115 results on '"Cytoplasm -- Genetic aspects"'
Search Results
2. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport
- Author
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Freibaum, Brian D., Lu, Yubing, Lopez-Gonzalez, Rodrigo, Kim, Nam Chul, Almeida, Sandra, Lee, Kyung-Ha, Badders, Nisha, Valentine, Marc, Miller, Bruce L., Wong, Philip C., Petrucelli, Leonard, Kim, Hong Joo, Gao, Fen-Biao, and Taylor, J. Paul
- Subjects
Cytoplasm -- Genetic aspects ,Biological transport -- Genetic aspects ,Trinucleotide repeats -- Research ,Genetic research ,Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
The GGGGCC ([G.sub.4][C.sub.2]) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia (1, 2). The basis for pathogenesis is unknown. To elucidate the consequences of [G.sub.4][C.sub.2] repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 [G.sub.4][C.sub.2]- repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. We show that these transgenic animals display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72-related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded [G.sub.4][C.sub.2] repeats in vitro and invivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded [G.sub.4][C.sub.2] repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72-related disease. These studies show that a primary consequence of [G.sub.4][C.sub.2] repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration., To explore pathogenic mechanisms of disease initiated by C9orf72 repeat expansion in a genetically tractable model organism, we used PhiC31 integrase-mediated insertion of 8, 28 or 58 copies of [G.sub.4][C.sub.2] [...]
- Published
- 2015
3. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport
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Zhang, Ke, Donnelly, Christopher J., Haeusler, Aaron R., Grima, Jonathan C., Machamer, James B., Steinwald, Peter, Daley, Elizabeth L., Miller, Sean J., Cunningham, Kathleen M., Vidensky, Svetlana, Gupta, Saksham, Thomas, Michael A., Hong, Ingie, Chiu, Shu-Ling, Huganir, Richard L., Ostrow, Lyle W., Matunis, Michael J., Wang, Jiou, Sattler, Rita, Lloyd, Thomas E., and Rothstein, Jeffrey D.
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Cytoplasm -- Genetic aspects ,Trinucleotide repeats -- Research ,Biological transport -- Genetic aspects ,Genetic research ,Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
The hexanucleotide repeat expansion (HRE) GGGGCC ([G.sub.4][C.sub.2]) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the [G.sub.4][C.sub.2] RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 [G.sub.4][C.sub.2] repeats identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention., The [G.sub.4][C.sub.2] HRE in the C9orf72 gene is found in as many as 40% of familial ALS and FTD cases, with additional reports in other neurodegenerative diseases (1-3). C9orf72 HRE-induced [...]
- Published
- 2015
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4. Addition of poly(A) and poly(A)-rich tails during RNA degradation in the cytoplasm of human cells
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Slomovic, Shimyn, Fremder, Ella, Staals, Raymond H.G., Pruijn, Ger J.M., and Schuster, Gadi
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Cytoplasm -- Genetic aspects ,Genetic transcription -- Physiological aspects ,RNA -- Synthesis ,RNA -- Research ,Science and technology - Abstract
Polyadenylation of RNA is a posttranscriptional modification that can play two somewhat opposite roles: stable polyadenylation of RNA encoded in the nuclear genomes of eukaryote cells contributes to nuclear export, translation initiation, and possibly transcript Iongevity as well. Conversely, transient polyadenylation targets RNA molecules to rapid exonucleolytic degradation. The latter role has been shown to take place in prokaryotes and organelles, as well as the nucleus of eukaryotic cells. Here we present evidence of hetero- and homopolymeric adenylation of truncated RNA molecules within the cytoplasm of human cells. RNAi-mediated silencing of the major RNA decay machinery of the cell resulted in the accumulation of these polyadenylated RNA fragments, indicating that they are degradation intermediates. Together, these results suggest that a mechanism of RNA decay, involving transient polyadenylation, is present in the cytoplasm of human cells. exosome | heteropolymeric tails | RNA polyadenylation doi/ 10.1073/pnas.0910621107
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- 2010
5. Flagellar formation in C-ring-defective mutants by overproduction of FliI, the ATPase specific for flagellar type III secretion
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Konishi, Manabu, Kanbe, Masaomi, McMurry, Jonathan L., and Aizawa, Shin-Ichi
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Flagella (Microbiology) -- Genetic aspects ,Flagella (Microbiology) -- Research ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Research ,Adenosine triphosphatase -- Genetic aspects ,Adenosine triphosphatase -- Research ,Gene mutations -- Research ,Biological sciences - Abstract
The flagellar cytoplasmic ring (C ring), which consists of three proteins, FliG, FliM, and FliN, is located on the cytoplasmic side of the flagellum. The C ring is a multifunctional structure necessary for flagellar protein secretion, torque generation, and switching of the rotational direction of the motor. The deletion of any one of the fliG, fliM, and fliN genes results in a [Fla.sup.-] phenotype. Here, we show that the overproduction of the flagellum-specific ATPase FliI overcomes the inability of basal bodies with partial C-ring structures to produce complete flagella. Flagella made upon FliI overproduction were paralyzed, indicating that an intact C ring is essential for motor function. In FliN-or FliM-deficient mutants, flagellum production was about 10% of the wild-type level, while it was only a few percent in FliG-deficient mutants, suggesting that the size of partial C rings affects the extent of flagellation. For flagella made in C-ring mutants, the hook length varied considerably, with many being markedly shorter or longer than that of the wild type. The broad distribution of hook lengths suggests that defective C rings cannot control the hook length as tightly as the wild type even though FliK and FlhB are both intact. doi: 10.1128/JB.00601-09
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- 2009
6. Mass spectrometry analysis of proteome-wide proteolytic post-translational degradation of proteins
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Shen, Yufeng, Hixson, Kim K., Tolic, Nikola, Camp, David G., Purvine, Samuel O., Moore, Ronald J., and Smith, Richard D.
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Proteolysis -- Research ,Mass spectrometry -- Methods ,Proteomics -- Research ,Genetic translation -- Research ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Properties ,Chemistry - Abstract
Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected ~1100 peptides and their ~200 protein-substrate origins we obtain evidence for new insights into the proteome-wide proteinselective degradation in yeast cells. This evidence shows that the yeast cytoplasm is the largest pool for the degradation of proteins with both biochemical and geometric specificities, whereas the yeast nucleus seems to be a proteolysis-inert organelle under the condition studied. Yeast V-ATPase subunits appear to be degraded during their disassembly, and yeast mitochondrial proteins functioning as precursors, transport carriers, and gates are preferentially degraded. Ubiquitylation may be unnecessary for the proteasomal degradation of yeast cytoplasmic regulatory and enzyme proteins according to our observations. This study shows that the intracellular peptides are informational targets for directly probing the protein degradation-involved molecular mechanisms and cell biology processes.
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- 2008
7. A viable Bacillus subtilis strain without functional extracytoplasmic function sigma genes
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Asai, Kei, Ishiwata, Keisuke, Matsuzaki, Kunihiko, and Sadaie, Yoshito
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Bacterial genetics -- Research ,Bacillus subtilis -- Genetic aspects ,Bacillus subtilis -- Physiological aspects ,Chromosome deletion -- Influence ,Gene mutations -- Influence ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Properties ,Biological sciences - Abstract
We constructed a Bacillus subtilis Marburg strain that harbors deletion mutations in all seven extracytoplasmic function (ECF) sigma genes. The strain shows wild-type growth at 37[degrees]C both in a complex and in a synthetic medium and exhibits wild-type sporulation. ECF sigma genes of B. subtilis are dispensable as long as no stress is imposed, although they seem to be required for quick response to stresses.
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- 2008
8. Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin
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Garzon, Ramiro, Garofalo, Michela, Martelli, Maria Paola, Briesewitz, Roger, Wang, Lisheng, Fernandez-Cymering, Cecilia, Volinia, Stefano, Liu, Chang-Gong, Schnittger, Susanne, Haferlach, Torsten, Liso, Arcangelo, Diverio, Daniela, Mancini, Marco, Meloni, Giovanna, Foa, Robin, Martelli, Massimo F., Mecucci, Cristina, Croce, Carlo M., and Falini, Brunangelo
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RNA -- Properties ,Cytoplasm -- Genetic aspects ,Gene mutations -- Influence ,Homeobox genes -- Properties ,Homeobox genes -- Influence ,Science and technology - Abstract
Acute myeloid leukemia (AML) carrying NPM1 mutations and cytoplasmic nucleophosmin (NPMc+ AML) accounts for about one-third of adult AML and shows distinct features, including a unique gene expression profile. MicroRNAs (miRNAs) are small noncoding RNAs of 19-25 nucleotides in length that have been linked to the development of cancer. Here, we investigated the role of miRNAs in the biology of NPMc+ AML. The miRNA expression was evaluated in 85 adult de novo AML patients characterized for subcellular localization/ mutation status of NPM1 and FLT3 mutations using a custom microarray platform. Data were analyzed by using univariate t test within BRB tools. We identified a strong miRNA signature that distinguishes NPMc+ mutated (n = 55) from the cytoplasmic-negative (NPM1 unmutated) cases (n = 30) and includes the up-regulation of miR-10a, miR-10b, several let-7 and miR-29 family members. Many of the down-regulated miRNAs including miR-204 and miR-128a are predicted to target several HOX genes. Indeed, we confirmed that miR-204 targets HOXA10 and MEIS1, suggesting that the HOX upregulation observed in NPMc+ AML may be due in part by loss of HOX regulators-miRNAs. FLT3-ITD+ samples were characterized by upregulation of miR-155. Further experiments demonstrated that the up-regulation of miR-155 was independent from FLT3 signaling. Our results identify a unique miRNA signature associated with NPMc+ AML and provide evidence that support a role for miRNAs in the regulation of HOX genes in this leukemia subtype. Moreover, we found that miR-155 was strongly but independently associated with FLT3-ITD mutations. FLT3-ITD | HOX | NPM1
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- 2008
9. Renal cystic diseases: diverse phenotypes converge on the cilium/centrosome complex
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Guay-Woodford, Lisa M.
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Chronic kidney failure -- Development and progression ,Chronic kidney failure -- Risk factors ,Cytoplasm -- Genetic aspects ,Kidney stones -- Complications and side effects ,Kidney stones -- Development and progression - Abstract
Abstract Inherited renal cystic diseases constitute an important set of single-gene disorders that frequently progress to end stage renal disease (ESRD). Transmitted as autosomal dominant, autosomal recessive, or X-linked traits, [...]
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- 2006
10. Targeted expression of the inositol 1,4,5-triphosphate receptor (I[P.sub.3]R)ligand-binding domain releases [Ca.sup.2+] via endogenous I[P.sub.3]R channels
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Varnai, Peter, Balla, Andras, Hunyady, Laszlo, and Balla, Tamas
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Cytoplasm -- Research ,Cytoplasm -- Genetic aspects ,Genetic research ,Science and technology - Abstract
Virtually all functions of a cell are influenced by cytoplasmic [[Ca.sup.2+]] increases. Inositol 1,4,5-trisphosphate receptor (I[P.sub.3]R) channels, located in the endoplasmic reticulum (ER), release [Ca.sup.2+] in response to binding of the second messenger, I[P.sub.3]. I[P.sub.3]Rs thus are part of the information chain interpreting external signals and transforming them into cytoplasmic [Ca.sup.2+] transients. I[P.sub.3]Rs function as tetramers, each unit comprising an N-terminal ligand-binding domain (LBD) and a C-terminal channel domain linked by a long regulatory region. It is not yet understood how the binding of I[P.sub.3] to the LBD regulates the gating properties of the channel. Here, we use the expression of I[P.sub.3] binding protein domains tethered to the surface of the endoplasmic reticulum (ER) to show that the all-helical domain of the I[P.sub.3]R LBD is capable of depleting the ER [Ca.sup.2+] pools by opening the endogenous I[P.sub.3]Rs, even without I[P.sub.3] binding. This effect requires the domain to be within 50 [Angstrom] of the ER membrane and is impaired by the presence of the N-terminal inhibitory segment on the LBD. These findings raise the possibility that the helical domain of the LBD functions as an effector module possibly interacting with the channel domain, thereby being part of the gating mechanisms by which the I[P.sub.3]-induced conformational change within the LBD regulates [Ca.sup.2+] release. [Ca.sup.2+] channel | endoplasmic reticulum | red fluorescent protein
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- 2005
11. SUMO: ligases, isopeptidases and nuclear pores
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Melchior, Frauke, Schergaut, Marion, and Pichler, Andrea
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Biochemistry -- Research ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Enzymes -- Genetic aspects ,Enzymes -- Physiological aspects ,Ligases -- Genetic aspects ,Ligases -- Physiological aspects ,Proteins -- Genetic aspects ,Proteins -- Physiological aspects ,Ubiquitin -- Physiological aspects ,Yeast fungi -- Genetic aspects ,Yeast fungi -- Physiological aspects ,Biological sciences ,Chemistry - Abstract
Small ubiquitin-related modifier (SUMO) proteins are reversibly coupled to numerous intracellular targets and modulate their interactions, localization, activity or stability. Recent advances in the SUMO field have uncovered the first SUMO E3 ligases and point to a complex family of isopeptidases. SUMO has been linked to many different pathways, including nucleocytoplasmic transport. Modifying enzymes and an isopeptidase have been detected at nuclear pore complexes. In addition, studies in yeast suggest a requirement of SUMO conjugation for nuclear protein import, and specific SUMO targets depend on modification for nuclear import or export.
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- 2003
12. APP processing is regulated by cytoplasmic phosphorylation
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Lee, Ming-Sum, Kao, Shih-Chu, Lemere, Cynthia A., Xia, Weiming, Tseng, Huang-Chun, Zhou, Ying, Neve, Rachael, Ahlijanian, Michael K., and Tsai, Li-Huei
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Cytoplasm -- Genetic aspects ,Genetic regulation -- Physiological aspects ,Genetic markers -- Physiological aspects ,Secretion -- Genetic aspects ,Viral antibodies -- Genetic aspects ,Antibodies -- Genetic aspects ,Phosphorylation -- Analysis ,Peptides -- Genetic aspects ,Peptides -- Physiological aspects ,Amyloid beta-protein -- Physiological aspects ,Amyloid beta-protein -- Genetic aspects ,Gene mutations -- Physiological aspects ,Cytology -- Research ,Biological sciences - Abstract
Amyloid-[beta] peptide (A[beta]) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the [beta]secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by [alpha]-secretase. The production of A[beta] is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase A[beta] generation.
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- 2003
13. Limitations of allotopic expression of mitochondrial genes in mammalian cells
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Oca-Cossio, Jose, Kenyon, Lesley, Hao, Huiling, and Moraes, Carlos T.
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Genetic research -- Analysis ,Polypeptides -- Genetic aspects ,Mitochondrial DNA -- Genetic aspects ,Cytoplasm -- Genetic aspects ,Gene expression -- Physiological aspects ,DNA -- Genetic aspects ,Biological sciences - Abstract
The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria.
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- 2003
14. Uncovering multiple axonal targeting pathways in hippocampal neurons
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Wisco, Dolora, Anderson, Eric D., Chang, Michael C., Norden, Caren, Boiko, Tatiana, Folsch, Heike, and Winckler, Bettina
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Axons -- Genetic aspects ,Genetic regulation -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Endocytosis -- Genetic aspects ,Endocytosis -- Analysis ,Cell membranes -- Genetic aspects ,Cell membranes -- Physiological aspects ,Cell adhesion -- Physiological aspects ,Cell adhesion -- Genetic aspects ,Neurons -- Genetic aspects ,Neurons -- Physiological aspects ,Membrane proteins -- Genetic aspects ,Membrane proteins -- Physiological aspects ,Cytology -- Research ,Biological sciences - Abstract
Neuronal polarity is, at least in part, mediated by the differential sorting of membrane proteins to distinct domains, such as axons and somata/dendrites. We investigated the pathways underlying the subcellular targeting of NgCAM, a cell adhesion molecule residing on the axonal plasma membrane. Following transport of NgCAM kinetically, surprisingly we observed a transient appearance of NgCAM on the somatodendritic plasma membrane. Down-regulation of endocytosis resulted in loss of axonal accumulation of NgCAM, indicating that the axonal localization of NgCAM was dependent on endocytosis. Our data suggest the existence of a dendrite-to-axon transcytotic pathway to achieve axonal accumulation. NgCAM mutants with a point mutation in a crucial cytoplasmic tail motif (YRSL) are unable to access the transcytotic route. Instead, they were found to travel to the axon on a direct route. Therefore, our results suggest that multiple distinct pathways operate in hippocampal neurons to achieve axonal accumulation of membrane proteins.
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- 2003
15. NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization
- Author
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Raemaekers, Tim, Ribbeck, Katharina, Beaudouin, Joel, Annaert, Wim, Van Camp, Mark, Stockmans, Ingrid, Smets, Nico, Bouillon, Roger, Ellenberg, Jan, and Carmeliet, Geert
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Chromosomes -- Genetic aspects ,Chromosomes -- Physiological aspects ,Cell proliferation -- Physiological aspects ,Cell proliferation -- Genetic aspects ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Microtubules -- Genetic aspects ,Microtubules -- Physiological aspects ,Cell division -- Physiological aspects ,Cell division -- Genetic aspects ,Gene expression -- Physiological aspects ,Proteins -- Physiological aspects ,Proteins -- Genetic aspects ,Cytology -- Research ,Biological sciences - Abstract
Here, we report on the identification of nucleolar spindle-associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.
- Published
- 2003
16. Nup358 integrates nuclear envelope breakdown with kinetochore assembly
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Salina, Davide, Enarson, Paul, Rattner, J.B., and Burke, Brian
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Proteins -- Physiological aspects ,Proteins -- Genetic aspects ,Metazoa -- Physiological aspects ,Metazoa -- Genetic aspects ,Mitosis -- Genetic aspects ,Mitosis -- Physiological aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Chromosomes -- Physiological aspects ,Chromosomes -- Genetic aspects ,Cell research -- Analysis ,Kinetochores ,Biological sciences - Abstract
Nuclear envelope breakdown (NEBD) and release of condensed chromosomes into the cytoplasm are key events in the early stages of mitosis in metazoans. NEBD involves the disassembly of all major structural elements of the nuclear envelope, including nuclear pore complexes (NPCs), and the dispersal of nuclear membrane components. The breakdown process is facilitated by microtubules of the mitotic spindle. After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation. Several NPC subunits relocate to kinetochores after NEBD. siRNA-mediated depletion of one of these proteins, Nup358, reveals that it is essential for kinetochore function. In the absence of Nup358, chromosome congression and segregation are severely perturbed. At the same time, the assembly of other kinetochore components is strongly inhibited, leading to aberrant kinetochore structure. The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function. Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.
- Published
- 2003
17. Centrosome positioning in interphase cells
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Burakov, Anton, Nadezhdina, Elena, Slepchenko, Boris, and Rodionov, Vladimir
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Dynein -- Physiological aspects ,Dynein -- Genetic aspects ,Microtubules -- Physiological aspects ,Microtubules -- Genetic aspects ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Centrosomes -- Physiological aspects ,Centrosomes -- Genetic aspects ,Cell research -- Analysis ,Biological sciences - Abstract
The position of the centrosome is actively maintained at the cell center, but the mechanisms of the centering force remain largely unknown. It is known that centrosome positioning requires a radial array of cytoplasmic microtubules (MTs) that can exert pushing or pulling forces involving MT dynamics and the activity of cortical MT motors. It has also been suggested that actomyosin can play a direct or indirect role in this process. To examine the centering mechanisms, we introduced an imbalance of forces acting on the centrosome by local application of an inhibitor of MT assembly (nocodazole), and studied the resulting centrosome displacement. Using this approach in combination with microinjection of function-blocking probes, we found that a MT-dependent dynein pulling force plays a key role in the positioning of the centrosome at the cell center, and that other forces applied to the centrosomal MTs, including actomyosin contractility, can contribute to this process.
- Published
- 2003
18. Overproduction of inactive variants of the murein synthase PBP1B causes lysis in Escherichia coli
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Meisel, Ute, Holtje, Joachim-Volker, and Vollmer, Waldemar
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Cells -- Genetic aspects ,Cytoplasm -- Genetic aspects ,Peptidoglycans -- Genetic aspects ,Binding proteins -- Genetic aspects ,Escherichia coli -- Genetic aspects ,Penicillin -- Physiological aspects ,Bacteriology -- Research ,Biological sciences - Abstract
Penicillin-binding protein 1B (PBP1B) of Escherichia coil is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms ([alpha], [beta], and [gamma]) which differ in the length of their N-terminal cytoplasmic region. Expression piilasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lyric transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a muitienzyme complex is discussed.
- Published
- 2003
19. Calcium gradient dependence of Neurospora crassa hyphal growth
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Silverman-Gavrila, Lorelei B. and Lew, Roger R.
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Neurospora -- Physiological aspects ,Neurospora -- Genetic aspects ,Calcium, Dietary -- Physiological aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Microbiology -- Research ,Biological sciences - Abstract
A tip-high cytoplasmic calcium gradient has been identified as a requirement for hyphal growth in the fungus Neurospora crassa. The [Ca.sup.2+] gradient is less steep compared to wall vesicle, wall incorporation and vesicular [Ca.sup.2+] gradients, but this can be explained by [Ca.sup.2+] diffusion. Analysis of the relation between the rate of hyphal growth and the spatial distribution of tip-localized calcium indicates that hyphal growth rates depend upon the tip-localized calcium concentration. It is not the steepness of the calcium gradient, but tip-localized calcium and the difference in tip-localized calcium versus subapical calcium concentration which correlate closely with hyphal growth rate. A minimal concentration difference between the apex and subapical region of 30 nM is required for growth to occur. The calcium concentration dependence of growth may relate directly to biochemical functions of calcium in hyphal extension, such as vesicle fusion and enzyme activation during cellular expansion. Initiation of tip growth may rely upon random [Ca.sup.2+] motions causing localized regions of elevated calcium. Continued hyphal expansion may activate a stretch-activated phospholipase C which would increase tip-localized inositol 1,4,5-trisphosphate (I[P.sub.3]). Hyphal expansion, induced by mild hypoosmotic treatment, does increase diacylglycerol, the other product of phospholipase C activity. This is consistent with evidence that I[P.sub.3]-activated [Ca.sup.2+] channels generate and maintain the tip-high calcium gradient.
- Published
- 2003
20. Regulation of the Bacillus subtilis extracytoplasmic function protein [[sigma].sup.y] and its target promoters
- Author
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Cao, Min, Salzberg, Letal, Tsai, Ching Sung, Mascher, Thorsten, Bonilla,Carla, Wang,Tao, Ye, Rick W., Marquez-Magana, Leticia, and Helmann, John D.
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Cytoplasm -- Genetic aspects ,Genetic transcription -- Physiological aspects ,Gene mutations -- Physiological aspects ,Genetic regulation -- Physiological aspects ,Gene expression -- Physiological aspects ,Operons -- Genetic aspects ,Bacillus subtilis -- Genetic aspects ,Bacteriology -- Research ,Biological sciences - Abstract
The Bacillus subtilis extracytoplasmic function sigma factor [[sigma].sup.Y] is of unknown function. We demonstrate that the sigY operon is expressed from an autoregulatory promoter site, [P.sub.Y]. We selected for transposon-induced mutations that upregulate [P.sub.Y] transcription in an attempt to identify genes involved in [sigma].sup.Y] regulation. The resulting insertions disrupted yxlC, the gene immediately downstream of sigY. However, the phenotype of the yxlC::Tn10 insertion was due to polarity on the downstream genes of the sigY operon; a nonpolar insertion in yxlC did not lead to derepression of [P.sub.y] Further analyses revealed that both yxlD and yxlE encoded proteins important for the negative regulation of [[sigma].sup.Y] activity. A comparison of the transcriptomes of wild-type and yxlC::Tn10 mutant strains revealed elevated expression of several operons. However, only one additional gene, ybgB, was unambiguously identified as a direct target for [[sigma].sup.y] This was supported by analysis of direct targets for [[sigma].sup.Y] transcription with whole-genome runoff transcription followed by macroarray analysis.
- Published
- 2003
21. Probing conservation of HAMP linker structure and signal transduction mechanism through analysis of hybrid sensor kinases
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Appleman, J. Alex, Chen, Li-Ling, and Stewart, Valley
- Subjects
Nitrites -- Physiological aspects ,Nitrates -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Escherichia coli -- Genetic aspects ,Chemotaxis -- Physiological aspects ,Chemotaxis -- Genetic aspects ,Ligands (Biochemistry) ,Bacteriology -- Research ,Biological sciences - Abstract
The HAMP linker, a predicted structural element observed in many sensor kinases and methyl-accepting chemotaxis proteins, transmits signals between sensory input modules and output modules. HAMP linkers are located immediately inside the cytoplasmic membrane and are predicted to form two short amphipathic [alpha]-helices (AS-1 and AS-2) joined by an unstructured connector. HAMP linkers are found in the Escherichia coli nitrate- and nitrite-responsive sensor kinases NarX and NarQ (which respond to ligand by increasing kinase activity) and the sensor kinase CpxA (which responds to ligand by decreasing kinase activity). We constructed a series of hybrids with fusion points throughout the HAMP linker, in which the sensory modules of NarX or NarQ are fused to the transmitter modules of NarX, NarQ, or CpxA. A hybrid of the NarX sensor module and the CpxA HAMP linker and transmitter module (NarX-CpxA. 1) responded to nitrate by decreasing kinase activity, whereas a hybrid in which the HAMP linker of NarX was replaced by that of CpxA (NarX-CpxA-NarX-1) responded to nitrate by increasing kinase activity. However, sequence variations between HAMP linkers do not allow free exchange of HAMP linkers or their components. Certain deletions in the NarX HAMP linker resulted in characteristic abnormal responses to ligand; similar deletions in the NarQ and NarX-CpxA-1 HAMP linkers resulted in responses to ligand generally similar to those seen in NarX. We conclude that the structure and action of the HAMP linker are conserved and that the HAMP linker transmits a signal to the output domain that ligand is bound.
- Published
- 2003
22. Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane
- Author
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Llamas, Maria A., Rodriguez-Herva, Jose J., Hancock, Robert E.W., Bitter, Wilbert, Tommassen, Jan, and Ramos, Juan L.
- Subjects
Membrane proteins -- Genetic aspects ,Glycerol -- Physiological aspects ,Glycerin -- Physiological aspects ,Arginine -- Physiological aspects ,Fructose -- Physiological aspects ,Pseudomonas -- Genetic aspects ,Cytoplasm -- Genetic aspects ,Cell membranes -- Genetic aspects ,Gram-negative bacteria -- Genetic aspects ,Gram-negative bacteria -- Growth ,Bacterial proteins -- Genetic aspects ,Bacteriology -- Research ,Company growth ,Biological sciences - Abstract
Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.
- Published
- 2003
23. Interplay of the Czc system and two p-type ATPases in conferring metal resistance to Ralstonia metallidurans
- Author
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Legatzki, Antje, Grass, Gregor, Anton, Andreas, Rensing, Christopher, and Nies, Dietrich H.
- Subjects
Cytoplasm -- Genetic aspects ,Adenosine triphosphatase -- Physiological aspects ,Gene expression -- Physiological aspects ,Escherichia coli -- Genetic aspects ,Cells -- Genetic aspects ,Zinc -- Physiological aspects ,Cadmium -- Physiological aspects ,Bacteriology -- Research ,Biological sciences - Abstract
Cadmium and zinc are removed from cells of Ralstonia metallidurans by the CzcCBA efflux pump and by two soft-metal-transporting P-type ATPases, CadA and ZntA. The czcCBA genes are located on plasmid pMOL30, and the cadA and zntA genes are on the bacterial chromosome. Expression of zntA from R. metallidurans in Escherichia coli predominantly mediated resistance to zinc, and expression of cadA predominantly mediated resistance to cadmium. Both transporters decreased the cellular content of zinc or cadmium in this host. In the plasmid-free R. metallidurans strain AE104, single gene deletions of cadA or zntA had only a moderate effect on cadmium and zinc resistance, but zinc resistance decreased 6-fold and cadmium resistance decreased 350-fold in double deletion strains. Neither single nor double gene deletions affected zinc resistance in the presence of czcCBA. In contrast, cadmium resistance of the cadA zntA double mutant could be elevated only partially by the presence of CzcCBA. lacZ reporter gene fusions indicated that expression of cadA was induced by cadmium but not by zinc in R. metallidurans strain AE104. In the absence of the zntA gene, expression of cadA occurred at lower cadmium concentrations and zinc now served as an inducer. In contrast, expression of zntA was induced by both zinc and cadmium, and the induction pattern did not change in the presence or absence of CadA. However, expression of both genes, zntA and cadA, was diminished in the presence of CzcCBA. This indicated that CzcCBA efficiently decreased cytoplasmic cadmium and zinc concentrations. It is discussed whether these data favor a model in which the cations are removed either from the cytoplasm or the periplasm by CzcCBA.
- Published
- 2003
24. The cytoplasmic domain of the plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking
- Author
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Gilberger, Tim-Wolf, Thompson, Jennifer K., Reed, Michael B., Good, Robert T., and Cowman, Alan F.
- Subjects
Gene expression -- Physiological aspects ,Erythrocytes -- Physiological aspects ,Erythrocytes -- Genetic aspects ,Plasmodium falciparum -- Physiological aspects ,Plasmodium falciparum -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Binding proteins -- Physiological aspects ,Binding proteins -- Genetic aspects ,Cytology -- Research ,Biological sciences - Abstract
The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein--protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.
- Published
- 2003
25. Critical roles for the COOH-terminal NITY and RGT sequences of the integrin [[beta].sub.3] cytoplasmic domain in inside-out and outside-in signaling
- Author
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Xi, Xiaodong, Bodnar, Richard J., Li, Zhenyu, Lam, Stephen C.-T., and Du, Xiaoping
- Subjects
Calcium compounds -- Physiological aspects ,Gene mutations -- Physiological aspects ,Glycoproteins -- Genetic aspects ,Glycoproteins -- Physiological aspects ,Cell adhesion -- Physiological aspects ,Cell adhesion -- Genetic aspects ,Gene expression -- Physiological aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Integrins -- Physiological aspects ,Integrins -- Genetic aspects ,Cytology -- Research ,Biological sciences - Abstract
Bidirectional signaling of integrin [[alpha].sub.[parallel]b][[beta].sub.3] requires the [[beta].sub.3] cytoplasmic domain. To determine the sequence in the [[beta].sup.3] cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein lb-IX, integrin [[alpha].sub.[parallel]b], and mutants of [[beta].sub.3] with truncations at sites COOH terminal to [T.sup.741], [Y.sup.747], [F.sup.754], and [Y.sup.759]. Truncation at [Y.sup.759] did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of [beta]3 is important for outside-in signaling but not inside-out signaling. In contrast, truncation at [F.sup.754], [Y.sup.747, or [T.sup.741] completely abolished integrin activation. A point mutation replacing [Y.sup.759 with alanine also abolished integrin activation. Thus, the [T.sup.755]NIT[Y.sup.759] sequence of [[beta].sub.3], containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at [Y.sup.759] in a population of [[beta].sub.3] during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.
- Published
- 2003
26. A novel role for dp115 in the organization of tER sites in Drosophila
- Author
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Kondylis, Vangelis and Rabouille, Catherine
- Subjects
Proteins -- Physiological aspects ,Proteins -- Genetic aspects ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Immunofluorescence -- Usage ,Fluorescent antibody technique -- Usage ,Golgi apparatus -- Genetic aspects ,Golgi apparatus -- Physiological aspects ,RNA -- Genetic aspects ,Drosophila -- Physiological aspects ,Drosophila -- Genetic aspects ,Cytology -- Research ,Biological sciences - Abstract
Here, we describe that depletion of the Drosophila homologue of p115 (dp115) by RNA interference in Drosophila S2 cells led to important morphological changes in the Golgi stack morphology and the transitional ER (tER) organization. Using conventional and immunoelectron microscopy and confocal immunofluorescence microscopy, we show that Golgi stacks were converted into clusters of vesicles and tubules, and that the tERs (marked by Sec23p) lost their focused organization and were now dispersed throughout the cytoplasm. However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays. The effects were specific for dp115. Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p. Taken together, these studies suggest that dp115 could be implicated in the architecture of both the Golgi stacks and the tER sites.
- Published
- 2003
27. Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells
- Author
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Tucker, Ward C., Edwardson, J. Michael, Bai, Jihong, Kim, Hyun-Jung, Martin, Thomas F.J., and Chapman, Edwin R.
- Subjects
Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Gene expression -- Physiological aspects ,Calcium compounds -- Physiological aspects ,Secretion -- Physiological aspects ,Secretion -- Genetic aspects ,Neurons -- Physiological aspects ,Neurons -- Genetic aspects ,Cell membranes -- Physiological aspects ,Cell membranes -- Genetic aspects ,Genetic regulation -- Physiological aspects ,Cytology -- Research ,Biological sciences - Abstract
The synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the [Ca.sup.2+]-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative [Ca.sup.2+]-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during [Ca.sup.2+]-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin-SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PI[P.sub.2])--binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as [Ca.sup.2+] sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their [Ca.sup.2+]-regulated interactions with t-SNAREs and PI[P.sub.2], target molecules known to play critical roles in exocytosis.
- Published
- 2003
28. rpoN, mmoR and mmoG, genes involved in regulating the expression of soluble methane monooxygenase in Methylosinus trichosporium OB3b
- Author
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Stafford, Graham P., Scanlan, Julie, McDonald, Ian R., and Murrell, J. Colin
- Subjects
Nitrogen -- Physiological aspects ,Genetic regulation -- Physiological aspects ,Gene expression -- Physiological aspects ,Operons -- Genetic aspects ,Operons -- Physiological aspects ,Copper -- Physiological aspects ,Genetic transcription -- Physiological aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Methanol -- Physiological aspects ,Methanobacteriaceae -- Physiological aspects ,Methane -- Physiological aspects ,Microbiology -- Research ,Biological sciences - Abstract
The methanotrophic bacterium Methylosinus trichosporium OB3b converts methane to methanol using two distinct forms of methane monooxygenase (MMO) enzyme: a cytoplasmic soluble form (sMMO) and a membrane-bound form (pMMO). The transcription of these two operons is known to proceed in a reciprocal fashion with sMMO expressed at Iow copper-to-biomass ratios and pMMO at high copper-to-biomass ratios. Transcription of the smmo operon is initiated from a [[sigma].sup.N] promoter 5' of mmoX. In this study the genes encoding [[sigma].sup.N] (rpoN) and a typical [[sigma].sup.N] -dependent transcriptional activator (mmoR) were cloned and sequenced, mmoR, a regulatory gene, and mmoG, a gene encoding a GroEL homologue, lie 5' of the structural genes for the sMMO enzyme. Subsequent mutation of rpoN and mmoR by marker-exchange mutagenesis resulted in strains Gm1 and JS1, which were unable to express functional sMMO or initiate transcription of mmoX. An rpoN mutant was also unable to fix nitrogen or use nitrate as sole nitrogen source, indicating that [[sigma].sup.N] plays a role in both nitrogen and carbon metabolism in Ms. trichosporium OB3b. The data also indicate that mmoG is transcribed in a [[sigma].sup.N] and MmoR-independent manner. Marker-exchange mutagenesis of mmoG revealed that MmoG is necessary for smmo gene transcription and activity and may be an MmoR-specific chaperone required for functional assembly of transcriptionally competent MmoR in vivo. The data presented allow the proposal of a more complete model for copper-mediated regulation of smmo gene expression.
- Published
- 2003
29. Small ubiquitin-like modifier conjugation regulates nuclear export of TEL, a putative tumor suppressor
- Author
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Wood, Lauren D., Irvin, Brenda J., Nucifora, Giuseppina J., Luce, K. Scott, and Hiebert, Scott W.
- Subjects
Genetic translation -- Physiological aspects ,Proteins -- Genetic aspects ,Proteins -- Physiological aspects ,Chromosomes -- Physiological aspects ,Chromosomes -- Genetic aspects ,B cells -- Growth ,B cells -- Genetic aspects ,B cells -- Physiological aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Lymphocytic leukemia -- Causes of ,Gene mutations -- Physiological aspects ,Company growth ,Science and technology - Abstract
Posttranslational modification by small ubiquitin-like modifier (SUMO) conjugation regulates the subnuclear localization of several proteins; however, SUMO modification has not been directly linked to nuclear export. The ETS (E-Twenty-Six) family member TEL (ETV6) is a transcriptional repressor that can inhibit Ras-dependent colony growth in soft agar and induce cellular aggregation of Ras-transformed cells. TEL is frequently disrupted by chromosomal translocations such as the t(12;21), which is associated with nearly one-fourth of pediatric B cell acute lymphoblastic leukemia. In the vast majority of t(12;21)-containing cases, the second allele of TEL is deleted, suggesting that inactivation of TEL contributes to the disease. Although TEL functions in the nucleus as a DNA-binding transcriptional repressor, it has also been detected in the cytoplasm. Here we demonstrate that TEL is actively exported from the nucleus in a leptomycin B-sensitive manner. TEL is posttranslationally modified by sumoylation at lysine 99 within a highly conserved domain (the 'pointed' domain). Mutation of the sumo-acceptor lysine or mutations within the pointed domain that affect sumoylation impair nuclear export of TEL. Mutation of lysine 99 also results in an increase in TEL transcriptional repression, presumably because of decreased nuclear export. We propose that the ability of TEL to repress transcription and suppress growth is regulated by sumoylation and nuclear export.
- Published
- 2003
30. PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure
- Author
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Reiken, Steven, Lacampagne, Alain, Zhou, Hua, Kherani, Aftab, Lehnart, Stephan E., Ward, Chris, Huang, Fannie, Gaburjakova, Marta, Gaburjakova, Jana, Rosemblit, Nora, Warren, Michelle S., He, Kun-lun, Yi, Geng-hua, Wang, Jie, Burkhoff, Daniel, Vassort, Guy, and Marks, Andrew R.
- Subjects
Cytology -- Research ,Calcium compounds -- Physiological aspects ,Sarcoplasmic reticulum -- Physiological aspects ,Sarcoplasmic reticulum -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Carrier proteins -- Genetic aspects ,Carrier proteins -- Physiological aspects ,Nervous system, Sympathetic -- Physiological aspects ,Phosphorylation -- Physiological aspects ,Muscles -- Physiological aspects ,Muscles -- Genetic aspects ,Biological sciences - Abstract
The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium ([Ca.sup.2+]) release channel required for skeletal muscle excitation--contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at [Ser.sup.2843] activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are 'leaky.' RyR1 PKA hyperphosphorylation correlated with impaired SR [Ca.sup.2+] release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.
- Published
- 2003
31. Formation of filopodia-like bundles in vitro from a dendritic network
- Author
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Vignjevic, Danijela, Yarar, Defne, Welch, Matthew D., Peloquin, John, Svitkina, Tatyana, and Borisy, Gary G.
- Subjects
Cytology -- Research ,Actin -- Physiological aspects ,Actin -- Genetic aspects ,Proteins -- Physiological aspects ,Proteins -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Chemical inhibitors -- Physiological aspects ,Biological sciences - Abstract
We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)--activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)--coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.
- Published
- 2003
32. The RasGAP-associated endoribonuclease G3BP assembles stress granules
- Author
-
Tourriere, Helene, Chebli, Karim, Zekri, Latifa, Courselaud, Brice, Blanchard, Jean Marie, Bertrand, Edouard, and Tazi, Jamal
- Subjects
Cytology -- Research ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Toxins -- Physiological aspects ,Messenger RNA -- Genetic aspects ,Metabolism -- Physiological aspects ,Phosphorylation -- Physiological aspects ,Gene expression -- Physiological aspects ,Gene mutations -- Physiological aspects ,Ribonuclease -- Genetic aspects ,Ribonuclease -- Physiological aspects ,Biological sciences - Abstract
Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents, and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149, whose dephosphorylation is induced by arsenite treatment. Critically, a phosphomimetic mutant (S149E) fails to oligomerize and to assemble SGs, whereas a nonphosphorylatable G3BP mutant (S149A) does both. These results suggest that G3BP is an effector of SG assembly, and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.
- Published
- 2003
33. Single-cell perforin and granzyme expression reveals the anatomical localization of effector CD[8.sup.+] T cells in influenza virus-infected mice
- Author
-
Johnson, Barbara J., Costelloe, Elaine O., Fitzpatrick, David R., Haanen, John B.A.G., Schumacher, Ton N.M., Brown, Lorena E., and Kelso, Anne
- Subjects
Medical research -- Analysis ,Medicine, Experimental ,Influenza viruses -- Physiological aspects ,Influenza viruses -- Research ,T cells -- Genetic aspects ,T cells -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Lymph nodes -- Physiological aspects ,Gene expression -- Physiological aspects ,Messenger RNA -- Genetic aspects ,Carrier proteins -- Physiological aspects ,Carrier proteins -- Genetic aspects ,Science and technology - Abstract
Influenza virus infection activates cytolytic T lymphocytes (CTL) that contribute to viral clearance by releasing perforin and granzymes from cytoplasmic granules. Virus-specific, perforin-dependent CD[8.sup.+] CTL were detected in freshly isolated cells from the mouse lung parenchyma but not from the mediastinal lymph nodes (MLN), where they are primed, or from the spleen during primary influenza virus infection. To determine whether this difference was due to the low frequency or incomplete maturation of effector CTL in MLN, we measured expression of perforin, granzymes A, B, and C, and IFN-[gamma] mRNAs in CD[8.sup.+] populations and single cells immediately after isolation from virus-infected mice. Quantitative PCR revealed significant expression of perforin, granzyme A, granzyme B, and IFN-[gamma] in activated CD[8.sup.+] cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramer-binding CD[8.sup.+] cells from the three tissues expressed diverse combinations of perforin, granzyme, and IFN-[gamma] mRNAs. Although cells from lung expressed granzymes A and B at higher frequency, each of the tissues contained cells that coexpressed perforin with granzymes A and/or B. The main difference between MLN and lung was the elevated frequency of activated CD[8.sup.+] T cells in the lung, rather than their perforin/granzyme expression profile. The data suggest that some CTL mature into perforin/granzyme-expressing effector cells in MLN but reach detectable frequencies only when they accumulate in the infected lung.
- Published
- 2003
34. Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in Drosophila
- Author
-
Kurucz, Eva, Zettervall, Carl-Johan, Sinka, Rita, Vilmos, Peter, Pivarcsi, Andor, Ekengren, Sophia, Hegedus, Zoltan, Ando, Istvan, and Hultmark, Dan
- Subjects
Membrane proteins -- Physiological aspects ,Membrane proteins -- Genetic aspects ,Blood cells -- Physiological aspects ,Blood cells -- Genetic aspects ,Drosophila -- Physiological aspects ,Drosophila -- Genetic aspects ,Antibodies -- Physiological aspects ,Antibodies -- Genetic aspects ,Cloning -- Genetic aspects ,Peptides -- Physiological aspects ,Peptides -- Genetic aspects ,Hematopoietic agents -- Physiological aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Gene expression -- Physiological aspects ,Viral antibodies ,Science and technology - Abstract
We have identified a previously undescribed transmembrane protein, Hemese, from Drosophila melanogaster blood cells (hemocytes), by using a monoclonal pan-hemocyte antibody. Heavy glycosylation is suggested by the heterogeneous size distribution, ranging between 37 and 70 kDa. Hemese expression is restricted to the cell surfaces of hemocytes of all classes, and to the hematopoietic organs. The sequence of the corresponding gene, Hemese (He), predicts a glycophorin-like protein of 15 kDa, excluding an N-terminal signal peptide, with a single hydrophobic transmembrane region. The extracellular region consists mainly of Ser/Thr-rich sequence of low complexity, with several potential O-glycosylation sites. Hemese contains phosphotyrosine and the cytoplasmic region has potential phosphorylation sites, suggesting an involvement in signal transduction. Depletion of Hemese by RNA interference has no obvious effect under normal conditions, but the cellular response to parasitic wasps is much enhanced. This finding indicates that Hemese plays a modulatory role in the activation or recruitment of the hemocytes.
- Published
- 2003
35. Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand
- Author
-
Luo, Bing-Hao, Springer, Timothy A., and Takagi, Junichi
- Subjects
Cells -- Physiological aspects ,Cells -- Genetic aspects ,Integrins -- Physiological aspects ,Ligands (Biochemistry) -- Genetic aspects ,Ligands (Biochemistry) -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Gene mutations -- Physiological aspects ,Science and technology - Abstract
The affinity of the extracellular domain of integrins for ligand is regulated by conformational changes signaled from the cytoplasm. Alternative types of conformational movement in the ligand-binding headpiece have been proposed. In one study, electron micrograph image averages of the headpiece of integrin aV[beta]3 show two different conformations. The open conformation of the headpiece is present when a ligand mimetic peptide is bound and differs from the closed conformation in the presence of an obtuse angle between the [beta]3 subunit hybrid and I-like domains. We tested the hypothesis that opening of the hybrid-I-like domain interface increases ligand-binding affinity by mutationally introducing an N-glycosylation site into it. Both [beta]3 and [beta]1 integrin glycan wedge mutants exhibit constitutively high affinity for physiological ligands. The data uniquely support one model of integrin activation and suggest that movement at the interface with the hybrid domain pulls down the C-terminal helix of the I-like domain and activates its metal ion-dependent adhesion site, analogously to activation of the integrin I domain.
- Published
- 2003
36. Integrin [beta] cytoplasmic domain interactions with phosphotyrosine-binding domains: a structural prototype for diversity in integrin signaling
- Author
-
Calderwood, David A., Fujioka, Yosuke, de Pereda, Jose M., Garcia-Alvarez, Begona, Nakamoto, Tetsuya, Margolis, Ben, McGlade, C. Jane, Liddington, Robert C., and Ginsberg, Mark H.
- Subjects
Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Cell migration -- Research ,Carrier proteins -- Genetic aspects ,Carrier proteins -- Physiological aspects ,Integrins -- Physiological aspects ,Ligands (Biochemistry) -- Physiological aspects ,Ligands (Biochemistry) -- Genetic aspects ,Gene mutations -- Physiological aspects ,Science and technology - Abstract
The cytoplasmic domains (tails) of heterodimeric integrin adhesion receptors mediate integrins' biological functions by binding to cytoplasmic proteins. Most integrin [beta] tails contain one or two NPXY/F motifs that can form [beta] turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, protein modules that are present in a wide variety of signaling and cytoskeletal proteins. Indeed, talin and ICAP1-[alpha] bind to integrin [beta] tails by means of a PTB domain-NPXY ligand interaction. To assess the generality of this interaction we examined the binding of a series of recombinant PTB domains to a panel of short integrin [beta] tails. In addition to the known integrin-binding proteins, we found that Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domain bound to integrin tails. Furthermore, Dok-1 physically associated with integrin [alpha]IIb[beta]3. Mutations of the integrin [beta] tails confirmed that these interactions are canonical PTB domain-ligand interactions. First, the interactions were blocked by mutation of an NPXY motif in the integrin tail. Second, integrin class-specific interactions were observed with the PTB domains of Dab, EPS8, and tensin. We used this specificity, and a molecular model of an integrin [beta] tail-PTB domain interaction to predict critical interacting residues. The importance of these residues was confirmed by generation of gain-and loss-of-function mutations in [beta]7 and [beta]3 tails. These data establish that short integrin [beta] tails interact with a large number of PTB domain-containing proteins through a structurally conserved mechanism.
- Published
- 2003
37. Crystal structure of Mycobacterium tuberculosis SecA, a preprotein translocating ATPase
- Author
-
Sharma, Vivek, Arockiasamy, Arulandu, Ronning, Donald R., Savva, Christos G., Holzenburg, Andreas, Braunstein, Miriam, Jacobs, William R., Jr., and Sacchettini, James C.
- Subjects
Membrane proteins -- Physiological aspects ,Membrane proteins -- Genetic aspects ,Adenosine triphosphate -- Physiological aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Mycobacterium tuberculosis -- Genetic aspects ,Mycobacterium tuberculosis -- Physiological aspects ,Hydrolysis -- Physiological aspects ,Science and technology - Abstract
In bacteria, the majority of exported proteins are translocated by the Sec system, which recognizes the signal sequence of a preprotein and uses ATP and the proton motive force to mediate protein translocation across the cytoplasmic membrane. SecA is an essential protein component of this system, containing the molecular motor that facilitates translocation, Here we report the three-dimensional structure of the SecA protein of Mycobacterium tuberculosis. Each subunit of the homodimer contains a 'motor' domain and a translocation domain. The structure predicts that SecA can interact with the SecYEG pore and function as a molecular ratchet that uses ATP hydrolysis for physical movement of the preprotein. Knowledge of this structure provides a framework for further elucidation of the translocation process.
- Published
- 2003
38. Tyrosine-phosphorylated and nonphosphorylated isoforms of [alpha]-dystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions
- Author
-
Grady, R. Mark, Akaaboune, Mohammed, Cohen, Alexander L., Maimone, Margaret M., Lichtman, Jeff W., and Sanes, Joshua R.
- Subjects
Cytology -- Research ,Tyrosine -- Genetic aspects ,Tyrosine -- Physiological aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Muscle cells -- Physiological aspects ,Muscle cells -- Genetic aspects ,Glycoproteins -- Physiological aspects ,Glycoproteins -- Genetic aspects ,Dystrophin -- Genetic aspects ,Dystrophin -- Physiological aspects ,Muscular dystrophy -- Causes of ,Genetic engineering -- Management ,Company restructuring/company reorganization ,Company organization ,Biological sciences - Abstract
[alpha]-Dystrobrevin (DB), a cytoplasmic component of the dystrophin--glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking [alpha]DB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main [alpha]DB isoforms, [alpha]DB1 and [alpha]DB2, with common N[H.sub.2]-terminal but distinct COOH-terminal domains. [alpha]DB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. [alpha]DB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in [alpha]D[B.sup.-/-] mice prevented muscle fiber degeneration; however, only [alpha]DB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of [alpha]DB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, [alpha]DB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, [alpha]DB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.
- Published
- 2003
39. A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo
- Author
-
Li, Hoi Yeung, Wirtz, Denis, and Zheng, Yixian
- Subjects
Cytology -- Research ,Cell cycle -- Physiological aspects ,Cell cycle -- Genetic aspects ,Mitosis -- Genetic aspects ,Mitosis -- Physiological aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Enzymes -- Genetic aspects ,Enzymes -- Physiological aspects ,Chromosomes -- Physiological aspects ,Chromosomes -- Genetic aspects ,Genetic regulation -- Physiological aspects ,Biological sciences - Abstract
The RanGTP gradient across the interphase nuclear envelope and on the condensed mitotic chromosomes is essential for many cellular processes, including nucleocytoplasmic transport and spindle assembly. Although the chromosome-associated enzyme RCC1 is responsible for RanGTP production, the mechanism of generating and maintaining the RanGTP gradient in vivo remains unknown. Here, we report that regulator of chromosome condensation (RCC1) rapidly associates and dissociates with both interphase and mitotic chromosomes in living cells, and that this mobility is regulated during the cell cycle. Our kinetic modeling suggests that RCC1 couples its catalytic activity to chromosome binding to generate a RanGTP gradient. Indeed, we have demonstrated experimentally that the interaction of RCC1 with the chromatin is coupled to the nucleotide exchange on Ran in vivo. The coupling is due to the stable binding of the binary complex of RCC1-Ran to chromatin. Successful nucleotide exchange dissociates the binary complex, permitting the release of RCC1 and RanGTP from the chromatin and the production of RanGTP on the chromatin surface.
- Published
- 2003
40. Evidence for involvement of the putative first extracellular loop in differential volume sensitivity of the Na (super)+/H (super)+ exchangers NHE1 and NHE2
- Author
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Xiaohua Su, Tianxiang Pang, Wakabayashi, Shigeo, and Shigekawa, Munekazu
- Subjects
Biochemistry -- Research ,Sodium channels -- Physiological aspects ,Hydrogen -- Physiological aspects ,Gene mutations -- Physiological aspects ,Cells -- Genetic aspects ,Hydrogen-ion concentration -- Physiological aspects ,Cytoplasm -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Biological sciences ,Chemistry - Abstract
Research has been conducted on PS120 cells expressing Na (super)+/H (super)+ exchanger isoforms and their mutants. The study of the hyperosmolarity-induced changes in cell volume and cytoplasmic pH in these cells is described.
- Published
- 2003
41. The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast
- Author
-
Lee, Wei-Lih, Oberle, Jessica R., and Cooper, John A.
- Subjects
Cell research -- Analysis ,Mitosis -- Physiological aspects ,Mitosis -- Genetic aspects ,Brewer's yeast -- Physiological aspects ,Brewer's yeast -- Genetic aspects ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Microtubules -- Genetic aspects ,Microtubules -- Physiological aspects ,Biological sciences - Abstract
During mitosis in Saccharomyces cerevisiae, the mitotic spindle moves into the mother-bud neck via dyneindependent sliding of cytoplasmic microtubules along the cortex of the bud. Here we show that Pac1, the yeast homologue of the human lissencephaly protein LIS1, plays a key role in this process. First, genetic interactions placed Pac1 in the dynein/dynactin pathway. Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Third, Pac1 localized to the plus ends (distal tips) of cytoplasmic microtubules in the bud. This localization did not depend on the dynein heavy chain Dyn1. Moreover, the Pac1 fluorescence intensity' at the microtubule end was enhanced in cells lacking dynactin or the cortical attachment molecule Num1. Fourth, dynein heavy chain Dyn1 also localized to the tips of cytoplasmic microtubules in wild-type cells. Dynein localization required Pac1 and, like Pac1, was enhanced in cells lacking the dynactin component Arp1 or the cortical attachment molecule Num1. Our results suggest that Pac1 targets dynein to microtubule tips, which is necessary for sliding of microtubules along the bud cortex. Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.
- Published
- 2003
42. Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process
- Author
-
Abrami, Laurence, Liu, Shihui, Cosson, Pierre, Leppla, Stephen H., and van der Goot, F. Gisou
- Subjects
Anthrax -- Physiological aspects ,Toxins -- Physiological aspects ,Antigens -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Endocytosis -- Genetic aspects ,Endocytosis -- Physiological aspects ,Cell research -- Analysis ,Biological sciences - Abstract
The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.
- Published
- 2003
43. Dynactin is required for bidirectional organelle transport
- Author
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Deacon, Sean W., Serpinskaya, Anna S., Vaughan, Patricia S., Fanarraga, Monica Lopez, Vernos, Isabelle, Vaughan, Kevin T., and Gelfand, Vladimir I.
- Subjects
Cell research -- Analysis ,Kinesin -- Genetic aspects ,Kinesin -- Physiological aspects ,Microtubules -- Genetic aspects ,Microtubules -- Physiological aspects ,Cell organelles -- Genetic aspects ,Cell organelles -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Biochemistry -- Research ,Proteins -- Genetic aspects ,Proteins -- Physiological aspects ,Biological sciences - Abstract
Kinesin II is a heterotrimeric plus end-directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II-mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II-associated protein (XKAP), binds directly to the p[150.sup.Glued] subunit of dynactin. This interaction occurs through aa 530-793 of XKAP and aa 600-811 of p[150.sup.Glued]. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.
- Published
- 2003
44. Regulation of the Bacillus subtilis bcrC bacitracin resistance gene by two extracytoplasmic function sigma factors
- Author
-
Cao, Min and Helmann, John D.
- Subjects
Bacteriology -- Research ,Bacillus subtilis -- Physiological aspects ,Bacillus subtilis -- Genetic aspects ,Genetic regulation -- Analysis ,Bacitracin -- Physiological aspects ,Bacitracin -- Genetic aspects ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Biological sciences - Abstract
Research has been conducted on Bacillus subtilis bcrC bacitracin resistance gene. Results demonstrate that this gene is important for bacitracin resistance, is controlled by sigma (super)x and sigma (super)m and is inducible by bacitracin.
- Published
- 2002
45. Associations between cytoplasmic and nuclear loci in hybridizing populations
- Author
-
Orive, Maria E. and Barton, Nicholas H.
- Subjects
Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Cell nuclei -- Genetic aspects ,Cell nuclei -- Physiological aspects ,Biological sciences - Abstract
We extend current multilocus models to describe the effects of migration, recombination, selection, and nonrandom mating on sets of genes in diploids with varied modes of inheritance, allowing us to consider the patterns of nuclear and cytonuclear associations (disequilibria) under various models of migration. We show the relationship between the multilocus notation recently presented by Kirkpatrick, Johnson, and Barton (developed from previous work by Barton and Turelli) and the cytonuclear parameterization of Asmussen, Arnold, and Avise and extend this notation to describe associations between cytoplasmic elements and multiple nuclear genes. Under models with sexual symmetry, both nuclear-nuclear and cytonuclear disequilibria are equivalent. They differ, however, in cases involving some type of sexual asymmetry, which is then reflected in the asymmetric inheritance of cytoplasmic markers. An example given is the case of different migration rates in males and females; simulations using 2, 3, 4, or 5 unlinked autosomal markers with a maternally inherited cytoplasmic marker illustrate how nuclear-nuclear and cytonuclear associations can be used to separately estimate female and male migration rates. The general framework developed here allows us to investigate conditions where associations between loci with different modes of inheritance are not equivalent and to use this nonequivalence to test for deviations from simple models of admixture.
- Published
- 2002
46. Karyopherin [beta]2B participates in mRNA export from the nucleus
- Author
-
Shamsher, Monee K., Ploski, Jonathan, and Radu, Aurelian
- Subjects
Messenger RNA -- Physiological aspects ,Cell nuclei -- Genetic aspects ,Cell nuclei -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Macromolecules -- Genetic aspects ,Macromolecules -- Physiological aspects ,Science and technology - Abstract
Transport of macromolecules between the cell nucleus and cytoplasm occurs through the nuclear pores and is mediated by soluble carriers known as karyopherins (Kaps), transportins, importins, or exportins. We report that Kap [beta]2B (transportin-2) forms complexes with the mRNA export factor TAP in the presence of RanGTP, as shown by coimmunoprecipitation from HeLa cells. The interaction strictly depends on the presence of RanGTP. In digitoninpermeabilized cells, Kap [beta]2B mediates TAP-GFP export from the nuclei in the presence of RanGTP. A TAP mutant that does not coimmunoprecipitate with Kap [beta]2B is also not exported by Kap [beta]2B. In the permeabilized cells assay, TAP is also exported independently of Kap [beta]2B by direct interaction with nucleoporins, in agreement with previous reports. The export rate is, however, significantly lower than the Kap [beta]2B-mediated pathway. Both Kap [beta]2B and TAP are present and enriched in the poly[(A).sup.+] RNA complexes isolated from HeLa cell nuclear lysates. Poly[(A).sup.+] RNA strongly accumulates in the nuclei of HeLa cells treated with Kap [beta]2B short interfering RNA, indicating that Kap[beta]2B is involved in the export of at least a large proportion of the mRNA species. The export of[beta]-actin and GAPDH mRNA is also inhibited, whereas 28S RNA is not affected. The data support the conclusion that Kap [beta]2B participates directly in the export of a large proportion of cellular mRNAs, and TAP connects Kap [beta]2B to the mRNAs to be exported.
- Published
- 2002
47. Evidence that Armadillo transduces Wingless by mediating nuclear export or cytosolic activation of pangolin
- Author
-
Chan, Siu-Kwong and Struhl, Gary
- Subjects
Cell research -- Analysis ,Glycoproteins -- Genetic aspects ,Secretion -- Genetic aspects ,Drosophila -- Genetic aspects ,DNA -- Genetic aspects ,Cytoplasm -- Genetic aspects ,Biological sciences - Abstract
Research has been conducted on the secreted proteins of the Wingless family. The experimental results suggest that beta-catenin may transduce Wingless protein signals via exporting TCF from the nucleus or via TCF activation in the cytoplasm.
- Published
- 2002
48. Histone chaperone ASF1 cooperates with the Brahma chromatin-remodeling machinery
- Author
-
Moshkin, Yuri M., Armstrong, Jennifer A., Maeda, Robert K., Tamkun, John W., Verrijzer, Peter, Kennison, James A., and Karch, Francois
- Subjects
Bacterial proteins -- Genetic aspects ,Histones -- Genetic aspects ,Gene mutations -- Physiological aspects ,Gene silencing -- Research ,Drosophila -- Genetic aspects ,Chromatin -- Genetic aspects ,Cell cycle -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Cytoplasm -- Physiological aspects ,Biological sciences - Abstract
Research has been conducted on histone chaperone, anti-silencing function 1 protein (ASF1). Results demonstrate that Drosophila asf1 gene mutations depress silencing at heterochromatin and that ASF1 protein has cell cycle-specific cytoplasmic and nuclear localization suggesting the crucial role of this protein in chromatin assembly and Brahma chromatin-remodeling complex-mediated chromatin remodelling.
- Published
- 2002
49. The local environment at the cytoplasmic end of TM6 of the mu opioid receptor differes from those of rhodopsin and monoamine receptors: introduction of an ionic lock between the cytoplasmic ends of helices 3 and 6 by a L6.30(275)E mutation inactivates the mu opioid receptor and reduces the constitutive activity of its T6.34(279)K mutant
- Author
-
Huang, Peng, Visiers, Irache, Weinstein, Harel, and Liu-Chen, Lee-Yuan
- Subjects
Biochemistry -- Research ,Cytoplasm -- Genetic aspects ,Opioids -- Receptors ,Rhodopsin -- Physiological aspects ,Amines -- Physiological aspects ,Gene mutations -- Physiological aspects ,G proteins -- Physiological aspects ,Biological sciences ,Chemistry - Abstract
Research has been conducted on the rhodopsin and monoamine G protein-coupled receptors. The structural differences between these receptors and the mu opioid receptor and the role of these differences in the rhodopsin-like family have been investigated and the details are presented.
- Published
- 2002
50. Integrins: bidirectional, allosteric signaling machines
- Author
-
Hynes, Richard O.
- Subjects
Cell research -- Comparative analysis ,Integrins -- Genetic aspects ,Cell adhesion -- Physiological aspects ,Ligands (Biochemistry) -- Genetic aspects ,Gene expression -- Physiological aspects ,Cytoplasm -- Genetic aspects ,Biological sciences - Abstract
The author reviews the publications on adhesion receptors integrins. The topics of interest include the ability of integrins to transmit signals between their cytoplasmic domains and their extracellular ligand binding adhesion sites.
- Published
- 2002
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