally in the form of protected areas) has created the most issues with local resource-dependent communities, often resulting in significant challenges for tiger conservation policy and management. Very little empirical research has, however, been done to understand and contextualize the local-level socio-political interactions that may influence the efficacy of tiger conservation in India. In this paper, we present the results of exploratory research into the ways in which local-stakeholder groups affect the management of Corbett Tiger Reserve (CTR). Using a combined grounded theory-case study research design, and the Institutional Analysis and Development framework for analysis, we identify the socio-political processes through which local-stakeholder groups are able to articulate their issues and elicit desirable actions from the management of CTR. Increasing our awareness of these processes can help inform the design and implementation of more effective tiger conservation management and policy strategies that have the potential to create more supportive coalitions of tiger conservation stakeholders at the local level.

  6. Sun/shade conditions affect recruitment and local adaptation of a columnar cactus in dry forests

    PubMed Central

    Miranda-Jácome, Antonio; Montaña, Carlos; Fornoni, Juan

    2013-01-01

    Background and Aims Facilitation among plants in water-limited environments (i.e. where evapotranspiration overcomes the availability of water during the growing season) has been considered a local adaptation to water and light conditions. Among cacti, early life-history stages can benefit from the facilitative effects of nurse plants that reduce solar radiation and water stress. However, whether light condition itself acts as an agent of selection through facilitation remains untested. The aim of this study was to determine (1) whether light conditions affect seedling recruitment, (2) whether the positive effect of shade on seedling recruitment is more intense under more stressful conditions and (3) whether shade condition (facilitation) reduces the magnitude of local adaptation on seedling recruitment relative to full sunlight conditions. Methods A reciprocal transplant experiment, coupled with the artificial manipulation of sun/shade conditions, was performed to test for the effects of local adaptation on germination, seedling survival and growth, using two demes of the columnar cactus Pilosocereus leucocephalus, representing different intensities of stressful conditions. Key Results Full sunlight conditions reduced recruitment success and supported the expectation of lower recruitment in more stressful environments. Significant local adaptation was mainly detected under full sunlight conditions, indicating that this environmental factor acts as an agent of selection at both sites. Conclusions The results supported the expectation that the magnitude of local adaptation, driven by the effects of facilitative nurse plants, is less intense under reduced stressful conditions. This study is the first to demonstrate that sun/shade conditions act as a selective agent accounting for local adaptation in water-limited environments, and that facilitation provided by nurse plants in these environments can attenuate the patterns of local adaptation among plants benefiting

  7. Bioavailability of purified subcellular metals to a marine fish.

    PubMed

    Guo, Feng; Yao, Jie; Wang, Wen-Xiong

    2013-09-01

    In the present study, the authors used a supply of naturally contaminated oysters to investigate how the subcellular metal distribution and the metal burden in prey affected the transfer of metals to a marine fish, the grunt Terapon jarbua. The oysters, Crassostrea hongkongensis, each with different contamination histories, were collected and separated into 3 subcellular fractions: 1) metal-rich granules, 2) cellular debris, and 3) a combined fraction of organelles, heat-denatured proteins, and metallothionein-like proteins, defined as the trophically available metal (TAM). These purified fractions showed a wide range of metal concentrations and were fed to the fish for a period of 7 d at a daily comparable feeding rate of 3% of fish body weight. After 7 d exposure, the newly absorbed metals were mainly distributed in the intestine and liver, indicating a significant tissue-specific trophic transfer, especially for Cd and Cu. The trophic transfer factors (TTFs) showed a sequence of cellular debris >TAM > metal-rich granules, suggesting the impact of subcellular distribution in prey on metal bioavailability. However, significant inverse relationships between the TTFs and the metal concentrations in diets were also found in the present study, especially for Cd and Zn. The subcellular metal compartmentalization might be less important than the metal concentration in prey influencing the trophic transfer. The authors' results have important implications for bioavailability and environmental assessment of dietary metals.

  8. SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations

    PubMed Central

    Hooper, Cornelia M.; Castleden, Ian R.; Tanz, Sandra K.; Aryamanesh, Nader; Millar, A. Harvey

    2017-01-01

    The SUBcellular location database for Arabidopsis proteins (SUBA4, http://suba.live) is a comprehensive collection of manually curated published data sets of large-scale subcellular proteomics, fluorescent protein visualization, protein-protein interaction (PPI) as well as subcellular targeting calls from 22 prediction programs. SUBA4 contains an additional 35 568 localizations totalling more than 60 000 experimental protein location claims as well as 37 new suborganellar localization categories. The experimental PPI data has been expanded to 26 327 PPI pairs including 856 PPI localizations from experimental fluorescent visualizations. The new SUBA4 user interface enables users to choose quickly from the filter categories: ‘subcellular location’, ‘protein properties’, ‘protein–protein interaction’ and ‘affiliations’ to build complex queries. This allows substantial expansion of search parameters into 80 annotation types comprising 1 150 204 new annotations to study metadata associated with subcellular localization. The ‘BLAST’ tab contains a sequence alignment tool to enable a sequence fragment from any species to find the closest match in Arabidopsis and retrieve data on subcellular location. Using the location consensus SUBAcon, the SUBA4 toolbox delivers three novel data services allowing interactive analysis of user data to provide relative compartmental protein abundances and proximity relationship analysis of PPI and coexpression partners from a submitted list of Arabidopsis gene identifiers. PMID:27899614

  9. SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations.

    PubMed

    Hooper, Cornelia M; Castleden, Ian R; Tanz, Sandra K; Aryamanesh, Nader; Millar, A Harvey

    2017-01-04

    The SUBcellular location database for Arabidopsis proteins (SUBA4, http://suba.live) is a comprehensive collection of manually curated published data sets of large-scale subcellular proteomics, fluorescent protein visualization, protein-protein interaction (PPI) as well as subcellular targeting calls from 22 prediction programs. SUBA4 contains an additional 35 568 localizations totalling more than 60 000 experimental protein location claims as well as 37 new suborganellar localization categories. The experimental PPI data has been expanded to 26 327 PPI pairs including 856 PPI localizations from experimental fluorescent visualizations. The new SUBA4 user interface enables users to choose quickly from the filter categories: 'subcellular location', 'protein properties', 'protein-protein interaction' and 'affiliations' to build complex queries. This allows substantial expansion of search parameters into 80 annotation types comprising 1 150 204 new annotations to study metadata associated with subcellular localization. The 'BLAST' tab contains a sequence alignment tool to enable a sequence fragment from any species to find the closest match in Arabidopsis and retrieve data on subcellular location. Using the location consensus SUBAcon, the SUBA4 toolbox delivers three novel data services allowing interactive analysis of user data to provide relative compartmental protein abundances and proximity relationship analysis of PPI and coexpression partners from a submitted list of Arabidopsis gene identifiers.

  10. 34 CFR 222.65 - How may a State aid program affect a local educational agency's eligibility for assistance under...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 1 2012-07-01 2012-07-01 false How may a State aid program affect a local educational agency's eligibility for assistance under section 8003(f)? 222.65 Section 222.65 Education Regulations of... Section 8003(f) of the Act § 222.65 How may a State aid program affect a local educational...

  11. Relative Preference and Localized Food Affect Predator Space Use and Consumption of Incidental Prey.

    PubMed

    Schartel, Tyler E; Schauber, Eric M

    2016-01-01

    Abundant, localized foods can concentrate predators and their foraging efforts, thus altering both the spatial distribution of predation risk and predator preferences for prey that are encountered incidentally. However, few investigations have quantified the spatial scale over which localized foods affect predator foraging behavior and consumption of incidental prey. In spring 2010, we experimentally tested how point-source foods altered how generalist predators (white-footed mice, Peromyscus leucopus) utilized space and depredated two incidental prey items: almonds (Prunus dulcis; highly profitable) and maple seeds (Acer saccharum; less profitable). We estimated mouse population densities with trapping webs, quantified mouse consumption rates of these incidental prey items, and measured local mouse activity with track plates. We predicted that 1) mouse activity would be elevated near full feeders, but depressed at intermediate distances from the feeder, 2) consumption of both incidental prey would be high near feeders providing less-preferred food and, 3) consumption of incidental prey would be contingent on predator preference for prey relative to feeders providing more-preferred food. Mouse densities increased significantly from pre- to post-experiment. Mean mouse activity was unexpectedly greatest in control treatments, particularly <15 m from the control (empty) feeder. Feeders with highly preferred food (sunflower seeds) created localized refuges for incidental prey at intermediate distances (15 to 25m) from the feeder. Feeders with less-preferred food (corn) generated localized high risk for highly preferred almonds <10 m of the feeder. Our findings highlight the contingent but predictable effects of locally abundant food on risk experienced by incidental prey, which can be positive or negative depending on both spatial proximity and relative preference.

  12. Relative Preference and Localized Food Affect Predator Space Use and Consumption of Incidental Prey

    PubMed Central

    Schartel, Tyler E.; Schauber, Eric M.

    2016-01-01

    Abundant, localized foods can concentrate predators and their foraging efforts, thus altering both the spatial distribution of predation risk and predator preferences for prey that are encountered incidentally. However, few investigations have quantified the spatial scale over which localized foods affect predator foraging behavior and consumption of incidental prey. In spring 2010, we experimentally tested how point-source foods altered how generalist predators (white-footed mice, Peromyscus leucopus) utilized space and depredated two incidental prey items: almonds (Prunus dulcis; highly profitable) and maple seeds (Acer saccharum; less profitable). We estimated mouse population densities with trapping webs, quantified mouse consumption rates of these incidental prey items, and measured local mouse activity with track plates. We predicted that 1) mouse activity would be elevated near full feeders, but depressed at intermediate distances from the feeder, 2) consumption of both incidental prey would be high near feeders providing less-preferred food and, 3) consumption of incidental prey would be contingent on predator preference for prey relative to feeders providing more-preferred food. Mouse densities increased significantly from pre- to post-experiment. Mean mouse activity was unexpectedly greatest in control treatments, particularly <15 m from the control (empty) feeder. Feeders with highly preferred food (sunflower seeds) created localized refuges for incidental prey at intermediate distances (15 to 25m) from the feeder. Feeders with less-preferred food (corn) generated localized high risk for highly preferred almonds <10 m of the feeder. Our findings highlight the contingent but predictable effects of locally abundant food on risk experienced by incidental prey, which can be positive or negative depending on both spatial proximity and relative preference. PMID:26978659

  13. Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility

    PubMed Central

    McIntosh, Victoria J.; Abou Samra, Abdul B.; Mohammad, Ramzi M.; Lasley, Robert D.

    2016-01-01

    Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter subcellular protein kinase activation, intracellular Ca2+ and contractility. Methods: Ventricular myocytes, isolated from adult Sprague Dawley rats, were treated with the cholesterol reducing agent methyl-β-cyclodextrin (MβCD, 5 mM, 1 hr, room temperature). Total cellular cholesterol levels, caveolin-3 localization, subcellular, ERK and p38 mitogen activated protein kinase (MAPK) signaling, contractility, and [Ca2+]i were assessed. Results: Treatment with MβCD reduced cholesterol levels by ~45 and shifted caveolin-3 from cytoskeleton and triton-insoluble fractions to the triton-soluble fraction, and increased ERK isoform phosphorylation in cytoskeletal, cytosolic, triton-soluble and triton-insoluble membrane fractions without altering their subcellular distributions. In contrast the primary effect of MβCD was on p38 subcellular distribution of p38α with little effect on p38 phosphorylation. Cholesterol depletion increased cardiomyocyte twitch amplitude and the rates of shortening and relaxation in conjunction with increased diastolic and systolic [Ca2+]i. Conclusions: These results indicate that acute reductions in membrane cholesterol levels differentially modulate basal cardiomyocyte subcellular MAPK signaling, as well as increasing [Ca2+]i and contractility. PMID:27441649

  14. Types and concentrations of metal ions affect local structure and dynamics of RNA

    NASA Astrophysics Data System (ADS)

    Wang, Jun; Xiao, Yi

    2016-10-01

    The roles that metal ions play in the structure and dynamics of RNA molecules are long-standing problems that have been studied extensively but are still not well understood. Here we show that metal ions have distributions around RNA molecules that strongly depend on the types and concentrations of the metal ions and also the electrostatic surface of the molecule. In particular, the ion distributions may not balance all the local electronegativity of the molecule. These ion distributions do not only greatly affect local structures but also lead to different local dynamics of RNA. We studied the effects of different ion solutions on the structure and dynamics of RNA by taking the pre Q1 riboswitch aptamer domain as an illustrative example and using molecular dynamics simulations. Since the local structures and dynamics of RNAs are important to their functions, our results also indicate that the selection of proper ion conditions is necessary to model them correctly, in contrast to the use of diverse ion solutions in current molecular dynamics simulations.

  15. Investigation of locally resonant absorption and factors affecting the absorption band of a phononic glass

    NASA Astrophysics Data System (ADS)

    Chen, Meng; Jiang, Heng; Feng, Yafei; Wang, Yuren

    2014-12-01

    We experimentally and theoretically investigated the mechanisms of acoustic absorption in phononic glass to optimize its properties. First, we experimentally studied its locally resonant absorption mechanism. From these results, we attributed its strong sound attenuation to its locally resonant units and its broadband absorption to its networked structure. These experiments also indicated that the porosity and thickness of the phononic glass must be tuned to achieve the best sound absorption at given frequencies. Then, using lumped-mass methods, we studied how the absorption bandgaps of the phononic glass were affected by various factors, including the porosity and the properties of the coating materials. These calculations gave optimal ranges for selecting the porosity, modulus of the coating material, and ratio of the compliant coating to the stiff matrix to achieve absorption bandgaps in the range of 6-30 kHz. This paper provides guidelines for designing phononic glasses with proper structures and component materials to work in specific frequency ranges.

  16. Small but Powerful: Top Predator Local Extinction Affects Ecosystem Structure and Function in an Intermittent Stream

    PubMed Central

    Rodríguez-Lozano, Pablo; Verkaik, Iraima; Rieradevall, Maria; Prat, Narcís

    2015-01-01

    Top predator loss is a major global problem, with a current trend in biodiversity loss towards high trophic levels that modifies most ecosystems worldwide. Most research in this area is focused on large-bodied predators, despite the high extinction risk of small-bodied freshwater fish that often act as apex consumers. Consequently, it remains unknown if intermittent streams are affected by the consequences of top-predators’ extirpations. The aim of our research was to determine how this global problem affects intermittent streams and, in particular, if the loss of a small-bodied top predator (1) leads to a ‘mesopredator release’, affects primary consumers and changes whole community structures, and (2) triggers a cascade effect modifying the ecosystem function. To address these questions, we studied the top-down effects of a small endangered fish species, Barbus meridionalis (the Mediterranean barbel), conducting an enclosure/exclosure mesocosm experiment in an intermittent stream where B. meridionalis became locally extinct following a wildfire. We found that top predator absence led to ‘mesopredator release’, and also to ‘prey release’ despite intraguild predation, which contrasts with traditional food web theory. In addition, B. meridionalis extirpation changed whole macroinvertebrate community composition and increased total macroinvertebrate density. Regarding ecosystem function, periphyton primary production decreased in apex consumer absence. In this study, the apex consumer was functionally irreplaceable; its local extinction led to the loss of an important functional role that resulted in major changes to the ecosystem’s structure and function. This study evidences that intermittent streams can be affected by the consequences of apex consumers’ extinctions, and that the loss of small-bodied top predators can lead to large ecosystem changes. We recommend the reintroduction of small-bodied apex consumers to systems where they have been

  17. Small but powerful: top predator local extinction affects ecosystem structure and function in an intermittent stream.

    PubMed

    Rodríguez-Lozano, Pablo; Verkaik, Iraima; Rieradevall, Maria; Prat, Narcís

    2015-01-01

    Top predator loss is a major global problem, with a current trend in biodiversity loss towards high trophic levels that modifies most ecosystems worldwide. Most research in this area is focused on large-bodied predators, despite the high extinction risk of small-bodied freshwater fish that often act as apex consumers. Consequently, it remains unknown if intermittent streams are affected by the consequences of top-predators' extirpations. The aim of our research was to determine how this global problem affects intermittent streams and, in particular, if the loss of a small-bodied top predator (1) leads to a 'mesopredator release', affects primary consumers and changes whole community structures, and (2) triggers a cascade effect modifying the ecosystem function. To address these questions, we studied the top-down effects of a small endangered fish species, Barbus meridionalis (the Mediterranean barbel), conducting an enclosure/exclosure mesocosm experiment in an intermittent stream where B. meridionalis became locally extinct following a wildfire. We found that top predator absence led to 'mesopredator release', and also to 'prey release' despite intraguild predation, which contrasts with traditional food web theory. In addition, B. meridionalis extirpation changed whole macroinvertebrate community composition and increased total macroinvertebrate density. Regarding ecosystem function, periphyton primary production decreased in apex consumer absence. In this study, the apex consumer was functionally irreplaceable; its local extinction led to the loss of an important functional role that resulted in major changes to the ecosystem's structure and function. This study evidences that intermittent streams can be affected by the consequences of apex consumers' extinctions, and that the loss of small-bodied top predators can lead to large ecosystem changes. We recommend the reintroduction of small-bodied apex consumers to systems where they have been extirpated, to restore

  18. Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs

    PubMed Central

    Wilk, Ronit; Hu, Jack; Blotsky, Dmitry; Krause, Henry M.

    2016-01-01

    In a previous analysis of 2300 mRNAs via whole-mount fluorescent in situ hybridization in cellularizing Drosophila embryos, we found that 70% of the transcripts exhibited some form of subcellular localization. To see whether this prevalence is unique to early Drosophila embryos, we examined ∼8000 transcripts over the full course of embryogenesis and ∼800 transcripts in late third instar larval tissues. The numbers and varieties of new subcellular localization patterns are both striking and revealing. In the much larger cells of the third instar larva, virtually all transcripts observed showed subcellular localization in at least one tissue. We also examined the prevalence and variety of localization mechanisms for >100 long noncoding RNAs. All of these were also found to be expressed and subcellularly localized. Thus, subcellular RNA localization appears to be the norm rather than the exception for both coding and noncoding RNAs. These results, which have been annotated and made available on a recompiled database, provide a rich and unique resource for functional gene analyses, some examples of which are provided. PMID:26944682

  19. Local to regional emission sources affecting mercury fluxes to New York lakes

    NASA Astrophysics Data System (ADS)

    Bookman, Revital; Driscoll, Charles T.; Engstrom, Daniel R.; Effler, Steven W.

    Lake-sediment records across the Northern Hemisphere show increases in atmospheric deposition of anthropogenic mercury (Hg) over the last 150 years. Most of the previous studies have examined remote lakes affected by the global atmospheric Hg reservoir. In this study, we present Hg flux records from lakes in an urban/suburban setting of central New York affected also by local and regional emissions. Sediment cores were collected from the Otisco and Skaneateles lakes from the Finger Lakes region, Cross Lake, a hypereutrophic lake on the Seneca River, and Glacial Lake, a small seepage lake with a watershed that corresponds with the lake area. Sediment accumulation rates and dates were established by 210Pb. The pre-anthropogenic regional atmospheric Hg flux was estimated to be 3.0 μg m -2 yr -1 from Glacial Lake, which receives exclusively direct atmospheric deposition. Mercury fluxes peaked during 1971-2001, and were 3 to more than 30 times greater than pre-industrial deposition. Land use change and urbanization in the Otisco and Cross watersheds during the last century likely enhanced sediment loads and Hg fluxes to the lakes. Skaneateles and Glacial lakes have low sediment accumulation rates, and thus are excellent indicators for atmospheric Hg deposition. In these lakes, we found strong correlations with emission records for the Great Lakes region that markedly increased in the early 1900s, and peaked during WWII and in the early 1970s. Declines in modern Hg fluxes are generally evident in the core records. However, the decrease in sediment Hg flux at Glacial Lake was interrupted and has increased since the early 1990s probably due to the operation of new local emission sources. Assuming the global Hg reservoir tripled since the pre-industrial period, the contribution of local and regional emission sources to central New York lakes was estimated to about 80% of the total atmospheric Hg deposition.

  20. Attention to local and global levels of hierarchical Navon figures affects rapid scene categorization.

    PubMed

    Brand, John; Johnson, Aaron P

    2014-01-01

    In four experiments, we investigated how attention to local and global levels of hierarchical Navon figures affected the selection of diagnostic spatial scale information used in scene categorization. We explored this issue by asking observers to classify hybrid images (i.e., images that contain low spatial frequency (LSF) content of one image, and high spatial frequency (HSF) content from a second image) immediately following global and local Navon tasks. Hybrid images can be classified according to either their LSF, or HSF content; thus, making them ideal for investigating diagnostic spatial scale preference. Although observers were sensitive to both spatial scales (Experiment 1), they overwhelmingly preferred to classify hybrids based on LSF content (Experiment 2). In Experiment 3, we demonstrated that LSF based hybrid categorization was faster following global Navon tasks, suggesting that LSF processing associated with global Navon tasks primed the selection of LSFs in hybrid images. In Experiment 4, replicating Experiment 3 but suppressing the LSF information in Navon letters by contrast balancing the stimuli examined this hypothesis. Similar to Experiment 3, observers preferred to classify hybrids based on LSF content; however and in contrast, LSF based hybrid categorization was slower following global than local Navon tasks.

  1. Attention to local and global levels of hierarchical Navon figures affects rapid scene categorization

    PubMed Central

    Brand, John; Johnson, Aaron P.

    2014-01-01

    In four experiments, we investigated how attention to local and global levels of hierarchical Navon figures affected the selection of diagnostic spatial scale information used in scene categorization. We explored this issue by asking observers to classify hybrid images (i.e., images that contain low spatial frequency (LSF) content of one image, and high spatial frequency (HSF) content from a second image) immediately following global and local Navon tasks. Hybrid images can be classified according to either their LSF, or HSF content; thus, making them ideal for investigating diagnostic spatial scale preference. Although observers were sensitive to both spatial scales (Experiment 1), they overwhelmingly preferred to classify hybrids based on LSF content (Experiment 2). In Experiment 3, we demonstrated that LSF based hybrid categorization was faster following global Navon tasks, suggesting that LSF processing associated with global Navon tasks primed the selection of LSFs in hybrid images. In Experiment 4, replicating Experiment 3 but suppressing the LSF information in Navon letters by contrast balancing the stimuli examined this hypothesis. Similar to Experiment 3, observers preferred to classify hybrids based on LSF content; however and in contrast, LSF based hybrid categorization was slower following global than local Navon tasks. PMID:25520675

  2. Analysis of Ras/ERK Compartmentalization by Subcellular Fractionation.

    PubMed

    Agudo-Ibañez, Lorena; Crespo, Piero; Casar, Berta

    2017-01-01

    A vast number of stimuli use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their cognate receptors, in order to regulate multiple cellular functions, including key processes such as proliferation, cell cycle progression, differentiation, and survival. The duration, intensity and specificity of the responses are, in part, controlled by the compartmentalization/subcellular localization of the signaling intermediaries. Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site-specific regulatory mechanisms, distinctively engaging effector pathways and switching-on diverse genetic programs to generate a multitude of biological responses. The Ras effector pathway leading to ERKs activation is also subject to space-related regulatory processes. About half of ERK1/2 substrates are found in the nucleus and function mainly as transcription factors. The other half resides in the cytosol and other cellular organelles. Such subcellular distribution enhances the complexity of the Ras/ERK cascade and constitutes an essential mechanism to endow variability to its signals, which enables their participation in the regulation of a broad variety of functions. Thus, analyzing the subcellular compartmentalization of the members of the Ras/ERK cascade constitutes an important factor to be taken into account when studying specific biological responses evoked by Ras/ERK signals. Herein, we describe methods for such purpose.

  3. Genetically Targeted Fluorogenic Macromolecules for Subcellular Imaging and Cellular Perturbation

    PubMed Central

    Magenau, Andrew J. D.; Saurabh, Saumya; Andreko, Susan K.; Telmer, Cheryl A.; Schmidt, Brigitte F.; Waggoner, Alan S.; Bruchez, Marcel P.

    2015-01-01

    The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration. PMID:26183934

  4. Measuring natural pest suppression at different spatial scales affects the importance of local variables.

    PubMed

    Bennett, A B; Gratton, C

    2012-10-01

    The role biodiversity plays in the provision of ecosystem services is widely recognized, yet few ecological studies have identified characteristics of natural systems that support and maintain ecosystem services. The purpose of this study was to identify landscape variables correlated with natural pest suppression carried out by arthropod natural enemies, predators and parasitoids. We conducted two field experiments, one observational and one experimental, where landscape variables at broad and local scales were measured and related to natural pest suppression. The first experiment measured natural pest suppression at 16 sites across an urban to rural landscape gradient in south central Wisconsin. We found natural enemy diversity positively affected natural pest suppression, whereas flower diversity negatively affected pest suppression. No relationship was found between natural pest suppression and broad scale variables, which measured the percentage of different land cover classes in the surrounding landscape. In the second experiment, we established small (2- by 3-m) replicated plots that experimentally varied flower diversity (0, 1, or 7 species) within a plot. We found no significant relationship between natural pest suppression and the different levels of flower diversity. The fact that we only found differences in natural pest suppression in our first experiment, which measured natural pest suppression at sites separated by larger distances than our second experiment, suggests the more appropriate scale for measuring ecosystem services performed by mobile organisms like insects, is across broad spatial scales where variation in natural enemies communities and the factors that affect them become more apparent.

  5. Imaging trace element distributions in single organelles and subcellular features

    SciTech Connect

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

    2016-02-25

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

  6. Imaging trace element distributions in single organelles and subcellular features

    PubMed Central

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

    2016-01-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies. PMID:26911251

  7. Imaging trace element distributions in single organelles and subcellular features

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

  8. 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

  9. DIRECT COMPARISON OF KINETIC AND LOCAL EQUILIBRIUM FORMULATIONS FOR SOLUTE TRANSPORT AFFECTED BY SURFACE REACTIONS.

    USGS Publications Warehouse

    Bahr, Jean M.; Rubin, Jacob

    1987-01-01

    Modeling transport of reacting solutes in porous media often requires a choice between models based on the local equilibrium assumption (LEA) and models involving reaction kinetics. Direct comparison of the mathematical formulations for these two types of transport models can aid in this choice. For cases of transport affected by surface reaction, such a comparison is made possible by a new derivation procedure. This procedure yields a kinetics-based formulation that is the sum of the LEA formulation and one or more kinetically influenced terms. The dimensionless form of the new kinetics-based formulation facilitates identification of critical parameter groupings which control the approach to transport behavior consistent with LEA model predictions. Results of numerical experiments demonstrate that criteria for LEA applicability can be expressed conveniently in terms of these parameter groupings. The derivation procedure is demonstrated for examples of surface reactions including first-order reversible sorption, Langmuir-type kinetics and binary, homovalent ion exchange.

  10. Subcellular distribution of potassium in striated muscles

    SciTech Connect

    Edelmann, L.

    1984-01-01

    Microanalytical experiments have been performed to answer the question whether the main cellular cation, K+, follows the water distribution in the striated muscle cell or whether K+ follows the distribution of negative fixed charges (beta- and gamma-carboxyl groups of aspartic and glutamic acid residues). Subcellular localization of K and/or of the K surrogates Rb, Cs, and Tl has been investigated by the following methods: Chemical precipitation of K with tetraphenylborate. Autoradiography of alkali-metals and Tl in air-dried and frozen-hydrated preparations. TEM visualization of electron dense Cs and Tl in sections of freeze-dried and plastic embedded muscle. X-ray microanalysis of air-dried myofibrils and muscle cryosections. The experiments consistently show that K, Rb, Cs, and Tl do not follow the water distribution but are mainly accumulated in the A band, especially in the marginal regions, and at Z lines. The same sites preferentially accumulate Cs or uranyl cations when sections of freeze-dried, embedded muscle are exposed to these electron microscopic stains. It is concluded that the detected uneven distribution of K, Rb, Cs, and Tl in muscle is neither a freeze-drying artifact nor an embedding artifact and may result from a weak ion binding to the beta- and gamma-carboxyl groups of cellular proteins.

  11. 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.

  12. Current Gaps in the Understanding of the Subcellular Distribution of Exogenous and Endogenous Protein TorsinA

    PubMed Central

    Harata, N. Charles

    2014-01-01

    Background An in-frame deletion leading to the loss of a single glutamic acid residue in the protein torsinA (ΔE-torsinA) results in an inherited movement disorder, DYT1 dystonia. This autosomal dominant disease affects the function of the brain without causing neurodegeneration, by a mechanism that remains unknown. Methods We evaluated the literature regarding the subcellular localization of torsinA. Results Efforts to elucidate the pathophysiological basis of DYT1 dystonia have relied partly on examining the subcellular distribution of the wild-type and mutated proteins. A typical approach is to introduce the human torsinA gene (TOR1A) into host cells and overexpress the protein therein. In both neurons and non-neuronal cells, exogenous wild-type torsinA introduced in this manner has been found to localize mainly to the endoplasmic reticulum, whereas exogenous ΔE-torsinA is predominantly in the nuclear envelope or cytoplasmic inclusions. Although these outcomes are relatively consistent, findings for the localization of endogenous torsinA have been variable, leaving its physiological distribution a matter of debate. Discussion As patients’ cells do not overexpress torsinA proteins, it is important to understand why the reported distributions of the endogenous proteins are inconsistent. We propose that careful optimization of experimental methods will be critical in addressing the causes of the differences among the distributions of endogenous (non-overexpressed) vs. exogenously introduced (overexpressed) proteins. PMID:25279252

  13. Local and regional factors affecting atmospheric mercury speciation at a remote location

    USGS Publications Warehouse

    Manolopoulos, H.; Schauer, J.J.; Purcell, M.D.; Rudolph, T.M.; Olson, M.L.; Rodger, B.; Krabbenhoft, D.P.

    2007-01-01

    Atmospheric concentrations of elemental (Hg0), reactive gaseous (RGM), and particulate (PHg) mercury were measured at two remote sites in the midwestern United States. Concurrent measurements of Hg0, PHg, and RGM obtained at Devil's Lake and Mt. Horeb, located approximately 65 km apart, showed that Hg0 and PHg concentrations were affected by regional, as well as local sources, while RGM was mainly impacted by local sources. Plumes reaching the Devil's Lake site from a nearby coal-fired power plant significantly impacted SO2 and RGM concentrations at Devil's Lake, but had little impact on Hg0. Our findings suggest that traditional modeling approaches to assess sources of mercury deposited that utilize source emissions and large-scale grids may not be sufficient to predict mercury deposition at sensitive locations due to the importance of small-scale sources and processes. We suggest the use of a receptor-based monitoring to better understand mercury source-receptor relationships. ?? 2007 NRC Canada.

  14. Local divergence of thermal reaction norms among amphibian populations is affected by pond temperature variation.

    PubMed

    Richter-Boix, Alex; Katzenberger, Marco; Duarte, Helder; Quintela, María; Tejedo, Miguel; Laurila, Anssi

    2015-08-01

    Although temperature variation is known to cause large-scale adaptive divergence, its potential role as a selective factor over microgeographic scales is less well-understood. Here, we investigated how variation in breeding pond temperature affects divergence in multiple physiological (thermal performance curve and critical thermal maximum [CTmax]) and life-history (thermal developmental reaction norms) traits in a network of Rana arvalis populations. The results supported adaptive responses to face two main constraints limiting the evolution of thermal adaptation. First, we found support for the faster-slower model, indicating an adaptive response to compensate for the thermodynamic constraint of low temperatures in colder environments. Second, we found evidence for the generalist-specialist trade-off with populations from colder and less thermally variable environments exhibiting a specialist phenotype performing at higher rates but over a narrower range of temperatures. By contrast, the local optimal temperature for locomotor performance and CTmax did not match either mean or maximum pond temperatures. These results highlight the complexity of the adaptive multiple-trait thermal responses in natural populations, and the role of local thermal variation as a selective force driving diversity in life-history and physiological traits in the presence of gene flow.

  15. Efficacy and Factors Affecting Outcome of Gemcitabine Concurrent Chemoradiotherapy in Patients With Locally Advanced Pancreatic Cancer

    SciTech Connect

    Huang, P.-I.; Chao, Yee; Li, C.-P.; Lee, R.-C.; Chi, K.-H.; Shiau, C.-Y.; Wang, L.-W.; Yen, S.-H.

    2009-01-01

    Purpose: To evaluate the efficacy and prognostic factors of gemcitabine (GEM) concurrent chemoradiotherapy (CCRT) in patients with locally advanced pancreatic cancer. Methods and Materials: Between January 2002 and December 2005, 55 patients with locally advanced pancreatic cancer treated with GEM (400 mg/m{sup 2}/wk) concurrently with radiotherapy (median dose, 50.4 Gy; range, 26-61.2) at Taipei Veterans General Hospital were enrolled. GEM (1,000 mg/m{sup 2}) was continued after CCRT as maintenance therapy once weekly for 3 weeks and repeated every 4 weeks. The response, survival, toxicity, and prognostic factors were evaluated. Results: With a median follow-up of 10.8 months, the 1- and 2-year survival rate was 52% and 19%, respectively. The median overall survival (OS) and median time to progression (TTP) was 12.4 and 5.9 months, respectively. The response rate was 42% (2 complete responses and 21 partial responses). The major Grade 3-4 toxicities were neutropenia (22%) and anorexia (19%). The median OS and TTP was 15.8 and 9.5 months in the GEM CCRT responders compared with 7.5 and 3.5 months in the nonresponders, respectively (both p < 0.001). The responders had a better Karnofsky performance status (KPS) (86 {+-} 2 vs. 77 {+-} 2, p = 0.002) and had received a greater GEM dose intensity (347 {+-} 13 mg/m{sup 2}/wk vs. 296 {+-} 15 mg/m{sup 2}/wk, p = 0.02) than the nonresponders. KPS and serum carbohydrate antigen 19-9 were the most significant prognostic factors of OS and TTP. Conclusion: The results of our study have shown that GEM CCRT is effective and tolerable for patients with locally advanced pancreatic cancer. The KPS and GEM dose correlated with response. Also, the KPS and CA 19-9 level were the most important factors affecting OS and TTP.

  16. Local Navon letter processing affects skilled behavior: a golf-putting experiment.

    PubMed

    Lewis, Michael B; Dawkins, Gemma

    2015-04-01

    Expert or skilled behaviors (for example, face recognition or sporting performance) are typically performed automatically and with little conscious awareness. Previous studies, in various domains of performance, have shown that activities immediately prior to a task demanding a learned skill can affect performance. In sport, describing the to-be-performed action is detrimental, whereas in face recognition, describing a face or reading local Navon letters is detrimental. Two golf-putting experiments are presented that compare the effects that these three tasks have on experienced and novice golfers. Experiment 1 found a Navon effect on golf performance for experienced players. Experiment 2 found, for experienced players only, that performance was impaired following the three tasks described above, when compared with reading or global Navon tasks. It is suggested that the three tasks affect skilled performance by provoking a shift from automatic behavior to a more analytic style. By demonstrating similarities between effects in face recognition and sporting behavior, it is hoped to better understand concepts in both fields.

  17. Plant sterol metabolism. Δ(7)-Sterol-C5-desaturase (STE1/DWARF7), Δ(5,7)-sterol-Δ(7)-reductase (DWARF5) and Δ(24)-sterol-Δ(24)-reductase (DIMINUTO/DWARF1) show multiple subcellular localizations in Arabidopsis thaliana (Heynh) L.

    PubMed

    Silvestro, Daniele; Andersen, Tonni Grube; Schaller, Hubert; Jensen, Poul Erik

    2013-01-01

    Sterols are crucial lipid components that regulate membrane permeability and fluidity and are the precursors of bioactive steroids. The plant sterols exist as three major forms, free sterols, steryl glycosides and steryl esters. The storage of steryl esters in lipid droplets has been shown to contribute to cellular sterol homeostasis. To further document cellular aspects of sterol biosynthesis in plants, we addressed the question of the subcellular localization of the enzymes implicated in the final steps of the post-squalene biosynthetic pathway. In order to create a clear localization map of steroidogenic enzymes in cells, the coding regions of Δ(7)-sterol-C(5)-desaturase (STE1/DWARF7), Δ(24)-sterol-Δ(24)-reductase (DIMINUTO/DWARF1) and Δ(5,7)-sterol-Δ(7)-reductase (DWARF5) were fused to the yellow fluorescent protein (YFP) and transformed into Arabidopsis thaliana mutant lines deficient in the corresponding enzymes. All fusion proteins were found to localize in the endoplasmic reticulum in functionally complemented plants. The results show that both Δ(5,7)-sterol-Δ(7)-reductase and Δ(24)-sterol-Δ(24)-reductase are in addition localized to the plasma membrane, whereas Δ(7)-sterol-C(5)-desaturase was clearly detected in lipid particles. These findings raise new challenging questions about the spatial and dynamic cellular organization of sterol biosynthesis in plants.

  18. How Are Local People Driving and Affected by Forest Cover Change? Opportunities for Local Participation in REDD+ Measurement, Reporting and Verification.

    PubMed

    Bong, Indah Waty; Felker, Mary Elizabeth; Maryudi, Ahmad

    2016-01-01

    Deforestation and forest degradation are complex and dynamic processes that vary from place to place. They are driven by multiple causes. Local communities are, to some extent, driving and also affected by some of these processes. Can their knowledge aid and add to place-specific assessment and monitoring of Deforestation and forest Degradation (DD) drivers? Our research was conducted in seven villages across three provinces of Indonesia (Papua, West Kalimantan and Central Java). Household surveys and focus group discussions were used to investigate how local community knowledge of DD drivers contributes to place-specific assessment and monitoring of DD drivers. We analyzed the link between drivers and local livelihoods to see how attempts to address deforestation and forest degradation might affect local communities and how this link might influence their participation in climate change mitigation measures such as Reducing Emissions from Deforestation and Forest Degradation (REDD+) and Measuring, Reporting and Verifying (MRV) activities. We found that local knowledge is fundamental to capturing the variety of drivers particularly in countries like Indonesia where forest and socio-economic conditions are diverse. Better understanding of drivers and their importance for local livelihoods will not only contribute to a more locally appropriate design of REDD+ and monitoring systems but will also foster local participation.

  19. How Are Local People Driving and Affected by Forest Cover Change? Opportunities for Local Participation in REDD+ Measurement, Reporting and Verification

    PubMed Central

    Bong, Indah Waty; Felker, Mary Elizabeth; Maryudi, Ahmad

    2016-01-01

    Deforestation and forest degradation are complex and dynamic processes that vary from place to place. They are driven by multiple causes. Local communities are, to some extent, driving and also affected by some of these processes. Can their knowledge aid and add to place-specific assessment and monitoring of Deforestation and forest Degradation (DD) drivers? Our research was conducted in seven villages across three provinces of Indonesia (Papua, West Kalimantan and Central Java). Household surveys and focus group discussions were used to investigate how local community knowledge of DD drivers contributes to place-specific assessment and monitoring of DD drivers. We analyzed the link between drivers and local livelihoods to see how attempts to address deforestation and forest degradation might affect local communities and how this link might influence their participation in climate change mitigation measures such as Reducing Emissions from Deforestation and Forest Degradation (REDD+) and Measuring, Reporting and Verifying (MRV) activities. We found that local knowledge is fundamental to capturing the variety of drivers particularly in countries like Indonesia where forest and socio-economic conditions are diverse. Better understanding of drivers and their importance for local livelihoods will not only contribute to a more locally appropriate design of REDD+ and monitoring systems but will also foster local participation. PMID:27806044

  20. Atrazine exposure affects the ability of crayfish (Orconectes rusticus) to localize a food odor source.

    PubMed

    Belanger, Rachelle M; Peters, Tyler J; Sabhapathy, Gita S; Khan, Sana; Katta, Juhi; Abraham, Noor K

    2015-05-01

    Environmental pollutants, found in aquatic ecosystems, have been shown to have an effect on olfactory-mediated behaviors including feeding, mate attraction, and other important social behaviors. Crayfish are polytrophic, meaning that they feed on and become prey for all levels of the aquatic food web as well as are also important for the transfer of energy between benthic and terrestrial food webs. Because crayfish are a keystone species, it is important to investigate any factors that may affect their population size. Crayfish are active at night and rely heavily on their sensory appendages (e.g., antennulues, maxillipeds, and pereopods) to localize food sources. In this experiment, we investigated the effects of atrazine (ATR) exposure on the chemosensory responses of male and female crayfish to food odors. We exposed crayfish to environmentally relevant, sublethal levels of ATR [80 ppb (µg/L)] for 72 h and then examined the behavioral responses of both ATR-treated and control crayfish to food odor delivered from one end of a test arena. We used Noldus Ethovision XT software to measure odor localization and locomotory behaviors of crayfish in response to food (fish) odor. We found that control crayfish spent more time in the proximal region of the test arena and at the odor source compared with ATR-treated crayfish. Furthermore, there were no differences in the time spent moving and not moving, total distance travelled in the tank, and walking speed (cm/s) when control and ATR-treated crayfish were compared. Overall, this indicates that acute ATR exposure alters chemosensory abilities of crayfish, whereas overall motor function remains unchanged.

  1. Localized β-adrenergic receptor blockade does not affect sweating during exercise.

    PubMed

    Buono, Michael J; Tabor, Brian; White, Ailish

    2011-05-01

    The purpose of the current study was to determine the effect of a locally administered nonselective β-adrenergic antagonist on sweat gland function during exercise. Systemically administered propranolol has been reported to increase, decrease, or not alter sweat production during exercise. To eliminate the confounding systemic effects associated with orally administered propranolol, we used iontophoresis to deliver it to the eccrine sweat glands within a localized area on one forearm prior to exercise. This allowed for determination of the direct effect of β-adrenergic receptor blockade on sweating during exercise. Subjects (n = 14) reported to the laboratory (23 ± 1°C, 35 ± 3% relative humidity) after having refrained from exercise for ≥12 h. Propranolol (1% solution) was administered to a 5-cm(2) area of the flexor surface of one forearm via iontophoresis (1.5 mA) for 5 min. A saline solution was administered to the opposing arm via iontophoresis. Each subject then exercised on a motor-driven treadmill at 75% of their age-predicted maximal heart rate for 20 min, while sweat rate was measured simultaneously in both forearms. Immediately after cessation of exercise, the number of active sweat glands was measured by application of iodine-impregnated paper to each forearm. The sweat rate for the control and propranolol-treated forearm was 0.62 ± 41 and 0.60 ± 0.44 (SD) mg·cm(-2)·min(-1), respectively (P = 0.86). The density of active sweat glands for the control and propranolol-treated forearm was 130 ± 6 and 134 ± 5 (SD) glands/cm(2), respectively, (P = 0.33). End-exercise skin temperature was 32.9 ± 0.2 and 33.1 ± 0.3°C for the control and propranolol-treated forearm, respectively (P = 0.51). Results of the current study show that when propranolol is administered locally, thus eliminating the potential confounding systemic effects of the drug, it does not directly affect sweating during the initial stages of high-intensity exercise in young, healthy

  2. A Benchtop Fractionation Procedure for Subcellular Analysis of the Plant Metabolome

    PubMed Central

    Fürtauer, Lisa; Weckwerth, Wolfram; Nägele, Thomas

    2016-01-01

    Although compartmentation is a key feature of eukaryotic cells, biological research is frequently limited by methods allowing for the comprehensive subcellular resolution of the metabolome. It has been widely accepted that such a resolution would be necessary in order to approximate cellular biochemistry and metabolic regulation, yet technical challenges still limit both the reproducible subcellular fractionation and the sample throughput being necessary for a statistically robust analysis. Here, we present a method and a detailed protocol which is based on the non-aqueous fractionation technique enabling the assignment of metabolites to their subcellular localization. The presented benchtop method aims at unraveling subcellular metabolome dynamics in a precise and statistically robust manner using a relatively small amount of tissue material. The method is based on the separation of cellular fractions via density gradients consisting of organic, non-aqueous solvents. By determining the relative distribution of compartment-specific marker enzymes together with metabolite profiles over the density gradient it is possible to estimate compartment-specific metabolite concentrations by correlation. To support this correlation analysis, a spreadsheet is provided executing a calculation algorithm to determine the distribution of metabolites over subcellular compartments. The calculation algorithm performs correlation of marker enzyme activity and metabolite abundance accounting for technical errors, reproducibility and the resulting error propagation. The method was developed, tested and validated in three natural accessions of Arabidopsis thaliana showing different ability to acclimate to low temperature. Particularly, amino acids were strongly shuffled between subcellular compartments in a cold-sensitive accession while a cold-tolerant accession was characterized by a stable subcellular metabolic homeostasis. Finally, we conclude that subcellular metabolome analysis is

  3. Cytogenetic and molecular localization of tipE: A gene affecting sodium channels in Drosophila melanogaster

    SciTech Connect

    Feng, G.; Deak, P.; Hall, L.M.

    1995-04-01

    Voltage-sensitive sodium channels play a key role in nerve cells where they are responsible for the increase in sodium permeability during the rising phase of action potentials. In Drosophila melanogaster a subset of temperature-sensitive paralytic mutations affect sodium channel function. One such mutation is temperature-induced paralysis locus E (tipE), which has been shown by electrophysiology and ligand binding studies to reduce sodium channel numbers. Three new {gamma}-ray-induced tipE alleles associated with either visible deletions in 64AB or a translocation breakpoint within 64B2 provide landmarks for positional cloning of tipE. Beginning with the flanking cloned gene Ras2, a 140-kb walk across the translocation breakpoint was completed. Germline transformation using a 42-kb cosmid clone and successively smaller subclones localized the tipE gene within a 7.4-kb genomic DNA segment. Although this chromosome region is rich in transcripts, only three overlapping mRNAs (5.4, 4.4, and 1.7 kb) lie completely within the smallest rescuing construct. The small sizes of the rescuing construct and transcripts suggests that tipE does not encode a standard sodium channel {alpha}-subunit with four homologous repeats. Sequencing these transcripts will elucidate the role of the tipE gene product in sodium channel functional regulation. 55 refs., 4 figs., 2 tabs.

  4. Cytogenetic and molecular localization of tipE: a gene affecting sodium channels in Drosophila melanogaster.

    PubMed

    Feng, G; Deák, P; Kasbekar, D P; Gil, D W; Hall, L M

    1995-04-01

    Voltage-sensitive sodium channels play a key role in nerve cells where they are responsible for the increase in sodium permeability during the rising phase of action potentials. In Drosophila melanogaster a subset of temperature-sensitive paralytic mutations affect sodium channel function. One such mutation is temperature-induced paralysis locus E (tipE), which has been shown by electrophysiology and ligand binding studies to reduce sodium channel numbers. Three new gamma-ray-induced tipE alleles associated with either visible deletions in 64AB or a translocation breakpoint within 64B2 provide landmarks for positional cloning of tipE. Beginning with the flanking cloned gene Ras2, a 140-kb walk across the translocation breakpoint was completed. Germline transformation using a 42-kb cosmid clone and successively smaller subclones localized the tipE gene within a 7.4-kb genomic DNA segment. Although this chromosome region is rich in transcripts, only three overlapping mRNAs (5.4, 4.4, and 1.7 kb) lie completely within the smallest rescuing construct. The small sizes of the rescuing construct and transcripts suggest that tipE does not encode a standard sodium channel alpha-subunit with four homologous repeats. Sequencing these transcripts will elucidate the role of the tipE gene product in sodium channel functional regulation.

  5. Seasonality and Locality Affect the Diversity of Anopheles gambiae and Anopheles coluzzii Midgut Microbiota from Ghana

    PubMed Central

    Gendrin, Mathilde; Pels, Nana Adjoa P.; Yeboah-Manu, Dorothy; Christophides, George K.; Wilson, Michael D.

    2016-01-01

    Symbiotic bacteria can have important implications in the development and competence of disease vectors. In Anopheles mosquitoes, the composition of the midgut microbiota is largely influenced by the larval breeding site, but the exact factors shaping this composition are currently unknown. Here, we examined whether the proximity to urban areas and seasons have an impact on the midgut microbial community of the two major malaria vectors in Africa, An. coluzzii and An. gambiae. Larvae and pupae were collected from selected habitats in two districts of Ghana during the dry and rainy season periods. The midgut microbiota of adults that emerged from these collections was determined by 454-pyrosequencing of the 16S ribosomal DNA. We show that in both mosquito species, Shewanellaceae constituted on average of 54% and 73% of the midgut microbiota from each site in the dry and rainy season, respectively. Enterobacteriaceae was found in comparatively low abundance below 1% in 22/30 samples in the dry season, and in 25/38 samples in the rainy season. Our data indicate that seasonality and locality significantly affect both the diversity of microbiota and the relative abundance of bacterial families with a positive impact of dry season and peri-urban settings. PMID:27322614

  6. Sub-cellular proteomics of Medicago truncatula

    PubMed Central

    Lee, Jeonghoon; Lei, Zhentian; Watson, Bonnie S.; Sumner, Lloyd W.

    2013-01-01

    Medicago truncatula is a leading model species and substantial molecular, genetic, genomics, proteomics, and metabolomics resources have been developed for this species to facilitate the study of legume biology. Currently, over 60 proteomics studies of M. truncatula have been published. Many of these have focused upon the unique symbiosis formed between legumes and nitrogen fixing rhizobia bacteria, while others have focused on seed development and the specialized proteomes of distinct tissues/organs. These include the characterization of sub-cellular organelle proteomes such as nuclei and mitochondria, as well as proteins distributed in plasma or microsomal membranes from various tissues. The isolation of sub-cellular proteins typically requires a series of steps that are labor-intensive. Thus, efficient protocols for sub-cellular fractionation, purification, and enrichment are necessary for each cellular compartment. In addition, protein extraction, solubilization, separation, and digestion prior to mass spectral identification are important to enhance the detection of low abundance proteins and to increase the overall detectable proportion of the sub-cellular proteome. This review summarizes the sub-cellular proteomics studies in M. truncatula. PMID:23641248

  7. CHARACTERIZATION OF RAT LIVER SUBCELLULAR MEMBRANES

    PubMed Central

    DeHeer, David H.; Olson, Merle S.; Pinckard, R. Neal

    1974-01-01

    The induction of acute hepatocellular necrosis in rats resulted in the production of complement fixing, IgM autoantibodies directed toward inner and outer mitochondrial membranes, microsomal membrane, lysosomal membrane, nuclear membrane, cytosol, but not to plasma membrane. Utilizing selective absorption procedures it was demonstrated that each subcellular membrane fraction possessed unique autoantigenic activity with little or no cross-reactivity between the various membrane fractions. It is proposed that the development of membrane-specific autoantibodies may provide an immunological marker useful in the differential characterization of various subcellular membranes. PMID:4813214

  8. Subcellular Neural Probes from Single-Crystal Gold Nanowires

    PubMed Central

    2014-01-01

    Size reduction of neural electrodes is essential for improving the functionality of neuroprosthetic devices, developing potent therapies for neurological and neurodegenerative diseases, and long-term brain–computer interfaces. Typical neural electrodes are micromanufactured devices with dimensions ranging from tens to hundreds of micrometers. Their further miniaturization is necessary to reduce local tissue damage and chronic immunological reactions of the brain. Here we report the neural electrode with subcellular dimensions based on single-crystalline gold nanowires (NWs) with a diameter of ∼100 nm. Unique mechanical and electrical properties of defect-free gold NWs enabled their implantation and recording of single neuron-activities in a live mouse brain despite a ∼50× reduction of the size compared to the closest analogues. Reduction of electrode dimensions enabled recording of neural activity with improved spatial resolution and differentiation of brain activity in response to different social situations for mice. The successful localization of the epileptic seizure center was also achieved using a multielectrode probe as a demonstration of the diagnostics potential of NW electrodes. This study demonstrated the realism of single-neuron recording using subcellular-sized electrodes that may be considered a pivotal point for use in diverse studies of chronic brain diseases. PMID:25112683

  9. The Push and Pull of Standards-Based Reform: How Does It Affect Local School Districts and Students with Disabilities?

    ERIC Educational Resources Information Center

    Raber, Suzanne; Roach, Virginia; Fraser, Katherine, Ed.

    This report discusses findings from case studies in California, Missouri, New Mexico, and Pennsylvania that examined ways in which state-level general and special education reforms interact, impact local districts, and affect the educational programs for students with disabilities. Chapter 1 reviews two major state-level priorities for education…

  10. Manipulating volatile emission in tobacco leaves by expressing Aspergillus nigerbeta-glucosidase in different subcellular compartments.

    PubMed

    Wei, Shu; Marton, Ira; Dekel, Mara; Shalitin, Dror; Lewinsohn, Efraim; Bravdo, Ben-Ami; Shoseyov, Oded

    2004-07-01

    Expression of the Aspergillus nigerbeta-glucosidase gene, BGL1, in Nicotiana tabacum plants (cv. Xanthi) had a profound effect on the volatile emissions of intact and crushed leaves. BGL1 was expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter and targeted to the cytoplasm, cell wall, lytic vacuole (LV), chloroplast or endoplasmic reticulum (ER). Subcellular localization was confirmed by gold immunolabelling, followed by transmission electron microscopy (TEM). Significant beta-glucosidase activity was observed in transgenic plants expressing BGL1 in the cell wall, LV and ER. Compared with controls, all intact transgenic leaves were found to emit increased levels of 2-ethylhexanol, as determined by gas chromatography-mass spectrometry (GC-MS) analysis of the headspace volatiles. Plants expressing BGL1 in the cell wall (Tcw) emitted more trans-caryophyllene than did non-transgenic controls, whereas plants expressing BGL1 in the ER (Ter) and LV (Tvc) emitted more cembrene than did non-transgenic controls. Volatiles released from crushed transgenic leaves and collected with solid-phase microextraction (SPME) polydimethylsiloxane fibre were distinctly enhanced. Significant increases in linalool, nerol, furanoid cis-linalool oxide, 4-methyl-1-pentanol, 6-methyl-hept-5-en-2-ol and 2-ethylhexanol were detected in transgenic plants when compared with wild-type controls. 3-Hydroxyl-beta-ionone levels were increased in crushed Tcw and Ter leaves, but were undetectable in Tvc leaves. The addition of glucoimidazole, a beta-glucosidase inhibitor, abolished the increased emission of these volatiles. These results indicate that the expression of a fungal beta-glucosidase gene in different subcellular compartments has the potential to affect the emission of plant volatiles, and thereby to modify plant-environment communication and aroma of agricultural products.

  11. Cell-permeable peptide-based disruption of endogenous PKA-AKAP complexes: a tool for studying the molecular roles of AKAP-mediated PKA subcellular anchoring.

    PubMed

    Faruque, Omar M; Le-Nguyen, Dung; Lajoix, Anne-Dominique; Vives, Eric; Petit, Pierre; Bataille, Dominique; Hani, El-Habib

    2009-02-01

    Stimulation of numerous G protein-coupled receptors leads to the elevation of intracellular concentrations of cAMP, which subsequently activates the PKA pathway. Specificity of the PKA signaling module is determined by a sophisticated subcellular targeting network that directs the spatiotemporal activation of the kinase. This specific compartmentalization mechanism occurs through high-affinity interactions of PKA with A-kinase anchoring proteins (AKAPs), the role of which is to target the kinase to discrete subcellular microdomains. Recently, a peptide designated "AKAPis" has been proposed to competitively inhibit PKA-AKAP interactions in vitro. We therefore sought to characterize a cell-permeable construct of the AKAPis inhibitor and use it as a tool to characterize the impact of PKA compartmentalization by AKAPs. Using insulin-secreting pancreatic beta-cells (INS-1 cells), we showed that TAT-AKAPis (at a micromolar range) dose dependently disrupted a significant fraction of endogenous PKA-AKAP interactions. Immunoflurescent analysis also indicated that TAT-AKAPis significantly affected PKA subcellular localization. Furthermore, TAT-AKAPis markedly attenuated glucagon-induced phosphorylations of p44/p42 MAPKs and cAMP response element binding protein, which are downstream effectors of PKA. In parallel, TAT-AKAPis dose dependently inhibited the glucagon-induced potentiation of insulin release. Therefore, AKAP-mediated subcellular compartmentalization of PKA represents a key mechanism for PKA-dependent phosphorylation events and potentiation of insulin secretion in intact pancreatic beta-cells. More interestingly, our data highlight the effectiveness of the cell-permeable peptide-mediated approach to monitoring in cellulo PKA-AKAP interactions and delineating PKA-dependent phosphorylation events underlying specific cellular responses.

  12. Probing cell mechanics with subcellular laser dissection of actomyosin networks in the early developing Drosophila embryo.

    PubMed

    Rauzi, M; Lenne, P-F

    2015-01-01

    Laser dissection is a useful tool in developmental biology to probe mechanical forces from the subcellular to the tissue/embryo scale. During tissue morphogenesis, cells are equipped with networks of actomyosin that generate forces. Here we present a technique based on near-infrared (NIR) femtosecond (fs) pulsed laser dissection that allows subcellular ablation of actomyosin networks. This technique allows to selectively ablate actomyosin networks while preserving cell plasma membrane. The resulting relaxation of the remaining network after laser dissection is imaged and analyzed to deduce local forces responsible for tissue morphogenesis in the developing Drosophila embryo.

  13. Socioeconomic Factors Affecting Local Support for Black Bear Recovery Strategies(AED)

    EPA Science Inventory

    There is global interest in recovering locally extirpated carnivore species. Successful efforts to recover Louisiana black bear in Louisiana have prompted interest in recovery throughout the species’ historical range. We evaluated support for three potential black bear recovery s...

  14. A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. Characterization and subcellular localization of platelet activation-dependent granule-external membrane protein.

    PubMed Central

    Berman, C L; Yeo, E L; Wencel-Drake, J D; Furie, B C; Ginsberg, M H; Furie, B

    1986-01-01

    We have identified and purified a platelet integral membrane protein (140,000 mol wt), using the KC4 monoclonal antibody specific for activated platelets, that is internal in resting platelets but exposed on activated platelets (Hsu-Lin S.-C., C.L. Berman, B.C. Furie, D. August, and B. Furie, 1984, J. Biol. Chem. 259: 9121-9126.). The expression of the protein on the platelet surface is secretion-dependent. This protein has been named platelet activation-dependent granule-external membrane (PADGEM) protein. PADGEM protein is distinct from the surface glycoproteins of resting platelets, but identical to the S12 antigen, GMP-140. Using immunofluorescent staining, resting platelets failed to stain for PADGEM protein with the KC4 antibody, but after permeabilization showed a punctate staining of the cell interior. Thrombin-stimulated intact platelets stained with a peripheral rim pattern thus demonstrating the translocation of PADGEM protein from an internal location to the cell surface. PADGEM protein expression on the platelet surface at varying thrombin concentrations correlated with alpha granule release, as measured by the secretion of platelet factor 4. Further evidence for an alpha granule localization of PADGEM protein was provided by nitrogen cavitation of resting platelets followed by metrizamide density gradient centrifugation; PADGEM protein codistributed with platelet factor 4. Using immunoelectron microscopy, the protein was localized to the alpha granule in frozen ultrathin sections of resting platelets labeled using rabbit anti-PADGEM protein antibodies, whereas in thrombin-activated platelets, the plasma membrane was labeled. These studies indicate that PADGEM protein is a component of the alpha granule membrane of resting platelets and is incorporated into the plasma membrane upon activation and secretion. Images PMID:2941452

  15. How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides

    PubMed Central

    Swiecicki, Jean-Marie; Thiebaut, Frédéric; Di Pisa, Margherita; Gourdin -Bertin, Simon; Tailhades, Julien; Mansuy, Christelle; Burlina, Fabienne; Chwetzoff, Serge; Trugnan, Germain; Chassaing, Gérard; Lavielle, Solange

    2016-01-01

    Confocal laser scanning microscopy (CLSM) is the most popular technique for mapping the subcellular distribution of a fluorescent molecule and is widely used to investigate the penetration properties of exogenous macromolecules, such as cell-penetrating peptides (CPPs), within cells. Despite the membrane-association propensity of all these CPPs, the signal of the fluorescently labeled CPPs did not colocalize with the plasma membrane. We studied the origin of this fluorescence extinction and the overall consequence on the interpretation of intracellular localizations from CLSM pictures. We demonstrated that this discrepancy originated from fluorescence self-quenching. The fluorescence was unveiled by a “dilution” protocol, i.e. by varying the ratio fluorescent/non-fluorescent CPP. This strategy allowed us to rank with confidence the subcellular distribution of several CPPs, contributing to the elucidation of the penetration mechanism. More generally, this study proposes a broadly applicable and reliable method to study the subcellular distribution of any fluorescently labeled molecules. PMID:26839211

  16. Understanding Locally, Culturally, and Contextually Relevant Mental Health Problems among Rwandan Children and Adolescents Affected by HIV/AIDS

    PubMed Central

    Betancourt, Theresa Stichick; Rubin-Smith, Julia E.; Beardslee, William R.; Stulac, Sara N.; Fayida, Ildephonse; Safren, Steven

    2011-01-01

    In assessing the mental health of HIV/AIDS-affected children and adolescents in Sub-Saharan Africa, researchers often employ mental health measures developed in other settings. However, measures derived from standard Western psychiatric criteria are frequently based on conceptual models of illness or terminology that may or may not be an appropriate for diverse populations. Understanding local perceptions of mental health problems can aid in the selection or creation of appropriate measures. This study used qualitative methodologies (Free Listing [FL], Key Informant [KI] interviews, and Clinician Interviews [C-KIs]) to understand local perceptions of mental health problems facing HIV/AIDS-affected youth in Rwinkwavu, Rwanda. Several syndrome terms were identified by participants: agahinda kenshi, kwiheba, guhangayika, ihahamuka, umushiha and uburara. While these local syndromes share some similarities with Western mood, anxiety, and conduct disorders, they also contain important culture-specific features and gradations of severity. Our findings underscore the importance of understanding local manifestations of mental health syndromes when conducting mental health assessments and when planning interventions for HIV/AIDS-affected children and adolescents in diverse settings. PMID:21271393

  17. Both population size and patch quality affect local extinctions and colonizations.

    PubMed

    Franzén, Markus; Nilsson, Sven G

    2010-01-07

    Currently, the habitat of many species is fragmented, resulting in small local populations with individuals occasionally dispersing between the remaining habitat patches. In a solitary bee metapopulation, extinction probability was related to both local bee population sizes and pollen resources measured as host plant population size. Patch size, on the other hand, had no additional predictive power. The turnover rate of local bee populations in 63 habitat patches over 4 years was high, with 72 extinction events and 31 colonization events, but the pollen plant population was stable with no extinctions or colonizations. Both pollen resources and bee populations had strong and independent effects on extinction probability, but connectivity was not of importance. Colonizations occurred more frequently within larger host plant populations. For metapopulation survival of the bee, large pollen plant populations are essential, independent of current bee population size.

  18. Madness or sadness? Local concepts of mental illness in four conflict-affected African communities

    PubMed Central

    2013-01-01

    Background Concepts of ‘what constitutes mental illness’, the presumed aetiology and preferred treatment options, vary considerably from one cultural context to another. Knowledge and understanding of these local conceptualisations is essential to inform public mental health programming and policy. Methods Participants from four locations in Burundi, South Sudan and the Democratic Republic of the Congo, were invited to describe ‘problems they knew of that related to thinking, feeling and behaviour?’ Data were collected over 31 focus groups discussions (251 participants) and key informant interviews with traditional healers and health workers. Results While remarkable similarities occurred across all settings, there were also striking differences. In all areas, participants were able to describe