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Actin Capping
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Alla S Kostyukova – One of the best experts on this subject based on the ideXlab platform.
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alteration of tropomyosin binding properties of tropomodulin 1 affects its Capping ability and localization in skeletal myocytes
Journal of Biological Chemistry, 2013Co-Authors: Natalia Moroz, Alla S Kostyukova, Stefanie M. Novak, Carol C Gregorio, Ricardo Azevedo, Mert Colpan, Vladimir N UverskyAbstract:Tropomodulin (Tmod) is an Actin–Capping protein that binds to the two tropomyosins (TM) at the pointed end of the Actin filament to prevent further Actin polymerization and depolymerization. Therefore, understanding the role of Tmod is very important when studying Actin filament dependent processes such as muscle contraction and intracellular transport. The Capping ability of Tmod is highly influenced by TM and is 1000-fold greater in the presence of TM. There are four Tmod isoforms (Tmod1–4), three of which, Tmod1, Tmod3, and Tmod4, are expressed in skeletal muscles. The affinity of Tmod1 to skeletal striated TM (stTM) is higher than that of Tmod3 and Tmod4 to stTM. In this study, we tested mutations in the TM-binding sites of Tmod1, using circular dichdichroism (CD) and prediction analysis (PONDR). The mutations R11K, D12N, and Q144K were chosen because they decreased the affinity of Tmod1 to stTM, making it similar to that of affinity of Tmod3 and Tmod4 to stTM. Significant reduction of inhibition of Actin pointed-end polymerization in the presence of stTM was shown for Tmod1 (R11K/D12N/Q144K) as compared with WT Tmod1. When GFP-Tmod1 and mutants were expressed in primary chicken skeletal myocytes, decreased assembly of Tmod1 mutants was revealed. This indicates a direct correlation between TM-binding and the Actin–Capping abilities of Tmod. Our data confirmed the hypothesis that assembly of Tmod at the pointed-end of the Actin filament depends on its TM-binding affinity.
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alteration of tropomyosin binding properties of tropomodulin 1 affects its Capping ability and localization
, 2013Co-Authors: Natalia Moroz, Stefanie M. Novak, Carol C Gregorio, Ricardo Azevedo, Mert Colpan, Vladimir N Uversky, Alla S KostyukovaAbstract:Tropomodulin (Tmod) is an Actin–Capping protein that binds to the two tropomyosins (TM) at the pointed end of the Actin filament to prevent further Actin polymerization and depolymerization. Therefore, understanding the role of Tmod is very important when studying Actin filament dependent processes such as muscle contraction and intracellular transport. The Capping ability of Tmod is highly influenced by TM and is 1000-fold greater in the presence of TM. There are four Tmod isoforms (Tmod1– 4), three of which, Tmod1, Tmod3, and Tmod4, are expressed in skeletal muscles. The affinity of Tmod1 to skeletal striated TM (stTM) is higher than that of Tmod3 and Tmod4 to stTM. In this study, we tested mutations in the TM-binding sites of Tmod1, using circular dichdichroism (CD) and prediction analysis (PONDR). The mutations R11K, D12N, and Q144K were chosen because they decreased the affinity of Tmod1 to stTM, making it similar to that of affinity of Tmod3 and Tmod4 to stTM. Significant reduction of inhibition of Actin pointedend polymerization in the presence of stTM was shown for Tmod1 (R11K/D12N/Q144K) as compared with WT Tmod1. When GFP-Tmod1 and mutants were expressed in primary chicken skeletal myocytes, decreased assembly of Tmod1 mutants was revealed. This indicates a direct correlation between TM-binding and the Actin–Capping abilities of Tmod. Our data confirmed the hypothesis that assembly of Tmod at the pointed-end of the Actin filament depends on
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Identification of Residues within Tropomodulin-1 Responsible for Its Localization at the Pointed Ends of the Actin Filaments in Cardiac Myocytes
Journal of Biological Chemistry, 2010Co-Authors: Takehiro Tsukada, Lucy Kotlyanskaya, Robert Huynh, Brinda Desai, Stefanie M. Novak, Andrey V. Kajava, Carol C Gregorio, Alla S KostyukovaAbstract:Tropomodulin is a tropomyosin-dependent Actin filament Capping protein involved in the structural formation of thin filaments and in the regulation of their lengths through its localization at the pointed ends of Actin filaments. The disordered N-terminal domain of tropomodulin contains three functional sites: two tropomyosin-binding and one tropomyosin-dependent Actin–Capping sites. The C-terminal half of tropomodulin consists of one compact domain containing a tropomyosin-independent Actin–Capping site. Here we determined the structural properties of tropomodulin-1 that affect its roles in cardiomyocytes. To explore the significance of individual tropomyosin-binding sites, GFP-tropomodulin-1 with single mutations that destroy each tropomyosin-binding site was expressed in cardiomyocytes. We demonstrated that both sites are necessary for the optimal localization of tropomodulin-1 at thin filafilament pointed ends, with site 2 Acting as the major determinant. To investigate the functional properties of the tropomodulin C-terminal domain, truncated versions of GFP-tropomodulin-1 were expressed in cardiomyocytes. We discovered that the leucine-rich repeat (LRR) fold and the C-terminal helix are required for its proper targeting to the pointed ends. To investigate the structural significance of the LRR fold, we generated three mutations within the C-terminal domain (V232D, F263D, and L313D). Our results show that these mutations affect both tropomyosin-independent Actin–Capping activity and pointed end localization, most likely by changing local conformations of either loops or side chains of the surfaces involved in the interactions of the LRR domain. Studying the influence of these mutations individually, we concluded that, in addition to the tropomyosin-independent Actin–Capping site, there appears to be another regulatory site within the tropomodulin C-terminal domain.
Marc Ladanyi – One of the best experts on this subject based on the ideXlab platform.
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expression of f Actin Capping protein subunit beta capzb is associated with cell growth and motility in epithelioid sarcoma
BMC Cancer, 2016Co-Authors: Kenta Mukaihara, Yoshiyuki Suehara, Shinji Kohsaka, Daisuke Kubota, Midori Todaishii, Keisuke Akaike, Tsutomu Fujimura, Eisuke Kobayashi, Takashi Yao, Marc LadanyiAbstract:Background A previous proteomics study demonstrated the overexpression of F-Actin Capping protein subunit beta (CAPZB) in tissue specimens of epithelioid sarcoma (EpiS). The aim of the present study was to elucidate the function of CAPZB in EpiS.
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Expression of F-Actin–Capping protein subunit beta, CAPZB, is associated with cell growth and motility in epithelioid sarcoma
BMC Cancer, 2016Co-Authors: Kenta Mukaihara, Yoshiyuki Suehara, Shinji Kohsaka, Daisuke Kubota, Keisuke Akaike, Tsutomu Fujimura, Eisuke Kobayashi, Takashi Yao, Midori Toda-ishii, Marc LadanyiAbstract:Background A previous proteomics study demonstrated the overexpression of F-Actin Capping protein subunit beta (CAPZB) in tissue specimens of epithelioid sarcoma (EpiS). The aim of the present study was to elucidate the function of CAPZB in EpiS. Methods Cellular functional assays were performed in two EpiS cell lines using CAPZB siRNAs. In addition, comparative protein expression analyses using Isobaric Tags for Relative and Absolute Quantitation (i-TRAQ) method were performed to identify the specific proteins whose expression was dysregulated by CAPZB, and analysed the data with the Ingenuity Pathways Analysis (IPA) system using the obtained protein profiles to clarify the functional pathway networks associated with the oncogenic function of CAPZB in EpiS. Additionally, we performed functional assays of the INI1 protein using INI1-overexpressing EpiS cells. Results All 15 EpiS cases showed an immunohistochemical expression of CAPZB, and two EpiS cell lines exhibited a strong CAPZB expression. Silencing of CAPZB inhibited the growth, invasion and migration of the EpiS cells. Analysis of protein profiles using the IPA system suggested that SWI/SNF chromatin-remodeling complexes including INI1 may function as a possible upstream regulator of CAPZB. Furthermore, silencing of CAPZB resulted in a decreased expression of INI1 proteins in the INI1-positive EpiS cells, whereas the induction of INI1 in the INI1-deficient EpiS cells resulted in an increased CAPZB mRNA expression. Conclusions CAPZB is involved in tumor progression in cases of EpiS, irrespective of the INI1 expression, and may be a potential therapeutic target. The paradoxical relationship between the tumor suppressor INI1 and the oncoprotein CAPZB in the pathogenesis of EpiS remains to be clarified.
Jeffrey S. Simske – One of the best experts on this subject based on the ideXlab platform.
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the minus end Actin Capping protein unc 94 tropomodulin regulates development of the caenorhabditis elegans intestine
Developmental Dynamics, 2014Co-Authors: Elisabeth Coxpaulson, Vincent L. Cannataro, Thomas Gallagher, Corey M. Hoffman, Gary Mantione, Matthew Mcintosh, Malan Silva, Nicole Vissichelli, Rachel Walker, Jeffrey S. SimskeAbstract:Background: Tropomodulins are Actin–Capping proteins that regulate the stability of the slow-growing, minus-ends of Actin filaments. The C. elegans tropomodulin homolog, UNC-94, has sequence and functional similarity to vertebrate tropomodulins. We investigated the role of UNC-94 in C. elegans intestinal morphogenesis. Results: In the embryonic C. elegans intestine, UNC-94 localizes to the terminal web, an Actin– and intermediate filafilament-rich structure that underlies the apical membrane. Loss of UNC-94 function results in areas of flattened intestinal lumelumen. In worms homozygous for the strong loss-of-function allele, unc-94(tm724), the terminal web is thinner and the amount of F-Actin is reduced, pointing to a role for UNC-94 in regulating the structure of the terminal web. The non-muscle myosin, NMY-1, also localizes to the terminal web, and we present evidence that increasing actomyosin contractility by depleting the myosin phosphatase regulatory subunit, mel-11, can rescue the flattened lumen phenotype of unc-94 mutants. Conclusions: The data support a model in which minus-end Actin Capping by UNC-94 promotes proper F-Actin structure and contraction in the terminal web, yielding proper shape of the intestinal lumelumen. This establishes a new role for a tropomodulin in regulating lumen shape during tubulogenesis. Developmental Dynamics 243:753–764, 2014. V C 2014 Wiley Periodicals, Inc.
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The Minus-End Actin Capping Protein, UNC-94/Tropomodulin, Regulates Development of the Caenorhabditis elegans Intestine
Developmental Dynamics, 2014Co-Authors: Elisabeth Cox-paulson, Vincent L. Cannataro, Thomas Gallagher, Corey M. Hoffman, Gary Mantione, Matthew Mcintosh, Malan Silva, Nicole Vissichelli, Rachel L. Walker, Jeffrey S. SimskeAbstract:Background: Tropomodulins are Actin–Capping proteins that regulate the stability of the slow-growing, minus-ends of Actin filaments. The C. elegans tropomodulin homolog, UNC-94, has sequence and functional similarity to vertebrate tropomodulins. We investigated the role of UNC-94 in C. elegans intestinal morphogenesis. Results: In the embryonic C. elegans intestine, UNC-94 localizes to the terminal web, an Actin– and intermediate filafilament-rich structure that underlies the apical membrane. Loss of UNC-94 function results in areas of flattened intestinal lumelumen. In worms homozygous for the strong loss-of-function allele, unc-94(tm724), the terminal web is thinner and the amount of F-Actin is reduced, pointing to a role for UNC-94 in regulating the structure of the terminal web. The non-muscle myosin, NMY-1, also localizes to the terminal web, and we present evidence that increasing actomyosin contractility by depleting the myosin phosphatase regulatory subunit, mel-11, can rescue the flattened lumen phenotype of unc-94 mutants. Conclusions: The data support a model in which minus-end Actin Capping by UNC-94 promotes proper F-Actin structure and contraction in the terminal web, yielding proper shape of the intestinal lumelumen. This establishes a new role for a tropomodulin in regulating lumen shape during tubulogenesis. Developmental Dynamics 243:753–764, 2014. V C 2014 Wiley Periodicals, Inc.
John A. Cooper – One of the best experts on this subject based on the ideXlab platform.
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comparative analysis of cpi motif regulation of biochemical functions of Actin Capping protein
Biochemistry, 2020Co-Authors: Patrick Mcconnell, Marlene Mekel, Alexander G Kozlov, Olivia L Mooren, Timothy M Lohman, John A. CooperAbstract:The heterodimeric Actin Capping protein (CP) is regulated by a set of proteins that contain CP-interActing (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and …
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comparative analysis of cpi motif regulation of biochemical functions of Actin Capping protein
bioRxiv, 2020Co-Authors: Patrick Mcconnell, Marlene Mekel, Alexander G Kozlov, Olivia L Mooren, Timothy M Lohman, John A. CooperAbstract:The heterodimeric Actin Capping protein (CP) is regulated by a set of proteins that contain CP-interActing (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families to a) bind to CP, b) allosterically inhibit barbed-end Capping by CP, and c) allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino-acid residues that are conserved but are not part of the strictly defined consensus, as it was originally identified in comparisons of sequences of CPI motifs(1, 2) across all protein families (1, 2). These biochemical differences may be important for conserved distinct functions of CPI-motif protein families in cells with respect to the regulation of CP activity and Actin assembly near membranes.
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Capping protein regulators fine tune Actin assembly dynamics
Nature Reviews Molecular Cell Biology, 2014Co-Authors: Marc Edwards, David Sept, Adam Zwolak, Dorothy A Schafer, Roberto Dominguez, John A. CooperAbstract:The Actin Capping activity of Capping protein (CP) is indirectly regulated by competing with other factors for filament binding, or directly by factors that bind CP and sterically inhibit its interactions with filaments. Other proteins interact with CP through their ‘Capping protein interaction’ (CPI) motif and modulate its activity via allosteric effects.
Sofia Khaitlina – One of the best experts on this subject based on the ideXlab platform.
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1 REGULATION OF SODIUM CHANNEL ACTIVITY BY Capping OF Actin FILAMENTS
, 2013Co-Authors: Ekaterina Shumilina, Yuri A. Negulyaev, Elena A. Morachevskaya, Sofia KhaitlinaAbstract:cell. 2 Ion transport in various tissues can be regulated by the cortical Actin cytoskeleton. Specifically, involvement of Actin dynamics in the regulation of non-voltage-gated sodium channels has been shown. Here, inside-out patch clamp experiments were performed to study the effect of the heterodimeric Actin Capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasininduced disruption of Actin filaments and inactivated by G-Actin under ionic conditions promoting rapid Actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-Actin, CapZ prevented Actin-induced channel inactivation, and this effect occurred at CapZ/Actin molar ratios from 1:5 to 1:100. When Actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of Actin filaments at the plasma membrane due to the modification of Actin dynamics by CapZ. No effect on the channel activity was observed in response to F-Actin confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that Actin–Capping protein can participate in the cytoskeletonassociated regulation of sodium transport in nonexcitable cells
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Regulation of Sodium Channel Activity by Capping of Actin Filaments
Molecular biology of the cell, 2003Co-Authors: Ekaterina Shumilina, Yuri A. Negulyaev, Elena A. Morachevskaya, Horst Hinssen, Sofia KhaitlinaAbstract:Ion transport in various tissues can be regulated by the cortical Actin cytoskeleton. Specifically, involvement of Actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric Actin Capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of Actin filaments and inactivated by G-Actin under ionic conditions promoting rapid Actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-Actin, CapZ prevented Actin-induced channel inactivation, and this effect occurred at CapZ/Actin molar ratios from 1:5 to 1:100. When Actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of Actin filaments at the plasma membrane due to the modification of Actin dynamics by CapZ. No effect on the channel activity was observed in response to F-Actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that Actin–Capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells.
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Regulation of sodium channel activity by Capping of Actin filaments
'American Society for Cell Biology (ASCB)', 2003Co-Authors: Ekaterina Shumilina, Yuri A. Negulyaev, Elena A. Morachevskaya, Hinssen Horst, Sofia KhaitlinaAbstract:Shumilina EV, Negulyaev YA, Morachevskaya EA, Hinssen H, Khaitlina SY. Regulation of sodium channel activity by Capping of Actin filaments. MOLECULAR BIOLOGY OF THE CELL. 2003;14(4):1709-1716.Ion transport in various tissues can be regulated by the cortical Actin cytoskeleton. Specifically, involvement of Actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric Actin Capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of Actin filaments and inactivated by G-Actin under ionic conditions promoting rapid Actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-Actin, CapZ prevented Actin-induced channel inactivation, and this effect occurred at CapZ/Actin molar ratios from 1:5 to 1:100. When Actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of Actin filaments at the plasma membrane due to the modification of Actin dynamics by CapZ. No effect on the channel activity was observed in response to F-Actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that Actin–Capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells