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1
Huang, Xiaolan, Rongmei Qu, Yan Peng, Yuchao Yang, Tingyu Fan, Bing Sun, Asmat Ullah Khan et al. "Mechanical Sensing Element PDLIM5 Promotes Osteogenesis of Human Fibroblasts by Affecting the Activity of Microfilaments". Biomolecules 11, n.º 5 (19 de mayo de 2021): 759. http://dx.doi.org/10.3390/biom11050759.
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Human skin fibroblasts (HSFs) approximate the multidirectional differentiation potential of mesenchymal stem cells, so they are often used in differentiation, cell cultures, and injury repair. They are an important seed source in the field of bone tissue engineering. However, there are a few studies describing the mechanism of osteogenic differentiation of HSFs. Here, osteogenic induction medium was used to induce fibroblasts to differentiate into osteoblasts, and the role of the mechanical sensitive element PDLIM5 in microfilament-mediated osteogenic differentiation of human fibroblasts was evaluated. The depolymerization of microfilaments inhibited the expression of osteogenesis-related proteins and alkaline phosphatase activity of HSFs, while the polymerization of microfilaments enhanced the osteogenic differentiation of HSFs. The evaluation of potential protein molecules affecting changes in microfilaments showed that during the osteogenic differentiation of HSFs, the expression of PDLIM5 increased with increasing induction time, and decreased under the state of microfilament depolymerization. Lentivirus-mediated PDLIM5 knockdown by shRNA weakened the osteogenic differentiation ability of HSFs and inhibited the expression and morphological changes of microfilament protein. The inhibitory effect of knocking down PDLIM5 on HSF osteogenic differentiation was reversed by a microfilament stabilizer. Taken together, these data suggest that PDLIM5 can mediate the osteogenic differentiation of fibroblasts by affecting the formation and polymerization of microfilaments.
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Ashworth, Sharon L., Sarah E. Wean, Silvia B. Campos, Constance J. Temm-Grove, Erica L. Southgate, Bernadette Vrhovski, Peter Gunning, Ron P. Weinberger y Bruce A. Molitoris. "Renal ischemia induces tropomyosin dissociation-destabilizing microvilli microfilaments". American Journal of Physiology-Renal Physiology 286, n.º 5 (mayo de 2004): F988—F996. http://dx.doi.org/10.1152/ajprenal.00168.2003.
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Ischemic-induced cell injury results in rapid duration-dependent actin-depolymerizing factor (ADF)/cofilin-mediated disruption of the apical microvilli microfilament cores. Because intestinal microvillar microfilaments are bound and stabilized in the terminal web by the actin-binding protein tropomyosin, we questioned whether a protective effect of tropomyosin localization to the terminal web of the proximal tubule microfilament cores is disrupted during ischemic injury. With tropomyosin-specific antibodies, we examined rat cortical sections under physiological conditions and following ischemic injury by confocal microscopy. In addition, Western blot analysis of cortical extracts and urine was undertaken. Our studies demonstrated the presence of tropomyosin isoforms in the proximal tubule microvillar terminal web under physiological conditions and their dissociation in response to 25 min of ischemic injury. This correlated with the excretion of tropomyosin-containing plasma membrane vesicles in urine from ischemic rats. In addition, we noted increased tropomyosin Triton X-100 solubility following ischemia in cortical extracts. These studies suggest tropomyosin binds to and stabilizes the microvillar microfilament core in the terminal web under physiological conditions. With the onset of ischemic injury, we propose that tropomyosin dissociates from the microfilament core providing access to microfilaments in the terminal web for F-actin binding, severing and depolymerizing actions of ADF/cofilin proteins.
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Collings, David A. y Geoffrey O. Wasteneys. "Actin microfilament and microtubule distribution patterns in the expanding root of Arabidopsis thaliana". Canadian Journal of Botany 83, n.º 6 (1 de junio de 2005): 579–90. http://dx.doi.org/10.1139/b05-032.
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Determination of the precise role(s) of actin microfilaments in the control of cell shape and elongation in the root tips of the model genetic system Arabidopsis thaliana (L.) Heynh is frustrated by inadequate microscopy imaging techniques. In this paper, we documented both microfilaments and microtubules in the root tips of Arabidopsis by double immunofluorescence labelling and computer-generated reconstruction of confocal image series. Our procedure, which complements the use of recently developed fluorescent reporter proteins, revealed hitherto undescribed aspects of the Arabidopsis microfilament cytoskeleton that may provide important clues about mechanisms behind cell elongation. We found that preservation of extensive arrays of transverse cortical microfilaments depends on unperturbed microtubule organization. Compared with ordinary epidermal cells, cells situated in the trichoblast or hair-forming cell files were comparatively devoid of endoplasmic microfilaments when in the distal elongation zone, well before hair formation begins. Computer-aided reconstructions also revealed that the nonexpanding end walls of cells in the distal elongation zone have radially oriented microtubules and randomly arranged microfilaments. In dividing cells, microfilaments became more prominent in the cell cortex, and subtle differences between microtubule and microfilament organization were seen within the phragmoplast. These observations will form the basis of understanding the roles of the cytoskeleton in controlling elongation in root tissues. In light of the many Arabidopsis mutants with altered root morphology, our methods offer a reliable approach to assess the function of cytoskeletal proteins and signalling systems in root morphogenesis.Key words: actin microfilaments, Arabidopsis thaliana, distal elongation zone, microtubules, phragmoplast, roots.
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Lakkakorpi, P. T. y H. K. Väänänen. "Calcitonin, prostaglandin E2, and dibutyryl cyclic adenosine 3',5'-monophosphate disperse the specific microfilament structure in resorbing osteoclasts." Journal of Histochemistry & Cytochemistry 38, n.º 10 (octubre de 1990): 1487–93. http://dx.doi.org/10.1177/38.10.2169493.
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Bone resorbing osteoclasts form a specific microfilament structure at the attachment area, in which vinculin and talin appear as a double-circle structure and F-actin fills the space between these circles. This distribution of microfilaments is associated with the resorption lacunae, and F-actin, vinculin, and talin zones correspond roughly to the edges of the lacunae. In the present work, we examined by immunofluorescence the effects of calcitonin (CT) and prostaglandin E2 (PGE2), inhibitors of osteoclastic activity, as well as dibutyryl cyclic AMP (Bt2cAMP) and cytochalasin B, on the microfilament organization in resorbing osteoclasts. CT, PGE2, and Bt2cAMP rapidly dispersed the specific microfilament structure in resorbing osteoclasts. All microfilament proteins studied (vinculin, talin, and F-actin) spread to the central areas of the original circles. The effect of CT was dose dependent. The effects of CT and PGE2 could be reversed, but recovery was slower after CT treatment than after PGE2 treatment. Cytochalasin B entirely destroyed the F-actin organization but only partially the vinculin organization. The results suggest that one structural change leading to the inactivation of the osteoclasts caused by CT and PGE2 is the disintegration of the microfilament structure at the attachment area of resorbing osteoclasts.
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Samuelsson, Steven J., Paul W. Luther, David W. Pumplin y Robert J. Bloch. "Macromolecular specializations that mediate lateral interactions between microfilaments and the membrane at focal contacts". Proceedings, annual meeting, Electron Microscopy Society of America 50, n.º 1 (agosto de 1992): 724–25. http://dx.doi.org/10.1017/s0424820100124021.
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Focal contacts are membrane specializations of cultured cells where stress fibers terminate and where the cell is most closely applied to the substrate. The organization of this cytoskeletal-membrane-extracellular matrix assembly has been well characterized. Immunofluorescence microscopy has shown that two focal contact-specific proteins, vinculin and talin, colocalize with microfilaments for several microns before the stress fiber terminates. This result raises the question of whether microfilament-membrane interactions are limited to the ends of microfilaments, or if lateral interactions also occur. We addressed this question by examining the cytoplasmic surface of isolated focal contacts in detail.
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Schevzov, Galina, Bernadette Vrhovski, Nicole S. Bryce, Sarah Elmir, Min Ru Qiu, Geraldine M. O'Neill, Nan Yang, Nicole M. Verrills, Maria Kavallaris y Peter W. Gunning. "Tissue-specific Tropomyosin Isoform Composition". Journal of Histochemistry & Cytochemistry 53, n.º 5 (mayo de 2005): 557–70. http://dx.doi.org/10.1369/jhc.4a6505.2005.
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Four distinct genes encode tropomyosin (Tm) proteins, integral components of the actin microfilament system. In non-muscle cells, over 40 Tm isoforms are derived using alternative splicing. Distinct populations of actin filaments characterized by the composition of these Tm isoforms are found differentially sorted within cells ( Gunning et al. 1998b ). We hypothesized that these distinct intracellular compartments defined by the association of Tm isoforms may allow for independent regulation of microfilament function. Consequently, to understand the molecular mechanisms that give rise to these different microfilaments and their regulation, a cohort of fully characterized isoform-specific Tm antibodies was required. The characterization protocol initially involved testing the specificity of the antibodies on bacterially produced Tm proteins. We then confirmed that these Tm antibodies can be used to probe the expression and subcellular localization of different Tm isoforms by Western blot analysis, immunofluorescence staining of cells in culture, and immunohistochemistry of paraffin wax-embedded mouse tissues. These Tm antibodies, therefore, have the capacity to monitor specific actin filament populations in a range of experimental systems.
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Mykkänen, Olli-Matti, Mikaela Grönholm, Mikko Rönty, Maciej Lalowski, Paula Salmikangas, Heli Suila y Olli Carpén. "Characterization of Human Palladin, a Microfilament-associated Protein". Molecular Biology of the Cell 12, n.º 10 (octubre de 2001): 3060–73. http://dx.doi.org/10.1091/mbc.12.10.3060.
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Actin-containing microfilaments control cell shape, adhesion, and contraction. In striated muscle, α-actinin and other Z-disk proteins coordinate the organization and functions of actin filaments. In smooth muscle and nonmuscle cells, periodic structures termed dense bodies and dense regions, respectively, are thought to serve functions analogous to Z-discs. We describe here identification and characterization of human palladin, a protein expressed mainly in smooth muscle and nonmuscle and distributed along microfilaments in a periodic manner consistent with dense regions/bodies. Palladin contains three Ig-domains most homologous to the sarcomeric Z-disk protein myotilin. The N terminus includes an FPPPP motif recognized by the Ena-Vasp homology domain 1 domain in Ena/vasodilatator-stimulated phosphoprotein (VASP)/Wiscott-Aldrich syndrome protein (WASP) protein family. Cytoskeletal proteins with FPPPP motif target Ena/VASP/WASP proteins to sites of actin modulation. We identified palladin in a yeast two-hybrid search as an ezrin-associated protein. An interaction between palladin and ezrin was further verified by affinity precipitation and blot overlay assays. The interaction was mediated by the α-helical domain of ezrin and by Ig-domains 2–3 of palladin. Ezrin is typically a component of the cortical cytoskeleton, but in smooth muscle cells it is localized along microfilaments. These cells express palladin abundantly and thus palladin may be involved in the microfilament localization of ezrin. Palladin expression was up-regulated in differentiating dendritic cells (DCs), coinciding with major cytoskeletal and morphological alterations. In immature DCs, palladin localized in actin-containing podosomes and in mature DCs along actin filaments. The regulated expression and localization suggest a role for palladin in the assembly of DC cytoskeleton.
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Ji, Jun-Yuan, Marjan Haghnia, Cory Trusty, Lawrence S. B. Goldstein y Gerold Schubiger. "A Genetic Screen for Suppressors and Enhancers of the Drosophila Cdk1-Cyclin B Identifies Maternal Factors That Regulate Microtubule and Microfilament Stability". Genetics 162, n.º 3 (1 de noviembre de 2002): 1179–95. http://dx.doi.org/10.1093/genetics/162.3.1179.
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Abstract Coordination between cell-cycle progression and cytoskeletal dynamics is important for faithful transmission of genetic information. In early Drosophila embryos, increasing maternal cyclin B leads to higher Cdk1-CycB activity, shorter microtubules, and slower nuclear movement during cycles 5-7 and delays in nuclear migration to the cortex at cycle 10. Later during cycle 14 interphase of six cycB embryos, we observed patches of mitotic nuclei, chromosome bridges, abnormal nuclear distribution, and small and large nuclei. These phenotypes indicate disrupted coordination between the cell-cycle machinery and cytoskeletal function. Using these sensitized phenotypes, we performed a dosage-sensitive genetic screen to identify maternal proteins involved in this process. We identified 10 suppressors classified into three groups: (1) gene products regulating Cdk1 activities, cdk1 and cyclin A; (2) gene products interacting with both microtubules and microfilaments, Actin-related protein 87C; and (3) gene products interacting with microfilaments, chickadee, diaphanous, Cdc42, quail, spaghetti-squash, zipper, and scrambled. Interestingly, most of the suppressors that rescue the astral microtubule phenotype also reduce Cdk1-CycB activities and are microfilament-related genes. This suggests that the major mechanism of suppression relies on the interactions among Cdk1-CycB, microtubule, and microfilament networks. Our results indicate that the balance among these different components is vital for normal early cell cycles and for embryonic development. Our observations also indicate that microtubules and cortical microfilaments antagonize each other during the preblastoderm stage.
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Temm-Grove, C. J., B. M. Jockusch, M. Rohde, K. Niebuhr, T. Chakraborty y J. Wehland. "Exploitation of microfilament proteins by Listeria monocytogenes: microvillus-like composition of the comet tails and vectorial spreading in polarized epithelial sheets". Journal of Cell Science 107, n.º 10 (1 de octubre de 1994): 2951–60. http://dx.doi.org/10.1242/jcs.107.10.2951.
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Effective cell-to-cell spreading of the facultative intracellular pathogen Listeria monocytogenes requires the interaction between bacteria and the microfilament system of the host cell. By recruiting actin filaments into a ‘comet tail’ localized at one pole of the bacterial cell wall, Listeria become mobile and propel themselves through the cytoplasm. They create protrusions at the plasma membrane that can invaginate adjacent cells. In this work, we have analysed the structural composition of Listeria-recruited microfilaments in various epithelial cell lines by immunofluorescence microscopy. The microfilament-crosslinking proteins alpha-actinin, fimbrin and villin were localized around bacteria as soon as actin filaments could be detected on the bacterial surface. Surprisingly, the same was found for ezrin/radixin, proteins involved in linking microfilaments to the plasma membrane. We found that in a polarized cell line derived from brush border kidney epithelium (LLC-PK1), the actin filaments surrounding intracytoplasmic motile bacteria show the same immunoreactivity as the brush border-like microvilli, when analysed by a specific actin antibody. The successful invasion of polarized LLC-PK1 islets is vectorial, i.e. it progresses predominantly from the periphery of the islets towards the centre. Infection of the peripheral cells is sufficient for infiltration of the entire cellular islets, without any further contact with the extracellular milieu. This is in contrast to nonpolarized epithelial sheets, which can be invaded from the apical surface of any individual cell. The importance of active bacterial motility in this vectorial spreading is emphasized by our finding that an isogenic Listeria mutant that is unable to recruit actin filaments cannot colonize polarized epithelial layers but accumulates in the peripheral cells of the islets.
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Hüttelmaier, Stefan, Susanne Illenberger, Irina Grosheva, Manfred Rüdiger, Robert H. Singer y Brigitte M. Jockusch. "Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins". Journal of Cell Biology 155, n.º 5 (26 de noviembre de 2001): 775–86. http://dx.doi.org/10.1083/jcb.200105044.
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By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and α-actinin and colocalizes with vinculin/metavinculin and α-actinin at microfilament attachment sites, such as cell–cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.
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Sgorbissa, A., R. Benetti, S. Marzinotto, C. Schneider y C. Brancolini. "Caspase-3 and caspase-7 but not caspase-6 cleave Gas2 in vitro: implications for microfilament reorganization during apoptosis". Journal of Cell Science 112, n.º 23 (1 de diciembre de 1999): 4475–82. http://dx.doi.org/10.1242/jcs.112.23.4475.
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Apoptosis is characterized by proteolysis of specific cellular proteins by a family of cystein proteases known as caspases. Gas2, a component of the microfilament system, is cleaved during apoptosis and the cleaved form specifically regulates microfilaments and cell shape changes. We now demonstrate that Gas2 is a substrate of caspase-3 but not of caspase-6. Proteolytic processing both in vitro and in vivo is dependent on aspartic residue 279. Gas2 cleavage was only partially impaired in apoptotic MCF-7 cells which lack caspase-3, thus indicating that different caspases can process Gas2 in vivo. In vitro Gas2 was processed, albeit with low affinity, by caspase-7 thus suggesting that this caspase could be responsible for the incomplete Gas2 processing observed in UV treated MCF-7 cells. In vivo proteolysis of Gas2 was detected at an early stage of the apoptotic process when the cells are still adherent on the substrate and it was coupled to the specific rearrangement of the microfilament characterizing cell death. Finally we also demonstrated that Gas2 in vitro binds to F-actin, but this interaction was unaffected by the caspase-3 dependent proteolytic processing.
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Sun, Qing-Yuan y Heide Schatten. "Regulation of dynamic events by microfilaments during oocyte maturation and fertilization". Reproduction 131, n.º 2 (febrero de 2006): 193–205. http://dx.doi.org/10.1530/rep.1.00847.
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Actin filaments (microfilaments) regulate various dynamic events during oocyte meiotic maturation and fertilization. In most species, microfilaments are not required for germinal vesicle breakdown and meiotic spindle formation, but they mediate peripheral nucleus (chromosome) migration, cortical spindle anchorage, homologous chromosome separation, cortex development/maintenance, polarity establishment, and first polar body emission during oocyte maturation. Peripheral cortical granule migration is controlled by microfilaments, while mitochondria movement is mediated by microtubules. During fertilization, microfilaments are involved in sperm incorporation, spindle rotation (mouse), cortical granule exocytosis, second polar body emission and cleavage ring formation, but are not required for pronuclear apposition (except for the mouse). Many of the events are driven by the dynamic interactions between myosin and actin filaments whose polymerization is regulated by RhoA, Cdc42, Arp2/3 and other signaling molecules. Studies have also shown that oocyte cortex organization and polarity formation mediated by actin filaments are regulated by mitogen-activated protein kinase, myosin light-chain kinase, protein kinase C and its substrate p-MARKS as well as PAR proteins. The completion of several dynamic events, including homologous chromosome separation, spindle anchorage, spindle rotation, vesicle organelle transport and pronuclear apposition (mouse), requires interactions between microfilaments and microtubules, but determination of how the two systems of the cytoskeleton precisely cross-link, and which proteins link microfilaments to microtubules to perform functions in eggs, requires further studies. Finally, the meaning of microfilament-mediated oocyte polarity versus embryo polarity and embryo development in different species (Drosophila, Xenopus and mouse) is discussed.
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Dalby-Payne, Jacqueline Rae, Edward Vincent O'Loughlin y Peter Gunning. "Polarization of Specific Tropomyosin Isoforms in Gastrointestinal Epithelial Cells and Their Impact on CFTR at the Apical Surface". Molecular Biology of the Cell 14, n.º 11 (noviembre de 2003): 4365–75. http://dx.doi.org/10.1091/mbc.e03-03-0169.
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Microfilaments have been reported to be polarized in a number of cell types based both on function and isoform composition. There is evidence that microfilaments are involved in the movement of vesicles and the polarized delivery of proteins to specialized membrane domains. We have investigated the composition of actin microfilaments in gastrointestinal epithelial cells and their role in the delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) into the apical membrane using cultured T84 cells as a model. We identified a specific population of microfilaments containing the tropomyosin (Tm) isoforms Tm5a and/or Tm5b, which are polarized in T84 cell monolayers. Polarization of this microfilament population occurs very rapidly in response to cell-cell and cell-substratum contact and is not inhibited by jasplakinolide, suggesting this involves the movement of intact filaments. Colocalization of Tm5a and/or Tm5b and CFTR was observed in long-term cultures. A reduction in Tm5a and Tm5b expression, induced using antisense oligonucleotides, resulted in an increase in both CFTR surface expression and chloride efflux in response to cAMP stimulation. We conclude that Tm isoforms Tm5a and/or Tm5b mark an apical population of microfilaments that can regulate the insertion and/or retention of CFTR into the plasma membrane.
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Rokutan, K., R. B. Johnston y K. Kawai. "Oxidative stress induces S-thiolation of specific proteins in cultured gastric mucosal cells". American Journal of Physiology-Gastrointestinal and Liver Physiology 266, n.º 2 (1 de febrero de 1994): G247—G254. http://dx.doi.org/10.1152/ajpgi.1994.266.2.g247.
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Oxidative stress induces the formation of protein-mixed disulfides with low-molecular-weight thiols, especially glutathione. We analyzed this process, termed S-thiolation, in cultured gastric mucosal cells from guinea pigs by gel electrophoresis and autoradiography after radiolabeling of the intracellular glutathione pool with 35S. Hydrogen peroxide (H2O2) or diamide initiated rapid and reversible S-thiolation of specific proteins with molecular masses of 42, 30, 29, 28, and 22 kDa. Diamide caused particularly prominent S-thiolation of the 42 kDa protein. This protein was identified as actin by immunoblot analysis and actin-myosin precipitation. Fluorescence microscopy revealed that diamide caused a disappearance of normal stress fibers and a concomitant increase in actin polymerization in association with contraction of the cells. These morphological changes were completely reversible within minutes. With cells depleted of glutathione by incubation with DL-buthionine-[S,R]-sulfoximine, diamide caused severe contraction and rounding, and the cells detached from the culture plates. S-thiolation of actin could help protect gastric mucosal cells against irreversible organization of microfilaments by preserving microfilament dynamics under oxidative stress.
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Higgins, P. J. y M. P. Ryan. "p52(PAI-1) and actin expression in butyrate-induced flat revertants of v-ras-transformed rat kidney cells". Biochemical Journal 279, n.º 3 (1 de noviembre de 1991): 883–90. http://dx.doi.org/10.1042/bj2790883.
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Flat revertants of v-ras-transformed (KNRK) rat kidney cells, which express elevated levels of p21ras protein, were generated to high efficiencies with sodium butyrate (NaB). Overall protein synthesis in revertants was not different from parental cells, although changes were evident in expression and distribution of specific microfilament-associated cytoskeletal proteins. Quantitative two-dimensional electrophoresis revealed revertant-associated 3-4-fold increases in cytoskeletal deposition of the microfilament-associated proteins gelsolin and vinculin correlating with microfilament reorganization and focal-contact formation respectively. Similar increases in actin content were evident at both the total-cellular- and cytoskeletal-associated-protein levels. In contrast, intermediate-filament family elements (vimentin, lamins) remained unaltered. The only unique protein resolved in flat revertants was p52, a 52 kDa extracellular-matrix-associated protein previously identified as plasminogen-activator inhibitor type 1 (PAI-1). p52(PAI-1) expression was induced early during generation of the revertant phenotype and preceded development of focal-contact structures. NaB-induced p52(PAI-1) synthesis and generation of early morphological reversion in KNRK cells required ongoing RNA synthesis, since exposure to actinomycin D before addition of NaB inhibited both events. p52(PAI-1) induction by NaB was regulated at the level of mRNA abundance; in contrast, actin mRNA levels were the same in parental and revertant cells, suggesting that the increased actin content which typified the revertant phenotype was due to augmented actin microfilament stability.
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Higaki, Takumi, Toshio Sano y Seiichiro Hasezawa. "Actin microfilament dynamics and actin side-binding proteins in plants". Current Opinion in Plant Biology 10, n.º 6 (diciembre de 2007): 549–56. http://dx.doi.org/10.1016/j.pbi.2007.08.012.
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Franck, Z., M. Footer y A. Bretscher. "Microinjection of villin into cultured cells induces rapid and long-lasting changes in cell morphology but does not inhibit cytokinesis, cell motility, or membrane ruffling." Journal of Cell Biology 111, n.º 6 (1 de diciembre de 1990): 2475–85. http://dx.doi.org/10.1083/jcb.111.6.2475.
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Villin, a Ca2(+)-regulated F-actin bundling, severing, capping, and nucleating protein, is a major component of the core of microvilli of the intestinal brush border. Its actin binding properties, tissue specificity, and expression during cell differentiation suggest that it might be involved in the organization of the microfilaments in intestinal epithelial cells to form a brush border. Recently, Friederich et al., (Friederich, E., C. Huet, M. Arpin, and D. Louvard. 1989. Cell. 59:461-475) showed that villin expression in transiently transfected fibroblasts resulted in the loss of stress fibers and the appearance of large cell surface microvilli on some cells. Here, we describe the effect of villin microinjection into cells that normally lack this protein, which has allowed us to examine the immediate and long-term effects of introducing different concentrations of villin on microfilament organization and function. Microinjected cells rapidly lost their stress fibers and the actin was reorganized into abundant villin containing cortical structures, including microspikes and, in about half the cells, large surface microvilli. This change in actin organization persisted in cells for at least 24 h, during which time they had gone through two or three cell divisions. Microinjection of villin core, that lacks the bundling activity of villin but retains all the Ca2(+)-dependent properties, disrupted the stress fiber system and had no effect on cell surface morphology. Thus, the Ca2(+)-dependent activities of villin are responsible for stress fiber disruption, and the generation of cell surface structures is a consequence of its bundling activity. Microinjection of villin led to the reorganization of myosin, tropomyosin, and alpha-actinin, proteins normally associated with stress fibers, whereas both fimbrin and ezrin, which are also components of microvillar core filaments, were readily recruited into the induced surface structures. Vinculin was also redistributed from its normal location in focal adhesions. Despite these changes in the actin cytoskeleton, cells were able to divide and undergo cytokinesis, move, spread on a substratum, and ruffle. Thus, we show that a single microfilament-associated protein can reorganize the entire microfilament structure of a cell, without interfering with general microfilament-based functions like cytokinesis, cell locomotion, and membrane ruffling.
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Volberg, T., B. Geiger, J. Kartenbeck y W. W. Franke. "Changes in membrane-microfilament interaction in intercellular adherens junctions upon removal of extracellular Ca2+ ions." Journal of Cell Biology 102, n.º 5 (1 de mayo de 1986): 1832–42. http://dx.doi.org/10.1083/jcb.102.5.1832.
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EGTA-induced depletion of Ca2+ ions from the culture medium of Madin-Darby bovine kidney epithelial cells results in rapid splitting of adherens-type junctions and the detachment of the vinculin- and actin-containing filament bundle from the cytoplasmic faces of the plasma membrane of the zonula adhaerens. This process was monitored by phase-contrast microscopy, combined with electron microscopy and immunofluorescent localization of the two proteins. It is shown that shortly after extracellular free Ca2+ concentration is lowered to the micromolar range, the actin-containing, junction-associated belt of microfilaments, together with the vinculin-rich junctional plaque material, is irreversibly detached as one structural unit from the plasma membrane, contracts, and is displaced towards the perinuclear cytoplasm where it gradually disintegrates. Other actin- and vinculin-containing structures present in the same cells, notably the focal contacts at the substratum, are not similarly affected by the Ca2+ depletion and retain both the adhesion to the external surface and the association with the plaque and microfilament components. Electron microscopic examination has shown that the membrane domain of the zonulae adhaerentes, unlike that of desmosomes, is not endocytosed after Ca2+ removal and that the displaced actin- and vinculin-containing plaque and filament belt are not associated with a particular membrane. It is further shown that upon restoration of normal Ca2+ levels in the culture medium, new intercellular contacts are established gradually by accretion of both vinculin and actin into new belt-like plaque- and microfilament-containing structures.
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Senécal, J. L., S. Fortin, A. Roussin y F. Joyal. "Anticytoskeletal autoantibody to microfilament anchorage sites recognizes novel focal contact proteins." Journal of Clinical Investigation 80, n.º 3 (1 de septiembre de 1987): 778–85. http://dx.doi.org/10.1172/jci113134.
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Ravkov, Eugene V., Stuart T. Nichol, Clarence J. Peters y Richard W. Compans. "Role of Actin Microfilaments in Black Creek Canal Virus Morphogenesis". Journal of Virology 72, n.º 4 (1 de abril de 1998): 2865–70. http://dx.doi.org/10.1128/jvi.72.4.2865-2870.1998.
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ABSTRACT We have investigated the involvement of cytoskeletal proteins in the morphogenesis of Black Creek Canal virus (BCCV), a New World hantavirus. Immunofluorescent staining of BCCV-infected cells revealed a filamentous pattern of virus antigen, the appearance of which was sensitive to treatment with cytochalasin D, an actin microfilament-depolymerizing drug. Double immunofluorescence staining of BCCV-infected Vero cells with anti-BCCV nucleocapsid (N) monoclonal antibody and phalloidin revealed a colocalization of the BCCV N protein with actin microfilaments. A similar, though less prominent, filamentous pattern was observed in BHK21 cells transiently expressing the BCCV N protein alone but not in cells expressing the BCCV G1 and G2 glycoproteins. Moreover, the association of the N protein with actin microfilaments was confirmed by coimmunoprecipitation with β-actin-specific antibody. Treatment of the BCCV-infected Vero cells at 3 days postinfection with cytochalasin D decreased the yield of released BCCV by 94% relative to the yield from untreated cells. Pretreatment of Vero cells with cytochalasin D prior to and during BCCV adsorption and entry had no effect on the outcome of virus production. These results indicate that actin filaments may play an important role in hantavirus assembly and/or release.
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Chong, Audrey, Celia A. Lima, David S. Allan, Gheyath K. Nasrallah y Rafael A. Garduño. "The Purified and Recombinant Legionella pneumophila Chaperonin Alters Mitochondrial Trafficking and Microfilament Organization". Infection and Immunity 77, n.º 11 (17 de agosto de 2009): 4724–39. http://dx.doi.org/10.1128/iai.00150-09.
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ABSTRACT A portion of the total cellular pool of the Legionella pneumophila chaperonin, HtpB, is found on the bacterial cell surface, where it can mediate invasion of nonphagocytic cells. HtpB continues to be abundantly produced and released by internalized L. pneumophila and may thus have postinvasion functions. We used here two functional models (protein-coated beads and expression of recombinant proteins in CHO cells) to investigate the competence of HtpB in mimicking early intracellular trafficking events of L. pneumophila, including the recruitment of mitochondria, cytoskeletal alterations, the inhibition of phagosome-lysosome fusion, and association with the endoplasmic reticulum. Microscopy and flow cytometry studies indicated that HtpB-coated beads recruited mitochondria in CHO cells and U937-derived macrophages and induced transient changes in the organization of actin microfilaments in CHO cells. Ectopic expression of HtpB in the cytoplasm of transfected CHO cells also led to modifications in actin microfilaments similar to those produced by HtpB-coated beads but did not change the distribution of mitochondria. Association of phagosomes containing HtpB-coated beads with the endoplasmic reticulum was not consistently detected by either fluorescence or electron microscopy studies, and only a modest delay in the fusion of TrOv-labeled lysosomes with phagosomes containing HtpB-coated beads was observed. HtpB is the first Legionella protein and the first chaperonin shown to, by means of our functional models, induce mitochondrial recruitment and microfilament rearrangements, two postinternalization events that typify the early trafficking of virulent L. pneumophila.
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Sisson, John C., Christine Field, Richard Ventura, Anne Royou y William Sullivan. "Lava Lamp, a Novel Peripheral Golgi Protein, Is Required for Drosophila melanogaster Cellularization". Journal of Cell Biology 151, n.º 4 (13 de noviembre de 2000): 905–18. http://dx.doi.org/10.1083/jcb.151.4.905.
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Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti–Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.
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Friederich, E., T. E. Kreis y D. Louvard. "Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D". Journal of Cell Science 105, n.º 3 (1 de julio de 1993): 765–75. http://dx.doi.org/10.1242/jcs.105.3.765.
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Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca(2+)-dependent manner. In the absence of Ca2+, villin organizes microfilaments into bundles. Transfection of a villin-specific cDNA into cultured cells that do not produce this protein results in the growth of long surface microvilli and the reorganization of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plasma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the association of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structures, which were randomly distributed throughout the cytoplasm. The formation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy terminus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging revealed that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the important role of the plasma membrane in the organization of the actin cytoskeleton and suggest that the extension of the microvillar plasma membrane is dependent on the elongation of microfilaments at their fast-growing end. Inhibition of microfilament elongation near the plasma membrane by cytochalasin D may result in the ‘random’ nucleation of actin filaments throughout the cytoplasm. On the basis of the present data, we propose that villin is involved in the assembly of the microvillar actin bundle by a mechanism that does not prevent monomer association with the preferred end of microfilaments. For instance, villin may stabilize actin filaments by lateral interactions. The functional importance of the carboxy-terminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emphasize the observations that the effects of cytochalasin D in living cells can be modulated by actin-binding proteins.
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Shirao, T. "The Roles of Microfilament-Associated Proteins, Drebrins, in Brain Morphogenesis: A Review". Journal of Biochemistry 117, n.º 2 (1 de febrero de 1995): 231–36. http://dx.doi.org/10.1093/jb/117.2.231.
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Rawe, Vanesa Y., Christopher Payne y Gerald Schatten. "Profilin and actin-related proteins regulate microfilament dynamics during early mammalian embryogenesis". Human Reproduction 21, n.º 5 (20 de enero de 2006): 1143–53. http://dx.doi.org/10.1093/humrep/dei480.
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Eubanks, Haleigh B., Elise G. Lavoie, Jessica Goree, Jeffrey A. Kamykowski, Neriman Gokden, Michel Fausther y Jonathan A. Dranoff. "Reduction in SNAP-23 Alters Microfilament Organization in Myofibrobastic Hepatic Stellate Cells". Gene Expression 20, n.º 1 (12 de junio de 2020): 25–37. http://dx.doi.org/10.3727/105221619x15742818049365.
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Hepatic stellate cells (HSC) are critical effector cells of liver fibrosis. In the injured liver, HSC differentiate into a myofibrobastic phenotype. A critical feature distinguishing myofibroblastic from quiescent HSC is cytoskeletal reorganization. Soluble NSF attachment receptor (SNARE) proteins are important in trafficking of newly synthesized proteins to the plasma membrane for release into the extracellular environment. The goals of this project were to determine the expression of specific SNARE proteins in myofibroblastic HSC and to test whether their alteration changed the HSC phenotype in vitro and progression of liver fibrosis in vivo. We found that HSC lack the t-SNARE protein, SNAP-25, but express a homologous protein, SNAP-23. Downregulation of SNAP-23 in HSC induced reduction in polymerization and disorganization of the actin cytoskeleton associated with loss of cell movement. In contrast, reduction in SNAP-23 in mice by monogenic deletion delayed but did not prevent progression of liver fibrosis to cirrhosis. Taken together, these findings suggest that SNAP-23 is an important regular of actin dynamics in myofibroblastic HSC, but that the role of SNAP-23 in the progression of liver fibrosis in vivo is unclear.
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Samuelsson, SJ, PW Luther, DW Pumplin y RJ Bloch. "Structures linking microfilament bundles to the membrane at focal contacts". Journal of Cell Biology 122, n.º 2 (15 de julio de 1993): 485–96. http://dx.doi.org/10.1083/jcb.122.2.485.
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We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin-membrane interactions at focal contacts, including beta 1-integrin, vinculin, and talin, but they appear to contain less alpha-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained beta 1-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and beta 1-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.
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Rijken, P. J., W. J. Hage, P. M. van Bergen en Henegouwen, A. J. Verkleij y J. Boonstra. "Epidermal growth factor induces rapid reorganization of the actin microfilament system in human A431 cells". Journal of Cell Science 100, n.º 3 (1 de noviembre de 1991): 491–99. http://dx.doi.org/10.1242/jcs.100.3.491.
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Double immunofluorescence microscopy reveals that epidermal growth factor (EGF) treatment of A431 cells results in more apparent co-localization of EGF receptor (EGFR) and actin filaments, as compared to control cells. This indicates that EGF induces actin polymerization as well as additional association of the EGFR with similar sites on the membrane-skeleton. We show that immunoprecipitation of the cytoskeleton-linked EGFR after fragmentation of the cytoskeleton results in specific co-precipitation of F-actin and a limited set of other unidentified proteins. Interestingly, EGF treatment of intact cells results in increased immunoprecipitation of cytoskeleton-associated EGFR as well as of F-actin, while actin does not co-precipitate with the non-ionic detergent-solubilized EGFR. These results demonstrate that the cytoskeleton-linked EGFR is associated with the actin microfilament system. EGF induces additional formation of protein complexes, containing the EGFR and F-actin and a limited set of other unidentified proteins. The increased co-precipitation of F-actin is most likely related to EGF-induced actin polymerization, which is specifically associated with the apical cortical microfilament system, as demonstrated by confocal laser scanning microscopy and a phallicidin-binding assay.
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Guenal, I., Y. Risler y B. Mignotte. "Down-regulation of actin genes precedes microfilament network disruption and actin cleavage during p53-mediated apoptosis". Journal of Cell Science 110, n.º 4 (15 de febrero de 1997): 489–95. http://dx.doi.org/10.1242/jcs.110.4.489.
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Inactivation of Simian Virus 40 large T antigen, in cells immortalized with conditional mutants, leads to activation of p53 and apoptosis. We used the mRNA differential display method to identify genes differentially expressed during this process. We found that steady-state levels of mRNA for cytoplasmic actins decreased early during apoptosis. We also showed that, although the steady-state level of the corresponding proteins is not profoundly affected, they are substrates for an interleukin 1-beta converting enzyme (ICE)-like protease activated during the process. However, only a very small fraction of actin is proteolysed during the early stages of apoptosis. The microfilament network is affected and non polymerized actin accumulates in apoptotic bodies after the decrease of mRNA levels, but before a significant amount of actin is cleaved. This suggests that down-regulation of actin genes may be involved in microfilament rearrangements during p53-mediated apoptosis.
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Bubeck, P., S. Pistor, J. Wehland y B. M. Jockusch. "Ligand recruitment by vinculin domains in transfected cells". Journal of Cell Science 110, n.º 12 (15 de junio de 1997): 1361–71. http://dx.doi.org/10.1242/jcs.110.12.1361.
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Vinculin, a prominent protein component of microfilament-membrane attachment sites, consists of three major domains: an N-terminal, compact head and a C-terminal rod-like tail that are connected by a flexible, proline-rich hinge. In vitro, the protein has been shown to interact with numerous ligands, including other components of the microfilament system. To characterize the ligand recruitment ability of the different vinculin domains in a cellular environment, we used a novel approach of comprising chimeric proteins of either the vinculin head, hinge or tail regions, fused to the membrane anchor sequence of ActA, a surface protein of the intracellular bacterial pathogen Listeria monocytogenes. When PtK2 cells were transfected with the corresponding constructs, the ActA membrane anchor directed the chimeric polypeptides to mitochondrial membranes. In this position, they accumulated microfilament proteins, as seen by immunofluorescence analysis. A chimera comprising the full length vinculin clone recruited a substantial amount of the cellular F-actin, the vasodilator stimulated phosphoprotein (VASP) and paxillin, but little alpha-actinin and talin. The presence of only the vinculin head directed some of the fusion protein to focal contacts, and alpha-actinin recruitment was still ineffective. Prominent recruitment of F-actin and of VASP required the presence of the tail and proline-rich hinge, respectively. Reducing the vinculin tail to short pieces harboring only one of the two F-actin binding sequences, which were defined by in vitro experiments, resulted in loss of activity, possibly by incorrect polypeptide folding. The proline-rich hinge domain could be exchanged for the analogous region of the ActA protein, and the number of such proline-clusters, containing an FPPPP motif, correlated with the extent of VASP recruitment. The results show that this system can be used to analyze in vivo the activity of vinculin domains responsible for the assembly of various cytoskeletal ligands.
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SANGER, J. M., B. MITTAL, M. POCHAPIN y J. W. SANGER. "Observations of Microfilament Bundles in Living Cells Microinjected with Fluorescently Labelled Contractile Proteins". Journal of Cell Science 1986, Supplement 5 (1 de febrero de 1986): 17–44. http://dx.doi.org/10.1242/jcs.1986.supplement_5.2.
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JOCKUSCH, B. M., B. ZUREK, R. ZAHN, A. WESTMEYER y A. FUCHTBAUER. "Antibodies against vertebrate microfilament proteins in the analysis of cellular motility and adhesion". Journal of Cell Science 1991, Supplement 14 (1 de enero de 1991): 41–47. http://dx.doi.org/10.1242/jcs.1991.supplement_14.9.
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Schüler, Herwig y Kai Matuschewski. "Regulation of Apicomplexan Microfilament Dynamics by a Minimal Set of Actin-Binding Proteins". Traffic 7, n.º 11 (31 de agosto de 2006): 1433–39. http://dx.doi.org/10.1111/j.1600-0854.2006.00484.x.
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Chasis, JA, M. Prenant, A. Leung y N. Mohandas. "Membrane assembly and remodeling during reticulocyte maturation". Blood 74, n.º 3 (15 de agosto de 1989): 1112–20. http://dx.doi.org/10.1182/blood.v74.3.1112.1112.
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Abstract Membrane skeletal and cytoskeletal remodeling occurs throughout erythroid maturation. Microtubules and microfilaments have been identified morphologically in the nucleated erythroblast but the functional capability of these cytoskeletal structures during reticulocyte maturation has not been studied. Reticulocytes are formed from orthochromatic normoblasts by the process of nuclear extrusion. Two recognizable stages of reticulocyte maturation follow. The least mature reticulocytes are motile and multilobular, while the more mature reticulocytes are cup-shaped and nonmotile. To study the respective roles of microtubules and microfilaments in nuclear extrusion and cell motility, experiments were performed with agents that perturb these structures. Following the injection into rats of colchicine, a microtubule-disrupting substance, the number of normoblasts arrested at the stage of nuclear extrusion increased linearly over four hours. Similar results were obtained when bone marrow cells were incubated in culture in the presence of colchicine. In contrast, cell motility was dramatically decreased by cytochalasin B, a microfilament-disrupting agent, but not by colchicine. These results imply that microtubules are essential for the nuclear extrusion process, while microfilaments are essential for cell motility. Simultaneous changes in membrane skeletal assembly were assessed by measuring membrane deformability and stability, two properties regulated by the skeletal proteins. In ektacytometric assays, membrane deformability and mechanical stability of immature reticulocytes were markedly decreased to approximately 10% of normal, while that of more mature reticulocytes were nearly normal. Since the skeletal protein organization regulates these membrane properties, our findings imply that substantial membrane skeletal remodeling occurs during reticulocyte maturation. Thus we have identified major remodeling of both skeletal and cytoskeletal components during reticulocyte maturation.
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Chasis, JA, M. Prenant, A. Leung y N. Mohandas. "Membrane assembly and remodeling during reticulocyte maturation". Blood 74, n.º 3 (15 de agosto de 1989): 1112–20. http://dx.doi.org/10.1182/blood.v74.3.1112.bloodjournal7431112.
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Membrane skeletal and cytoskeletal remodeling occurs throughout erythroid maturation. Microtubules and microfilaments have been identified morphologically in the nucleated erythroblast but the functional capability of these cytoskeletal structures during reticulocyte maturation has not been studied. Reticulocytes are formed from orthochromatic normoblasts by the process of nuclear extrusion. Two recognizable stages of reticulocyte maturation follow. The least mature reticulocytes are motile and multilobular, while the more mature reticulocytes are cup-shaped and nonmotile. To study the respective roles of microtubules and microfilaments in nuclear extrusion and cell motility, experiments were performed with agents that perturb these structures. Following the injection into rats of colchicine, a microtubule-disrupting substance, the number of normoblasts arrested at the stage of nuclear extrusion increased linearly over four hours. Similar results were obtained when bone marrow cells were incubated in culture in the presence of colchicine. In contrast, cell motility was dramatically decreased by cytochalasin B, a microfilament-disrupting agent, but not by colchicine. These results imply that microtubules are essential for the nuclear extrusion process, while microfilaments are essential for cell motility. Simultaneous changes in membrane skeletal assembly were assessed by measuring membrane deformability and stability, two properties regulated by the skeletal proteins. In ektacytometric assays, membrane deformability and mechanical stability of immature reticulocytes were markedly decreased to approximately 10% of normal, while that of more mature reticulocytes were nearly normal. Since the skeletal protein organization regulates these membrane properties, our findings imply that substantial membrane skeletal remodeling occurs during reticulocyte maturation. Thus we have identified major remodeling of both skeletal and cytoskeletal components during reticulocyte maturation.
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McWhirter, J. R. y J. Y. Wang. "Activation of tyrosinase kinase and microfilament-binding functions of c-abl by bcr sequences in bcr/abl fusion proteins". Molecular and Cellular Biology 11, n.º 3 (marzo de 1991): 1553–65. http://dx.doi.org/10.1128/mcb.11.3.1553-1565.1991.
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Chronic myelogenous leukemia and one type of acute lymphoblastic leukemia are characterized by a 9;22 chronosome translocation in which 5' sequences of the bcr gene become fused to the c-abl proto-oncogene. The resulting chimeric genes encode bcr/abl fusion proteins which have deregulated tyrosine kinase activity and appear to play an important role in induction of these leukemias. A series of bcr/abl genes were constructed in which nested deletions of the bcr gene were fused to the c-abl gene. The fusion proteins encoded by these genes were assayed for autophosphorylation in vivo and for differences in subcellular localization. Our results demonstrate that bcr sequences activate two functions of c-abl; the tyrosine kinase activity and a previously undescribed microfilament-binding function. Two regions of bcr which activate these functions to different degrees have been mapped: amino acids 1 to 63 were strongly activating and amino acids 64 to 509 were weakly activating. The tyrosine kinase and microfilament-binding functions were not interdependent, as a kinase defective bcr/abl mutant still associated with actin filaments and a bcr/abl mutant lacking actin association still had deregulated kinase activity. Modification of actin filament functions by the bcr/abl tyrosine kinase may be an important event in leukemogenesis.
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McWhirter, J. R. y J. Y. Wang. "Activation of tyrosinase kinase and microfilament-binding functions of c-abl by bcr sequences in bcr/abl fusion proteins." Molecular and Cellular Biology 11, n.º 3 (marzo de 1991): 1553–65. http://dx.doi.org/10.1128/mcb.11.3.1553.
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Chronic myelogenous leukemia and one type of acute lymphoblastic leukemia are characterized by a 9;22 chronosome translocation in which 5' sequences of the bcr gene become fused to the c-abl proto-oncogene. The resulting chimeric genes encode bcr/abl fusion proteins which have deregulated tyrosine kinase activity and appear to play an important role in induction of these leukemias. A series of bcr/abl genes were constructed in which nested deletions of the bcr gene were fused to the c-abl gene. The fusion proteins encoded by these genes were assayed for autophosphorylation in vivo and for differences in subcellular localization. Our results demonstrate that bcr sequences activate two functions of c-abl; the tyrosine kinase activity and a previously undescribed microfilament-binding function. Two regions of bcr which activate these functions to different degrees have been mapped: amino acids 1 to 63 were strongly activating and amino acids 64 to 509 were weakly activating. The tyrosine kinase and microfilament-binding functions were not interdependent, as a kinase defective bcr/abl mutant still associated with actin filaments and a bcr/abl mutant lacking actin association still had deregulated kinase activity. Modification of actin filament functions by the bcr/abl tyrosine kinase may be an important event in leukemogenesis.
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Ng, S. Y., H. Erba, G. Latter, L. Kedes y J. Leavitt. "Modulation of microfilament protein composition by transfected cytoskeletal actin genes". Molecular and Cellular Biology 8, n.º 4 (abril de 1988): 1790–94. http://dx.doi.org/10.1128/mcb.8.4.1790-1794.1988.
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HuT-14T is a highly tumorigenic fibroblast cell line which exhibits a reduced steady-state level of beta-actin due to coding mutations in one of two beta-actin alleles. The normal rate of total actin synthesis could be restored in some clones of cells following transfection of the functional beta-actin gene but not following transfection of the functional gamma-actin gene. In gamma-actin gene-transfected substrains that have increased rates of gamma-actin synthesis, beta-actin synthesis is further reduced in a manner consistent with an autoregulatory mechanism, resulting in abnormal ratios of actin isoforms. Thus, both beta- and gamma-actin proteins can apparently regulate the synthesis of their coexpressed isoforms. In addition, decreased synthesis of normal beta-actin seems to correlate with a concomitant down-regulation of tropomyosin isoforms.
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Ng, S. Y., H. Erba, G. Latter, L. Kedes y J. Leavitt. "Modulation of microfilament protein composition by transfected cytoskeletal actin genes." Molecular and Cellular Biology 8, n.º 4 (abril de 1988): 1790–94. http://dx.doi.org/10.1128/mcb.8.4.1790.
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HuT-14T is a highly tumorigenic fibroblast cell line which exhibits a reduced steady-state level of beta-actin due to coding mutations in one of two beta-actin alleles. The normal rate of total actin synthesis could be restored in some clones of cells following transfection of the functional beta-actin gene but not following transfection of the functional gamma-actin gene. In gamma-actin gene-transfected substrains that have increased rates of gamma-actin synthesis, beta-actin synthesis is further reduced in a manner consistent with an autoregulatory mechanism, resulting in abnormal ratios of actin isoforms. Thus, both beta- and gamma-actin proteins can apparently regulate the synthesis of their coexpressed isoforms. In addition, decreased synthesis of normal beta-actin seems to correlate with a concomitant down-regulation of tropomyosin isoforms.
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Deretic, D., L. A. Huber, N. Ransom, M. Mancini, K. Simons y D. S. Papermaster. "rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis". Journal of Cell Science 108, n.º 1 (1 de enero de 1995): 215–24. http://dx.doi.org/10.1242/jcs.108.1.215.
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Small GTP-binding protein rab8 regulates transport from the TGN to the basolateral plasma membrane in epithelial cells and to the dendritic plasma membrane in cultured hippocampal neurons. In our approach to identify proteins involved in rhodopsin transport and sorting in retinal photoreceptors, we have found, using [32P]GTP overlays of 2D gel blots, that six small GTP-binding proteins are tightly bound to the post-Golgi membranes immunoisolated with a mAb to the cytoplasmic domain of frog rhodopsin. We report here that one of these proteins is rab8. About 50% of photoreceptor rab8 is membrane associated and approximately 13% is tightly bound to the post-Golgi vesicles. By confocal microscopy, antibody to rab8 specifically labels calycal processes and the actin bundles of the photoreceptor inner segment that extend inward to the junctional complexes that comprise the outer limiting membrane. Anti-rab8 shows a striking periodicity of high density labeling at 1 +/- 0.12 microns intervals along the actin bundles. Rhodopsin-bearing post-Golgi membranes cluster around the base of the cilium where rab8 and actin are also co-localized, as revealed by confocal microscopy of retinal sections double labeled with anti-rab8 and phalloidin. Microfilaments have been implicated in rod outer segment (ROS) disk morphogenesis. Our data suggest that rab6, which we have previously localized to the post-Golgi compartment, and rab8 associate with the post-Golgi membranes sequentially at different stages of transport. rab8 may mediate later steps that involve interaction of transport membranes with actin filaments and may participate in microfilament-dependent ROS disk morphogenesis.
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Postner, M. A. y E. F. Wieschaus. "The nullo protein is a component of the actin-myosin network that mediates cellularization in Drosophila melanogaster embryos". Journal of Cell Science 107, n.º 7 (1 de julio de 1994): 1863–73. http://dx.doi.org/10.1242/jcs.107.7.1863.
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After the 13th nuclear division cycle of Drosophila embryogenesis, cortical microfilaments are reorganized into a hexagonal network that drives the subsequent cellularization of the syncytial embryo. Zygotic transcription of the nullo and serendipity-alpha genes is required for normal structuring of the microfilament network. When either gene is deleted, the network assumes an irregular configuration leading to the formation of multinucleate cells. To investigate the role of these genes during cellularization, we have made monoclonal antibodies to both proteins. The nullo protein is present from cycle 13 through the end of cellularization. During cycle 13, it localizes between interphase actin caps and within metaphase furrows. In cellularizing embryos, nullo co-localizes with the actin-myosin network and invaginates along with the leading edge of the plasma membrane. The serendipity-alpha (sry-alpha) protein co-localizes with nullo protein to the hexagonal network but, unlike the nullo protein, it localizes to the sides rather than the vertices of each hexagon. Mutant embryos demonstrate that neither protein translationally regulates the other, but the localization of the sry-alpha protein to the hexagonal network is dependent upon nullo.
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Bird, Christopher, Marion Callus, Lynne Trickett y Robin Thorpe. "Immunochemical characterization of a new platelet specific monoclonal antibody and its use to demonstrate the cytoskeletal association of the platelet glycoprotein IIb-IIIa complex". Bioscience Reports 6, n.º 3 (1 de marzo de 1986): 323–33. http://dx.doi.org/10.1007/bf01115162.
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We describe the production and characterization of a monoclonal antibody specific for platelets. This antibody reacts strongly with human and primate platelets, but does not recognise human monocytes, polymorphonuclear leucocytes, lymphocytes, erythrocytes, leukaemic nor fibroblast cell lines, nor rodent platelets. Immunoprecipitation studies using radiolabelled platelet membrane proteins showed that the monoclonal antibody binds to the platelet membrane glycoprotein IIb-IIIa complex. Affinity chromatography using immobilized monoclonal antibody allows purification of the antigen, but also co-purifies the cytoskeletal proteins actin and myosin.Our results demonstrate immunochemically that although the GP IIb-IIIa complex is an external structure, it is connected through the cell membrane to the microfilament system.
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Lakkakorpi, P. T., I. Nakamura, M. Young, L. Lipfert, G. A. Rodan y L. T. Duong. "Abnormal localisation and hyperclustering of (alpha)(V)(beta)(3) integrins and associated proteins in Src-deficient or tyrphostin A9-treated osteoclasts". Journal of Cell Science 114, n.º 1 (1 de enero de 2001): 149–60. http://dx.doi.org/10.1242/jcs.114.1.149.
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The non-receptor tyrosine kinase Src was shown to be essential for osteoclast function in vivo. We have previously reported that engagement of (alpha)(v)(beta)(3) integrin in osteoclasts induces tyrosine phosphorylation and activation of the adhesion kinase PYK2 and the adaptor protein p130(Cas) in a Src-dependent manner. The objective of this study was to analyse the role of c-Src in the (alpha)(v)(beta)(3) integrin-dependent recruitment of signalling and cytoskeletal molecules in osteoclasts during bone resorption. Using prefusion osteoclasts (pOCs) obtained from cocultures of osteoblasts and spleen cells isolated from Src(-/-) mice or their normal littermates, we found: (1) similar expression levels and ligand binding affinities of (alpha)(v)(beta)(3) integrins in Src(-/-) and Src(+/?) pOCs, (2) reduced adhesion and spreading of Src(-/-) pOCs, (3) defective organisation of the microfilament proteins, F-actin, vinculin and paxillin, and of PYK2 and p130(Cas) in the sealing zone of Src(-/-)OCLs, and (4) hyperclustering of (alpha)(v)(beta)(3) integrins together with microfilament and signalling proteins in the basal membrane of Src-deficient OCLs. In normal OCLs, the tyrosine kinase inhibitor tyrphostin A9 inhibits actin ring formation, bone resorption and tyrosine phosphorylation of several proteins, including c-Src. Furthermore, tyrphostin A9 induced similar hyperclustering of (alpha)(v)(beta)(3) integrins in osteoclasts as observed in Src(-/-) OCLs. Taken together, these findings suggest that normal localisation of (alpha)(v)(beta)(3) and recruitment of its downstream effectors to the appropriate compartments of the osteoclast during resorption depend on Src kinase activity.
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Birgbauer, E. y F. Solomon. "A marginal band-associated protein has properties of both microtubule- and microfilament-associated proteins." Journal of Cell Biology 109, n.º 4 (1 de octubre de 1989): 1609–20. http://dx.doi.org/10.1083/jcb.109.4.1609.
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The marginal band of nucleated erythrocytes is a microtubule organelle under rigorous quantitative and spatial control, with properties quite different from those of the microtubule organelles of cultured cells. Previous results suggest that proteins other than tubulin may participate in organizing the marginal band, and may interact with elements of the erythrocyte cytoskeleton in addition to microtubules. To identify such species, we raised mAbs against the proteins that assemble from chicken brain homogenates with tubulin. One such antibody binds to a single protein in chicken erythrocytes, and produces an immunofluorescence pattern colocalizing with marginal band microtubules. Several properties of this protein are identical to those of ezrin, a protein isolated from brush border and localized to motile elements of cultured cells. A significant proportion of the antigen is not soluble in erythrocytes, as determined by extraction with nonionic detergent. This cytoskeleton-associated fraction is unaffected by treatments that solubilize the marginal band microtubules. The protein has properties of both microtubule- and microfilament-associated proteins. In the accompanying manuscript (Goslin, K., E. Birgbauer, G. Banker, and F. Solomon. 1989. J. Cell Biol. 109:1621-1631), we show that the same antibody recognizes a component of growth cones with a similar dual nature. In early embryonic red blood cells, the antigen is dispersed throughout the cell and does not colocalize with assembled tubulin. Its confinement to the marginal band during development follows rather than precedes that of microtubules. These results, along with previous work, suggest models for the formation of the marginal band.
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Carraway, Coralie A. Carothers y Michael Weiss. "Phalloidin shift on velocity sedimentation sucrose gradient centrifugation for identification of microfilament-associated proteins". Experimental Cell Research 161, n.º 1 (noviembre de 1985): 150–60. http://dx.doi.org/10.1016/0014-4827(85)90499-9.
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Cotton, Sophie, Romain Grangeon, Karine Thivierge, Isabelle Mathieu, Christine Ide, Taiyun Wei, Aiming Wang y Jean-François Laliberté. "Turnip Mosaic Virus RNA Replication Complex Vesicles Are Mobile, Align with Microfilaments, and Are Each Derived from a Single Viral Genome". Journal of Virology 83, n.º 20 (5 de agosto de 2009): 10460–71. http://dx.doi.org/10.1128/jvi.00819-09.
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ABSTRACT Nicotiana benthamiana plants were agroinoculated with an infectious cDNA clone of Turnip mosaic virus (TuMV) that was engineered to express a fluorescent protein (green fluorescent protein [GFP] or mCherry) fused to the viral 6K2 protein known to induce vesicle formation. Cytoplasmic fluorescent discrete protein structures were observed in infected cells, corresponding to the vesicles containing the viral RNA replication complex. The vesicles were motile and aligned with microfilaments. Intracellular movement of the vesicles was inhibited when cells were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. It was also observed that viral accumulation in the presence of this drug was reduced. These data indicate that microfilaments are used for vesicle movement and are necessary for virus production. Biogenesis of the vesicles was further investigated by infecting cells with two recombinant TuMV strains: one expressed 6K2GFP and the other expressed 6K2mCherry. Green- and red-only vesicles were observed within the same cell, suggesting that each vesicle originated from a single viral genome. There were also vesicles that exhibited sectors of green, red, or yellow fluorescence, an indication that fusion among individual vesicles is possible. Protoplasts derived from TuMV-infected N. benthamiana leaves were isolated. Using immunofluorescence staining and confocal microscopy, viral RNA synthesis sites were visualized as punctate structures distributed throughout the cytoplasm. The viral proteins VPg-Pro, RNA-dependent RNA polymerase, and cytoplasmic inclusion protein (helicase) and host translation factors were found to be associated with these structures. A single-genome origin and presence of protein synthetic machinery components suggest that translation of viral RNA is taking place within the vesicle.
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Swan, K. A., A. F. Severson, J. C. Carter, P. R. Martin, H. Schnabel, R. Schnabel y B. Bowerman. "cyk-1: a C. elegans FH gene required for a late step in embryonic cytokinesis". Journal of Cell Science 111, n.º 14 (30 de julio de 1998): 2017–27. http://dx.doi.org/10.1242/jcs.111.14.2017.
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A maternally expressed Caenorhabditis elegans gene called cyk-1 is required for polar body extrusion during meiosis and for a late step in cytokinesis during embryonic mitosis. Other microfilament- and microtubule-dependent processes appear normal in cyk-1 mutant embryos, indicating that cyk-1 regulates a specific subset of cytoskeletal functions. Because cytokinesis initiates normally and cleavage furrows ingress extensively in cyk-1 mutant embryos, we propose that the wild-type cyk-1 gene is required for a late step in cytokinesis. Cleavage furrows regress after completion of mitosis in cyk-1 mutants, leaving multiple nuclei in a single cell. Positional cloning and sequence analysis of the cyk-1 gene reveal that it encodes an FH protein, a newly defined family of proteins that appear to interact with the cytoskeleton during cytokinesis and in the regulation of cell polarity. Consistent with cyk-1 function being required for a late step in embryonic cytokinesis, we show that the CYK-1 protein co-localizes with actin microfilaments as a ring at the leading edge of the cleavage furrow, but only after extensive furrow ingression. We discuss our findings in the context of other studies suggesting that FH genes in yeast and insects function early in cytokinesis to assemble a cleavage furrow.
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Erdmann, Constanze, Roua Hassoun, Sebastian Schmitt, Carlos Kikuti, Anne Houdusse, Antonina J. Mazur, Andreas Mügge et al. "Integration of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) into Cellular Structures". Antioxidants 10, n.º 7 (5 de julio de 2021): 1082. http://dx.doi.org/10.3390/antiox10071082.
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The human mutant cardiac α-actins p.A295S or p.R312H and p.E361G, correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by the baculovirus/Sf21 insect cell system and purified to homogeneity. The purified cardiac actins maintained their native state but showed differences in Ca2+-sensitivity to stimulate the myosin-subfragment1 ATPase. Here we analyzed the interactions of these c-actins with actin-binding and -modifying proteins implicated in cardiomyocyte differentiation. We demonstrate that Arp2/3 complex and the formin mDia3 stimulated the polymerization rate and extent of the c-actins, albeit to different degrees. In addition, we tested the effect of the MICAL-1 monooxygenase, which modifies the supramolecular actin organization during development and adaptive processes. MICAL-1 oxidized these c-actin variants and induced their de-polymerization, albeit at different rates. Transfection experiments using MDCK cells demonstrated the preferable incorporation of wild type and p.A295S c-actins into their microfilament system but of p.R312H and p.E361G actins into the submembranous actin network. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding HA-tagged c-actin variants showed their incorporation into microfilaments after one day in culture and thereafter into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferentially incorporated at the minus ends.
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Gao, Lina y Anthony Bretscher. "Analysis of Unregulated Formin Activity Reveals How Yeast Can Balance F-Actin Assembly between Different Microfilament-based Organizations". Molecular Biology of the Cell 19, n.º 4 (abril de 2008): 1474–84. http://dx.doi.org/10.1091/mbc.e07-05-0520.
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Formins are regulated actin-nucleating proteins that are widespread among eukaryotes. Overexpression of unregulated formins in budding yeast is lethal and causes a massive accumulation of disorganized cable-like filaments. To explore the basis of this lethality, a cDNA library was screened to identify proteins whose overexpression could rescue the lethality conferred by unregulated Bnr1p expression. Three classes of suppressors encoding actin-binding proteins were isolated. One class encodes proteins that promote the assembly of actin cables (TPM1, TPM2, and ABP140), suggesting that the lethality was rescued by turning disorganized filaments into functional cables. The second class encodes proteins that bind G-actin (COF1, SRV2, and PFY1), indicating that reduction of the pool of actin available for cable formation may also rescue lethality. Consistent with this, pharmacological or genetic reduction of available actin also protected the cell from overproduction of unregulated Bnr1p. The third class consists of Las17p, an activator of the formin-independent Arp2/3p-dependent actin nucleation pathway. These results indicate that proper assembly of actin cables is sensitive to the appropriate balance of their constituents and that input into one pathway for actin filament assembly can affect another. Thus, cells must have a way of ensuring a proper balance between actin assembly pathways.
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Girard, Denis y Jean-Luc Senécal. "Anti-microfilament IgG Antibodies in Normal Adults and in Patients with Autoimmune Diseases: Immunofluorescence and Immunoblotting Analysis of 201 Subjects Reveals Polyreactivity with Microfilament-Associated Proteins". Clinical Immunology and Immunopathology 74, n.º 2 (febrero de 1995): 193–201. http://dx.doi.org/10.1006/clin.1995.1028.
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