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Results: 1 to 10 of 16

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Microtubules regulate brush border formation.
Tonucci FM, Ferretti A, Almada E, Cribb P, Vena R, Hidalgo F, Favre C, Tyska MJ, Kaverina I, Larocca MC
(2018) J Cell Physiol 233: 1468-1480
MeSH Terms: Actin Cytoskeleton, Animals, Cell Polarity, Centromere, Colon, Dogs, Enterocytes, Epithelial Cells, Humans, Kidney, Madin Darby Canine Kidney Cells, Microtubule-Associated Proteins, Microtubules, Microvilli, Nocodazole, Time Factors, Tubulin Modulators
Show Abstract · Added April 10, 2018
Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity.
© 2017 Wiley Periodicals, Inc.
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MeSH Terms
Microtubule segment stabilization by RASSF1A is required for proper microtubule dynamics and Golgi integrity.
Arnette C, Efimova N, Zhu X, Clark GJ, Kaverina I
(2014) Mol Biol Cell 25: 800-10
MeSH Terms: Apoptosis, Cell Division, Cell Line, Transformed, Cell Polarity, Epithelial Cells, Gene Expression, Golgi Apparatus, Humans, Microtubules, Nocodazole, RNA, Small Interfering, Retinal Pigment Epithelium, Time-Lapse Imaging, Tubulin, Tubulin Modulators, Tumor Suppressor Proteins
Show Abstract · Added March 20, 2014
The tumor suppressor and microtubule-associated protein Ras association domain family 1A (RASSF1A) has a major effect on many cellular processes, such as cell cycle progression and apoptosis. RASSF1A expression is frequently silenced in cancer and is associated with increased metastasis. Therefore we tested the hypothesis that RASSF1A regulates microtubule organization and dynamics in interphase cells, as well as its effect on Golgi integrity and cell polarity. Our results show that RASSF1A uses a unique microtubule-binding pattern to promote site-specific microtubule rescues, and loss of RASSF1A leads to decreased microtubule stability. Furthermore, RASSF1A-associated stable microtubule segments are necessary to prevent Golgi fragmentation and dispersal in cancer cells and maintain a polarized cell front. These results indicate that RASSF1A is a key regulator in the fine tuning of microtubule dynamics in interphase cells and proper Golgi organization and cell polarity.
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16 MeSH Terms
Concerted effort of centrosomal and Golgi-derived microtubules is required for proper Golgi complex assembly but not for maintenance.
Vinogradova T, Paul R, Grimaldi AD, Loncarek J, Miller PM, Yampolsky D, Magidson V, Khodjakov A, Mogilner A, Kaverina I
(2012) Mol Biol Cell 23: 820-33
MeSH Terms: Cell Line, Cell Movement, Cell Polarity, Centrosome, Computer Simulation, Golgi Apparatus, Humans, Microtubules, Nocodazole
Show Abstract · Added December 10, 2013
Assembly of an integral Golgi complex is driven by microtubule (MT)-dependent transport. Conversely, the Golgi itself functions as an unconventional MT-organizing center (MTOC). This raises the question of whether Golgi assembly requires centrosomal MTs or can be self-organized, relying on its own MTOC activity. The computational model presented here predicts that each MT population is capable of gathering Golgi stacks but not of establishing Golgi complex integrity or polarity. In contrast, the concerted effort of two MT populations would assemble an integral, polarized Golgi complex. Indeed, while laser ablation of the centrosome did not alter already-formed Golgi complexes, acentrosomal cells fail to reassemble an integral complex upon nocodazole washout. Moreover, polarity of post-Golgi trafficking was compromised under these conditions, leading to strong deficiency in polarized cell migration. Our data indicate that centrosomal MTs complement Golgi self-organization for proper Golgi assembly and motile-cell polarization.
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9 MeSH Terms
Murine CENP-F regulates centrosomal microtubule nucleation and interacts with Hook2 at the centrosome.
Moynihan KL, Pooley R, Miller PM, Kaverina I, Bader DM
(2009) Mol Biol Cell 20: 4790-803
MeSH Terms: Animals, COS Cells, Cell Line, Centrosome, Cercopithecus aethiops, Chromosomal Proteins, Non-Histone, Humans, Mice, Mice, Knockout, Microfilament Proteins, Microtubule-Associated Proteins, Microtubule-Organizing Center, Microtubules, Nocodazole, Tubulin Modulators, Two-Hybrid System Techniques
Show Abstract · Added December 10, 2013
The microtubule (MT) network is essential in a broad spectrum of cellular functions. Many studies have linked CENP-F to MT-based activities as disruption of this protein leads to major changes in MT structure and function. Still, the basis of CENP-F regulation of the MT network remains elusive. Here, our studies reveal a novel and critical localization and role for CENP-F at the centrosome, the major MT organizing center (MTOC) of the cell. Using a yeast two-hybrid screen, we identify Hook2, a linker protein that is essential for regulation of the MT network at the centrosome, as a binding partner of CENP-F. With recently developed immunochemical reagents, we confirm this interaction and reveal the novel localization of CENP-F at the centrosome. Importantly, in this first report of CENP-F(-/-) cells, we demonstrate that ablation of CENP-F protein function eliminates MT repolymerization after standard nocodazole treatment. This inhibition of MT regrowth is centrosome specific because MT repolymerization is readily observed from the Golgi in CENP-F(-/-) cells. The centrosome-specific function of CENP-F in the regulation of MT growth is confirmed by expression of truncated CENP-F containing only the Hook2-binding domain. Furthermore, analysis of partially reconstituted MTOC asters in cells that escape complete repolymerization block shows that disruption of CENP-F function impacts MT nucleation and anchoring rather than promoting catastrophe. Our study reveals a major new localization and function of CENP-F at the centrosome that is likely to impact a broad array of MT-based actions in the cell.
2 Communities
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16 MeSH Terms
Microtubule-dependent association of AKAP350A and CCAR1 with RNA stress granules.
Kolobova E, Efimov A, Kaverina I, Rishi AK, Schrader JW, Ham AJ, Larocca MC, Goldenring JR
(2009) Exp Cell Res 315: 542-55
MeSH Terms: A Kinase Anchor Proteins, Apoptosis Regulatory Proteins, Arsenites, Carrier Proteins, Cell Cycle Proteins, Cytoplasmic Granules, Cytoskeletal Proteins, Cytosol, DNA Helicases, Golgi Apparatus, HeLa Cells, Humans, Microtubules, Nocodazole, Poly-ADP-Ribose Binding Proteins, Protein Transport, RNA Helicases, RNA Recognition Motif Proteins, RNA Stability, RNA, Messenger
Show Abstract · Added December 10, 2013
Recent investigations have highlighted the importance of subcellular localization of mRNAs to cell function. While AKAP350A, a multifunctional scaffolding protein, localizes to the Golgi apparatus and centrosomes, we have now identified a cytosolic pool of AKAP350A. Analysis of AKAP350A scaffolded complexes revealed two novel interacting proteins, CCAR1 and caprin-1. CCAR1, caprin-1 and AKAP350A along with G3BP, a stress granule marker, relocate to RNA stress granules after arsenite treatment. Stress also caused loss of AKAP350 from the Golgi and fragmentation of the Golgi apparatus. Disruption of microtubules with nocodazole altered stress granule formation and changed their morphology by preventing fusion of stress granules. In the presence of nocodazole, arsenite induced smaller granules with the vast majority of AKAP350A and CCAR1 separated from G3BP-containing granules. Similar to nocodazole treatment, reduction of AKAP350A or CCAR1 expression also altered the size and number of G3BP-containing stress granules induced by arsenite treatment. A limited set of 69 mRNA transcripts was immunoisolated with AKAP350A even in the absence of stress, suggesting the association of AKAP350A with mRNA transcripts. These results provide the first evidence for the microtubule dependent association of AKAP350A and CCAR1 with RNA stress granules.
1 Communities
3 Members
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20 MeSH Terms
AKAP350 modulates microtubule dynamics.
Larocca MC, Jin M, Goldenring JR
(2006) Eur J Cell Biol 85: 611-9
MeSH Terms: A Kinase Anchor Proteins, Adaptor Proteins, Signal Transducing, Centrosome, Cytoskeletal Proteins, Cytoskeleton, Down-Regulation, Gene Expression, HeLa Cells, Humans, Microtubules, Nocodazole, Protein Structure, Tertiary, RNA Interference, RNA, Small Interfering, Tubulin Modulators, cdc42 GTP-Binding Protein
Show Abstract · Added October 7, 2013
AKAP350 is a multiply spliced type II protein kinase A-anchoring protein that localizes to the centrosomes in most cells and the Golgi apparatus in epithelial cells. Multiple studies suggest that AKAP350 is involved in microtubule nucleation at the centrosome. Our previous studies demonstrated that AKAP350 was necessary for the maintenance of Golgi apparatus integrity. These data suggested that AKAP350 might be necessary for normal cytoskeletal interactions with the Golgi. To examine the relationship of AKAP350 with the microtubule cytoskeleton, we analyzed the effect of the depletion of AKAP350 on microtubule regrowth after nocodazole treatment in HeLa cells. The decrease in AKAP350 expression with short interfering RNA induced a delay in microtubule elongation with no effect on microtubule aster formation. In contrast, overexpression of the centrosomal targeting domain of AKAP350 elicited alterations in aster formation, but did not affect microtubule elongation. RNA interference for AKAP350 also induced an increase in cdc42 activity during microtubule regrowth. Our data suggest that AKAP350 has a role in the remodeling of the microtubule cytoskeleton.
1 Communities
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16 MeSH Terms
Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway.
Goodwin JS, Drake KR, Rogers C, Wright L, Lippincott-Schwartz J, Philips MR, Kenworthy AK
(2005) J Cell Biol 170: 261-72
MeSH Terms: Animals, COS Cells, Cell Membrane, Cercopithecus aethiops, Cycloheximide, Cytosol, Endoplasmic Reticulum, Golgi Apparatus, Green Fluorescent Proteins, Microtubules, Mutation, Nocodazole, Palmitates, Protein Transport, Signal Transduction, ras Proteins
Show Abstract · Added December 10, 2013
Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.
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16 MeSH Terms
Cytoplasmic LEK1 is a regulator of microtubule function through its interaction with the LIS1 pathway.
Soukoulis V, Reddy S, Pooley RD, Feng Y, Walsh CA, Bader DM
(2005) Proc Natl Acad Sci U S A 102: 8549-54
MeSH Terms: Animals, COS Cells, Carrier Proteins, Cercopithecus aethiops, Cytoplasm, Immunohistochemistry, Microtubule-Associated Proteins, Microtubules, Nocodazole, Oligonucleotides, Antisense, Two-Hybrid System Techniques
Show Abstract · Added September 28, 2015
LIS1 and nuclear distribution gene E (NudE) are partner proteins in a conserved pathway regulating the function of dynein and microtubules. Here, we present data that cytoplasmic LEK1 (cytLEK1), a large protein containing a spectrin repeat and multiple leucine zippers, is a component of this pathway through its direct interaction with NudE, as determined by a yeast two-hybrid screen. We identified the binding domains in each molecule, and coimmunoprecipitation and colocalization studies confirmed the specificity of the interaction between cytLEK1 and NudE. Confocal deconvolution analysis revealed that cytLEK1 exhibits colocalization with endogenous NudE and with the known NudE binding partners, LIS1 and dynein. By localizing the NudE-binding domain of cytLEK1 to a small domain within the molecule, we were able to disrupt cytLEK1 function by using a dominant negative approach in addition to LEK1 knockdown and, thus, examine the role of the cytLEK1-NudE interaction in cells. Consistent with a defect in the LIS1 pathway, disruption of cytLEK1 function resulted in alteration of microtubule organization and cellular shape. The microtubule network of cells became tightly focused around the nucleus and resulted in a rounded cell shape. Additionally, cells exhibited a severe inability to repolymerize their microtubule networks after nocodazole challenge. Taken together, our studies revealed that cytLEK1 is essential for cellular functions regulated by the LIS1 pathway.
1 Communities
2 Members
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11 MeSH Terms
Regulation of p120-catenin nucleocytoplasmic shuttling activity.
Roczniak-Ferguson A, Reynolds AB
(2003) J Cell Sci 116: 4201-12
MeSH Terms: Cadherins, Catenins, Cell Adhesion, Cell Adhesion Molecules, Cells, Cultured, Cloning, Molecular, Cytoskeleton, HT29 Cells, HeLa Cells, Humans, Microscopy, Fluorescence, Microtubules, Mutation, Nocodazole, Nuclear Localization Signals, Paclitaxel, Phosphoproteins, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Protein Transport
Show Abstract · Added March 5, 2014
P120-catenin is the prototypic member of a subfamily of Armadillo repeat domain (Arm domain) proteins involved in cell-cell adhesion. Interestingly, all members of the p120 subfamily have also been observed in the nucleus, suggesting that they have additional roles that have yet to be determined. Here, we have developed a novel model system for studying the nucleocytoplasmic shuttling capabilities of p120. We show that simultaneous deletion of both of the conventional nuclear localization sequences (NLSs) in p120 had little effect on its nuclear localization. Instead, the Armadillo repeat domain was essential, and deletion of Arm repeat 3 or Arm repeat 5 eliminated nuclear entry despite the presence of both NLSs. In addition, deletion of Arm repeat 8 resulted in constitutive nuclear localization of p120-3A in both E-cadherin-positive and -negative cell lines. Thus, the core shuttling functions are dependent on the Arm domain. We have also identified two regions within the N-terminus of p120 that modulate nuclear shuttling dynamics of p120. In cadherin-deficient cells, normal epithelial morphology could be restored by both WT-E-cadherin and p120 uncoupled E-cadherin mutants, but only WT-E-cadherin strongly reduced nuclear localization of p120. Moreover, structural changes in p120 that reduced its affinity for E-cadherin increased p120 nuclear localization. Thus, reduced shuttling in the presence of E-cadherin is principally due to sequestration, a condition that is probably dynamic under normal circumstances but completely lost in metastatic cells that have downregulated E-cadherin. Notably, Arm repeats 3 and 5 are necessary for both E-cadherin binding and nuclear translocation, indicating that these repeats have dual roles. Surprisingly, in the absence of E-cadherin there was significant colocalization of cytoplasmic p120 with elements of the tubulin cytoskeleton, particularly in perinuclear locations. Depolymerizing microtubules with nocodazole increased nuclear p120, whereas stabilizing tubulin with taxol reduced nuclear p120 and strongly increased p120 association with microtubules. Thus, p120 has intrinsic nucleocytoplasmic shuttling activity that is modulated, in part, by extrinsic factors such as cadherin binding and interactions with the microtubule network.
1 Communities
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21 MeSH Terms
Regulation of focal complex composition and disassembly by the calcium-dependent protease calpain.
Bhatt A, Kaverina I, Otey C, Huttenlocher A
(2002) J Cell Sci 115: 3415-25
MeSH Terms: Actinin, Animals, CHO Cells, Calcium, Calcium-Binding Proteins, Calpain, Cell Adhesion, Cell Movement, Cells, Cultured, Cricetinae, Cysteine Proteinase Inhibitors, Cytoskeletal Proteins, Fibroblasts, Focal Adhesions, Glycoproteins, Goldfish, Green Fluorescent Proteins, Humans, Luminescent Proteins, Metalloproteins, Microscopy, Video, Microtubules, Nocodazole, Recombinant Fusion Proteins, Vinculin, Zyxin
Show Abstract · Added December 10, 2013
Cell migration requires the regulated and dynamic turnover of adhesive complexes. We have previously demonstrated that the calcium-dependent protease, calpain, regulates the organization of adhesive complexes and cell detachment during cell migration. Evidence is now provided that inhibiting calpain through over-expression of the endogenous inhibitor of calpain, calpastatin, and pharmacological inhibitors results in an inhibition of adhesive complex disassembly with stabilization of GFP-vinculin and GFP/RFP-zyxin at the cell periphery. Calpain was also required for the microtubule-mediated turnover of adhesive complex sites after nocodazole wash-out, suggesting that calpain may mediate focal complex disassembly downstream of microtubules. Using dual imaging of RFP-zyxin and GFP-alpha-actinin, we observed a temporal and spatial relationship between alpha-actinin localization to focal contacts and the subsequent disassembly or translocation of RFP-zyxin containing focal complexes in areas of cell retraction. Calpain inhibition disrupted alpha-actinin localization to zyxin-containing focal contacts and focal complex disassembly or translocation to the cell center. In addition, disrupting alpha-actinin localization to focal complexes through expression of the alpha-actinin rod domain, but not the head domain, resulted in inhibition of focal adhesion disassembly similar to calpain inhibition. Our studies suggest a novel mechanism of action whereby calpain may modulate alpha-actinin localization into focal complexes and their subsequent disassembly or translocation.
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26 MeSH Terms