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Shuh Narumiya - One of the best experts on this subject based on the ideXlab platform.
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epha4 dependent axon retraction and midline localization of ephrin b3 are disrupted in the spinal cord of mice lacking MDia1 and mdia3 in combination
Genes to Cells, 2013Co-Authors: Yosuke Toyoda, D Thumkeo, Toshimasa Ishizaki, Ryota Shinohara, Tomoyuki Furuyashiki, Hiroshi Kamijo, Hiroshi Nishimaru, Hiroyuki Hioki, Takeshi Kaneko, Shuh NarumiyaAbstract:mDia is an actin nucleator and polymerization factor regulated by the small GTPase Rho and consists of three isoforms. Here, we found that mice lacking MDia1 and mDia3, two isoforms expressed in the brain, in combination (mDia-DKO mice) show impaired left–right limb coordination during locomotion and aberrant midline crossing of axons of corticospinal neurons and spinal cord interneurons. Given that mice lacking Ephrin-B3–EphA4 signaling show a similar impairment in locomotion, we examined whether mDia is involved in Ephrin-B3–EphA4 signaling for axon repulsion. In primary cultured neurons, mDia deficiency impairs growth cone collapse and axon retraction induced by chemo-repellants including EphA ligands. In mDia-DKO mice, the Ephrin-B3-expressing midline structure in the spinal cord is disrupted, and axons aberrantly cross the spinal cord midline preferentially through the region devoid of Ephrin-B3. Therefore, mDia plays multiple roles in the proper formation of the neural network in vivo.
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deficiency of mdia an actin nucleator disrupts integrity of neuroepithelium and causes periventricular dysplasia
PLOS ONE, 2011Co-Authors: D Thumkeo, Kiyoshi Tohyama, Toshimasa Ishizaki, Ryota Shinohara, Keisuke Watanabe, Hirohide Takebayashi, Yosuke Toyoda, Tomoyuki Furuyashiki, Shuh NarumiyaAbstract:During development of the central nervous system, the apical-basal polarity of neuroepithelial cells is critical for homeostasis of proliferation and differentiation of neural stem cells. While adherens junctions at the apical surface of neuroepithelial cells are important for maintaining the polarity, the molecular mechanism regulating integrity of these adherens junctions remains largely unknown. Given the importance of actin cytoskeleton in adherens junctions, we have analyzed the role of mDia, an actin nucleator and a Rho effector, in the integrity of the apical adherens junction. Here we show that MDia1 and mDia3 are expressed in the developing brain, and that mDia3 is concentrated in the apical surface of neuroepithelium. Mice deficient in both MDia1 and mDia3 develop periventricular dysplastic mass widespread throughout the developing brain, where neuroepithelial cell polarity is impaired with attenuated apical actin belts and loss of apical adherens junctions. In addition, electron microscopic analysis revealed abnormal shrinkage and apical membrane bulging of neuroepithelial cells in the remaining areas. Furthermore, perturbation of Rho, but not that of ROCK, causes loss of the apical actin belt and adherens junctions similarly to mDia-deficient mice. These results suggest that actin cytoskeleton regulated by Rho-mDia pathway is critical for the integrity of the adherens junctions and the polarity of neuroepithelial cells, and that loss of this signaling induces aberrant, ectopic proliferation and differentiation of neural stem cells.
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MDia1 targets v src to the cell periphery and facilitates cell transformation tumorigenesis and invasion
Molecular and Cellular Biology, 2010Co-Authors: Masahiro Tanji, Toshimasa Ishizaki, Saman Ebrahimi, Yuko Tsuboguchi, Taiko Sukezane, Tsuyoshi Akagi, Margaret C Frame, Nobuo Hashimoto, Susumu Miyamoto, Shuh NarumiyaAbstract:The small GTPase Rho regulates cell morphogenesis through remodeling of the actin cytoskeleton. While Rho is overexpressed in many clinical cancers, the role of Rho signaling in oncogenesis remains unknown. MDia1 is a Rho effector producing straight actin filaments. Here we transduced mouse embryonic fibroblasts from MDia1-deficient mice with temperature-sensitive v-Src and examined the involvement and mechanism of the Rho-MDia1 pathway in Src-induced oncogenesis. We showed that in v-Src-transduced MDia1-deficient cells, formation of actin filaments is suppressed, and v-Src in the perinuclear region does not move to focal adhesions upon a temperature shift. Consequently, membrane translocation of v-Src, v-Src-induced morphological transformation, and podosome formation are all suppressed in MDia1-deficient cells with impaired tyrosine phosphorylation. MDia1-deficient cells show reduced transformation in vitro as examined by focus formation and colony formation in soft agar and exhibit suppressed tumorigenesis and invasion when implanted in nude mice in vivo. Given overexpression of c-Src in various cancers, these findings suggest that Rho-MDia1 signaling facilitates malignant transformation and invasion by manipulating the actin cytoskeleton and targeting Src to the cell periphery.
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Rho signaling, ROCK and MDia1, in transformation, metastasis and invasion
Cancer and Metastasis Reviews, 2009Co-Authors: Shuh Narumiya, Masahiro Tanji, Toshimasa IshizakiAbstract:The Rho subgroup of the Rho GTPases consisting of RhoA, RhoB and RhoC induces a specific type of actin cytoskeleton and carry out a variety of functions in the cell. mDia and ROCK are downstream effectors of Rho mediating Rho action on the actin cytoskeleton; mDia produces actin filaments by nucleation and polymerization and ROCK activate myosin to cross-link them for induction of actomyosin bundles and contractility. mDia is potentially linked to Rac activation and membrane ruffle formation through c-Src-induced phosphorylation of focal adhesion proteins, and ROCK antagonizes this mDia action. Thus, cell morphogenesis, adhesion, and motility can be determined by the balance between mDia and ROCK activities. Though they are not oncogenes by themselves, overexpression of RhoA and RhoC are often found in clinical cancers, and RhoC has been repeatedly identified as a gene associated with metastasis. The Rho-ROCK pathway is implicated in Ras-mediated transformation, the amoeboid movement of tumor cells in the three-dimensional matrix, and transmigration of tumor cells through the mesothelial monolayer. On the other hand, the Rho-MDia1 pathway is implicated in Src-mediated remodeling of focal adhesions and migration of tumor cells. There is also an indication that the Rho pathway other than ROCK is involved in Src-mediated induction of podosome and regulation of matrix metalloproteases. Thus, Rho mediates various phenotypes of malignant transformation by Ras and Src through its effectors, ROCK and mDia.
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g actin regulates rapid induction of actin nucleation by MDia1 to restore cellular actin polymers
Journal of Cell Science, 2008Co-Authors: Chiharu Higashida, Shuh Narumiya, James Monypenny, Shiro Suetsugu, Takahiro Tsuji, Naoki WatanabeAbstract:MDia1 belongs to the formin family of proteins that share FH1 and FH2 domains. Although formins play a critical role in the formation of many actin-based cellular structures, the physiological regulation of formin-mediated actin assembly within the cell is still unknown. Here we show that cells possess an acute actin polymer restoration mechanism involving MDia1. By using single-molecule live-cell imaging, we found that several treatments including low-dose G-actin-sequestering drugs and unpolymerizable actin mutants activate MDia1 to initiate fast directional movement. The FH2 region, the core domain for actin nucleation, is sufficient to respond to latrunculin B (LatB) to increase its actin nucleation frequency. Simulation analysis revealed an unexpected paradoxical effect of LatB that leads to a several fold increase in free G-actin along with an increase in total G-actin. These results indicate that in cells, the actin nucleation frequency of MDia1 is enhanced not only by Rho, but also strongly through increased catalytic efficiency of the FH2 domain. Consistently, frequent actin nucleation by MDia1 was found around sites of vigorous actin disassembly. Another major actin nucleator, the Arp2/3 complex, was not affected by the G-actin increase induced by LatB. Taken together, we propose that transient accumulation of G-actin works as a cue to promote MDia1-catalyzed actin nucleation to execute rapid reassembly of actin filaments.
Toshimasa Ishizaki - One of the best experts on this subject based on the ideXlab platform.
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MDia1 3 dependent actin polymerization spatiotemporally controls lat phosphorylation by zap70 at the immune synapse
Science Advances, 2020Co-Authors: Yoshimoto Katsura, D Thumkeo, Kiyoshi Tohyama, Toshimasa Ishizaki, Shunsuke Kitajima, Pakorn Kanchanawong, Chiaki Takahashi, Y Nishimura, Takako HirataAbstract:The mechanism by which the cytosolic protein Zap70 physically interacts with and phosphorylates its substrate, the transmembrane protein LAT, upon T cell receptor (TCR) stimulation remains largely obscure. In this study, we found that the pharmacological inhibition of formins, a major class of actin nucleators, suppressed LAT phosphorylation by Zap70, despite TCR stimulation–dependent phosphorylation of Zap70 remaining intact. High-resolution imaging and three-dimensional image reconstruction revealed that localization of phosphorylated Zap70 to the immune synapse (IS) and subsequent LAT phosphorylation are critically dependent on formin-mediated actin polymerization. Using knockout mice, we identify MDia1 and mDia3, which are highly expressed in T cells and which localize to the IS upon TCR activation, as the critical formins mediating this process. Our findings therefore describe previously unsuspected roles for MDia1 and mDia3 in the spatiotemporal control of Zap70-dependent LAT phosphorylation at the IS through regulation of filamentous actin, and underscore their physiological importance in TCR signaling.
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epha4 dependent axon retraction and midline localization of ephrin b3 are disrupted in the spinal cord of mice lacking MDia1 and mdia3 in combination
Genes to Cells, 2013Co-Authors: Yosuke Toyoda, D Thumkeo, Toshimasa Ishizaki, Ryota Shinohara, Tomoyuki Furuyashiki, Hiroshi Kamijo, Hiroshi Nishimaru, Hiroyuki Hioki, Takeshi Kaneko, Shuh NarumiyaAbstract:mDia is an actin nucleator and polymerization factor regulated by the small GTPase Rho and consists of three isoforms. Here, we found that mice lacking MDia1 and mDia3, two isoforms expressed in the brain, in combination (mDia-DKO mice) show impaired left–right limb coordination during locomotion and aberrant midline crossing of axons of corticospinal neurons and spinal cord interneurons. Given that mice lacking Ephrin-B3–EphA4 signaling show a similar impairment in locomotion, we examined whether mDia is involved in Ephrin-B3–EphA4 signaling for axon repulsion. In primary cultured neurons, mDia deficiency impairs growth cone collapse and axon retraction induced by chemo-repellants including EphA ligands. In mDia-DKO mice, the Ephrin-B3-expressing midline structure in the spinal cord is disrupted, and axons aberrantly cross the spinal cord midline preferentially through the region devoid of Ephrin-B3. Therefore, mDia plays multiple roles in the proper formation of the neural network in vivo.
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deficiency of mdia an actin nucleator disrupts integrity of neuroepithelium and causes periventricular dysplasia
PLOS ONE, 2011Co-Authors: D Thumkeo, Kiyoshi Tohyama, Toshimasa Ishizaki, Ryota Shinohara, Keisuke Watanabe, Hirohide Takebayashi, Yosuke Toyoda, Tomoyuki Furuyashiki, Shuh NarumiyaAbstract:During development of the central nervous system, the apical-basal polarity of neuroepithelial cells is critical for homeostasis of proliferation and differentiation of neural stem cells. While adherens junctions at the apical surface of neuroepithelial cells are important for maintaining the polarity, the molecular mechanism regulating integrity of these adherens junctions remains largely unknown. Given the importance of actin cytoskeleton in adherens junctions, we have analyzed the role of mDia, an actin nucleator and a Rho effector, in the integrity of the apical adherens junction. Here we show that MDia1 and mDia3 are expressed in the developing brain, and that mDia3 is concentrated in the apical surface of neuroepithelium. Mice deficient in both MDia1 and mDia3 develop periventricular dysplastic mass widespread throughout the developing brain, where neuroepithelial cell polarity is impaired with attenuated apical actin belts and loss of apical adherens junctions. In addition, electron microscopic analysis revealed abnormal shrinkage and apical membrane bulging of neuroepithelial cells in the remaining areas. Furthermore, perturbation of Rho, but not that of ROCK, causes loss of the apical actin belt and adherens junctions similarly to mDia-deficient mice. These results suggest that actin cytoskeleton regulated by Rho-mDia pathway is critical for the integrity of the adherens junctions and the polarity of neuroepithelial cells, and that loss of this signaling induces aberrant, ectopic proliferation and differentiation of neural stem cells.
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rho MDia1 pathway is required for adhesion migration and t cell stimulation in dendritic cells
Blood, 2010Co-Authors: Hideaki Tanizaki, Toshimasa Ishizaki, Gyohei Egawa, Kayo Inaba, Tetsuya Honda, Saeko Nakajima, Catharina Sagita Moniaga, Atsushi Otsuka, Michio Tomura, Takeshi WatanabeAbstract:Dendritic cells (DCs) are essential for the initiation of acquired immune responses through antigen acquisition, migration, maturation, and T-cell stimulation. One of the critical mechanisms in this response is the process actin nucleation and polymerization, which is mediated by several groups of proteins, including mammalian Diaphanous-related formins (mDia). However, the role of mDia in DCs remains unknown. Herein, we examined the role of MDia1 (one of the isoforms of mDia) in DCs. Although the proliferation and maturation of bone marrow-derived DCs were comparable between control C57BL/6 and MDia1-deficient (MDia1−/−) mice, adhesion and spreading to cellular matrix were impaired in MDia1−/− bone marrow–derived DCs. In addition, fluorescein isothiocyanate-induced cutaneous DC migration to draining lymph nodes in vivo and invasive migration and directional migration to CCL21 in vitro were suppressed in MDia1−/− DCs. Moreover, sustained T-cell interaction and T-cell stimulation in lymph nodes were impaired by MDia1 deficiency. Consistent with this, the DC-dependent delayed hypersensitivity response was attenuated by MDia1-deficient DCs. These results suggest that actin polymerization, which is mediated by MDia1, is essential for several aspects of DC-initiated acquired immune responses.
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MDia1 targets v src to the cell periphery and facilitates cell transformation tumorigenesis and invasion
Molecular and Cellular Biology, 2010Co-Authors: Masahiro Tanji, Toshimasa Ishizaki, Saman Ebrahimi, Yuko Tsuboguchi, Taiko Sukezane, Tsuyoshi Akagi, Margaret C Frame, Nobuo Hashimoto, Susumu Miyamoto, Shuh NarumiyaAbstract:The small GTPase Rho regulates cell morphogenesis through remodeling of the actin cytoskeleton. While Rho is overexpressed in many clinical cancers, the role of Rho signaling in oncogenesis remains unknown. MDia1 is a Rho effector producing straight actin filaments. Here we transduced mouse embryonic fibroblasts from MDia1-deficient mice with temperature-sensitive v-Src and examined the involvement and mechanism of the Rho-MDia1 pathway in Src-induced oncogenesis. We showed that in v-Src-transduced MDia1-deficient cells, formation of actin filaments is suppressed, and v-Src in the perinuclear region does not move to focal adhesions upon a temperature shift. Consequently, membrane translocation of v-Src, v-Src-induced morphological transformation, and podosome formation are all suppressed in MDia1-deficient cells with impaired tyrosine phosphorylation. MDia1-deficient cells show reduced transformation in vitro as examined by focus formation and colony formation in soft agar and exhibit suppressed tumorigenesis and invasion when implanted in nude mice in vivo. Given overexpression of c-Src in various cancers, these findings suggest that Rho-MDia1 signaling facilitates malignant transformation and invasion by manipulating the actin cytoskeleton and targeting Src to the cell periphery.
Xiequn Chen - One of the best experts on this subject based on the ideXlab platform.
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pi3 kinase mediates thrombin induced platelet aggregation through MDia1 pathway
Chinese Journal of Hematology, 2010Co-Authors: Guangxun Gao, Hongjuan Dong, Ying Gao, Yaozhu Pan, Yiwei Wang, Yang Yang, Xiequn ChenAbstract:OBJECTIVE To investigate the expression of MDia1 (mammalian diaphanous 1)in platelet and the role of MDia1 or phosphatidylinositol 3-kinase (PI3K) in the process of thrombin-induced platelet aggregation. METHODS The extent of platelet aggregation was measured by a platelet aggregation system and the expression of MDia1 and its relation with F-actin in quiescent, spreading or aggregated platelets by Western blot. RESULTS There was no significant difference in MDia1 expression level between quiescent and activated platelets. MDia1 moved from a Triton-X100-soluble cytosolic fraction to insoluble cytoskeleton fraction after thrombin induced platelets aggregation. Anti-MDia1 antibody could inhibit this aggregation. PI3K inhibitor Wortmannin or Ly294002 inhibited the thrombin induced platelet aggregation and the above mentioned MDia1 translocation. CONCLUSION PI3-kinase mediates the thrombin-induced platelet aggregation through MDia1 pathway.
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pi3 kinase mediates activity of rhoa and interaction of rhoa with MDia1 in thrombin induced platelet aggregation
Journal of Experimental Hematology, 2009Co-Authors: Guangxun Gao, Hongjuan Dong, Ying Gao, Yaozhu Pan, Yang Yang, Xiequn ChenAbstract:The aim of this study was to investigate the role of RhoA/MDia1 pathway in the process of thrombin-induced platelet aggregation and regulatory effect of PI3K inhibitor on this process. The human platelets were isolated from peripheral blood, the activation of RhoA, Rac1 and Cdc42 in the platelet aggregation was detected by GST pull-down assay and immune co-precipitation, the interaction of RhoA, Rac1 and Cdc42 with MDia1 and the formation of complex in the process of platelet aggregation were determined by immune coprecipitation, and the effect of PI3K inhibitor (wortmannin) on above-mentioned process was assayed. The results showed that thrombin elevated the activity of RhoA and the binding capability of RhoA with MDia1 during thrombin-induced platelet aggregation and spreading on Fg coated coverslips. Wortmannin inhibited the rising of RhoA activity and the binding level of RhoA with MDia1 induced by thrombin. Thrombin elevated the activity of Rac1 and Cdc42 during thrombin-induced platelet aggregation, but could not induce binding of Rac1 or Cdc42 with MDia1. Wortmannin could not inhibit the rising of Rac1 and Cdc42 activity induced by thrombin. It is concluded that the PI3-kinase regulates the thrombin-induced actin cytoskeleton reconstitution in platelets by RhoA-MDia1 pathway.
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rhoa effector MDia1 is required for pi 3 kinase dependent actin remodeling and spreading by thrombin in platelets
Blood, 2009Co-Authors: Xiequn Chen, Guangxun Gao, Jishi WangAbstract:Abstract 5077 The RhoA effector MDia1 is involved in controlling the balance between filamentous and monomeric actin, but its role in modulating thrombin-induced actin remodeling and platelet spreading on fibrinogen matrices remains unclear. In this study, MDia1 was shown to translocate to the platelet cytoskeleton following thrombin stimulation, in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner. AntiMDia1 loading or pretreatment with PI 3-kinase inhibitors essentially abrogated thrombin-elicited actin stress fiber formation, with a corresponding decrease in the proportion of platelets exhibiting a fully spread morphology. We also investigated the mechanisms underlying the effects of MDia1 on thrombin-induced actin remodeling and platelet spreading, and found that these involved PI 3-kinase-mediated induction of MDia1 interaction with RhoA. Collectively, these results suggest that the PI 3-kinase/RhoA/MDia1 axis is a critical pathway for coupling thrombin signaling to actin cytoskeletal remodeling during platelet spreading. Disclosures No relevant conflicts of interest to declare.
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rhoa effector MDia1 is required for pi 3 kinase dependent actin remodeling and spreading by thrombin in platelets
Biochemical and Biophysical Research Communications, 2009Co-Authors: Guangxun Gao, Hongjuan Dong, Ying Gao, Yaozhu Pan, Liang Chen, Baoxia Dong, Xiequn ChenAbstract:The RhoA effector MDia1 is involved in controlling the balance between filamentous and monomeric actin, but its role in modulating thrombin-induced actin remodeling and platelet spreading on fibrinogen matrices remains unclear. In this study, MDia1 was shown to translocate to the platelet cytoskeleton following thrombin stimulation, in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner. Anti-MDia1 loading or pretreatment with PI 3-kinase inhibitors essentially abrogated thrombin-elicited actin stress fiber formation, with a corresponding decrease in the proportion of platelets exhibiting a fully spread morphology. We also investigated the mechanisms underlying the effects of MDia1 on thrombin-induced actin remodeling and platelet spreading, and found that these involved PI 3-kinase-mediated induction of MDia1 interaction with RhoA. Collectively, these results suggest that the PI 3-kinase/RhoA/MDia1 axis is a critical pathway for coupling thrombin signaling to actin cytoskeletal remodeling during platelet spreading.
Guangxun Gao - One of the best experts on this subject based on the ideXlab platform.
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pi3 kinase mediates thrombin induced platelet aggregation through MDia1 pathway
Chinese Journal of Hematology, 2010Co-Authors: Guangxun Gao, Hongjuan Dong, Ying Gao, Yaozhu Pan, Yiwei Wang, Yang Yang, Xiequn ChenAbstract:OBJECTIVE To investigate the expression of MDia1 (mammalian diaphanous 1)in platelet and the role of MDia1 or phosphatidylinositol 3-kinase (PI3K) in the process of thrombin-induced platelet aggregation. METHODS The extent of platelet aggregation was measured by a platelet aggregation system and the expression of MDia1 and its relation with F-actin in quiescent, spreading or aggregated platelets by Western blot. RESULTS There was no significant difference in MDia1 expression level between quiescent and activated platelets. MDia1 moved from a Triton-X100-soluble cytosolic fraction to insoluble cytoskeleton fraction after thrombin induced platelets aggregation. Anti-MDia1 antibody could inhibit this aggregation. PI3K inhibitor Wortmannin or Ly294002 inhibited the thrombin induced platelet aggregation and the above mentioned MDia1 translocation. CONCLUSION PI3-kinase mediates the thrombin-induced platelet aggregation through MDia1 pathway.
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pi3 kinase mediates activity of rhoa and interaction of rhoa with MDia1 in thrombin induced platelet aggregation
Journal of Experimental Hematology, 2009Co-Authors: Guangxun Gao, Hongjuan Dong, Ying Gao, Yaozhu Pan, Yang Yang, Xiequn ChenAbstract:The aim of this study was to investigate the role of RhoA/MDia1 pathway in the process of thrombin-induced platelet aggregation and regulatory effect of PI3K inhibitor on this process. The human platelets were isolated from peripheral blood, the activation of RhoA, Rac1 and Cdc42 in the platelet aggregation was detected by GST pull-down assay and immune co-precipitation, the interaction of RhoA, Rac1 and Cdc42 with MDia1 and the formation of complex in the process of platelet aggregation were determined by immune coprecipitation, and the effect of PI3K inhibitor (wortmannin) on above-mentioned process was assayed. The results showed that thrombin elevated the activity of RhoA and the binding capability of RhoA with MDia1 during thrombin-induced platelet aggregation and spreading on Fg coated coverslips. Wortmannin inhibited the rising of RhoA activity and the binding level of RhoA with MDia1 induced by thrombin. Thrombin elevated the activity of Rac1 and Cdc42 during thrombin-induced platelet aggregation, but could not induce binding of Rac1 or Cdc42 with MDia1. Wortmannin could not inhibit the rising of Rac1 and Cdc42 activity induced by thrombin. It is concluded that the PI3-kinase regulates the thrombin-induced actin cytoskeleton reconstitution in platelets by RhoA-MDia1 pathway.
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rhoa effector MDia1 is required for pi 3 kinase dependent actin remodeling and spreading by thrombin in platelets
Blood, 2009Co-Authors: Xiequn Chen, Guangxun Gao, Jishi WangAbstract:Abstract 5077 The RhoA effector MDia1 is involved in controlling the balance between filamentous and monomeric actin, but its role in modulating thrombin-induced actin remodeling and platelet spreading on fibrinogen matrices remains unclear. In this study, MDia1 was shown to translocate to the platelet cytoskeleton following thrombin stimulation, in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner. AntiMDia1 loading or pretreatment with PI 3-kinase inhibitors essentially abrogated thrombin-elicited actin stress fiber formation, with a corresponding decrease in the proportion of platelets exhibiting a fully spread morphology. We also investigated the mechanisms underlying the effects of MDia1 on thrombin-induced actin remodeling and platelet spreading, and found that these involved PI 3-kinase-mediated induction of MDia1 interaction with RhoA. Collectively, these results suggest that the PI 3-kinase/RhoA/MDia1 axis is a critical pathway for coupling thrombin signaling to actin cytoskeletal remodeling during platelet spreading. Disclosures No relevant conflicts of interest to declare.
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rhoa effector MDia1 is required for pi 3 kinase dependent actin remodeling and spreading by thrombin in platelets
Biochemical and Biophysical Research Communications, 2009Co-Authors: Guangxun Gao, Hongjuan Dong, Ying Gao, Yaozhu Pan, Liang Chen, Baoxia Dong, Xiequn ChenAbstract:The RhoA effector MDia1 is involved in controlling the balance between filamentous and monomeric actin, but its role in modulating thrombin-induced actin remodeling and platelet spreading on fibrinogen matrices remains unclear. In this study, MDia1 was shown to translocate to the platelet cytoskeleton following thrombin stimulation, in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner. Anti-MDia1 loading or pretreatment with PI 3-kinase inhibitors essentially abrogated thrombin-elicited actin stress fiber formation, with a corresponding decrease in the proportion of platelets exhibiting a fully spread morphology. We also investigated the mechanisms underlying the effects of MDia1 on thrombin-induced actin remodeling and platelet spreading, and found that these involved PI 3-kinase-mediated induction of MDia1 interaction with RhoA. Collectively, these results suggest that the PI 3-kinase/RhoA/MDia1 axis is a critical pathway for coupling thrombin signaling to actin cytoskeletal remodeling during platelet spreading.
Richard Treisman - One of the best experts on this subject based on the ideXlab platform.
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the diaphanous related formin MDia1 controls serum response factor activity through its effects on actin polymerization
Molecular Biology of the Cell, 2002Co-Authors: John W Copeland, Richard Treisman, John W Copeland, Richard TreismanAbstract:SRF-dependent transcription is regulated by the small GTPase RhoA via its effects on actin dynamics. The diaphanous-related formin (DRF) proteins have been identified as candidate RhoA effectors mediating signaling to SRF. Here we investigate the relationship between SRF activation and actin polymerization by the DRF MDia1. We show that the ability of MDia1 to potentiate SRF activity is strictly correlated with its ability to promote F-actin assembly. Both processes can occur independently of the MDia1 FH1 domain but require sequences in an extended C-terminal region encompassing the conserved FH2 domain. mDia-mediated SRF activation, but not F-actin assembly, can be blocked by a nonpolymerizable actin mutant, placing actin downstream of mDia in the signal pathway. The SRF activation assay was used to identify inactive MDia1 derivatives that inhibit serum- and LPA-induced signaling to SRF. We show that these interfering mutants also block F-actin assembly, whether induced by mDia proteins or extracellular signals. These results identify novel functional elements of MDia1 and show that it regulates SRF activity by inducing depletion of the cellular pool of G-actin.
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lim kinase and diaphanous cooperate to regulate serum response factor and actin dynamics
Journal of Cell Biology, 2002Co-Authors: O Geneste, John W Copeland, Richard TreismanAbstract:The small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. We show that in PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho kinase (ROCK)–LIM kinase (LIMK)–cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering MDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways.
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analysis of rhoa binding proteins reveals an interaction domain conserved in heterotrimeric g protein beta subunits and the yeast response regulator protein skn7
Journal of Biological Chemistry, 1998Co-Authors: Arthur S Alberts, Nicolas Bouquin, Leland H Johnston, Richard TreismanAbstract:Abstract To identify potential RhoA effector proteins, we conducted a two-hybrid screen for cDNAs encoding proteins that interact with a Gal4-RhoA.V14 fusion protein. In addition to the RhoA effector ROCK-I we identified cDNAs encoding Kinectin, mDia2 (a p140 mDia-related protein), and the guanine nucleotide exchange factor, mNET1. ROCK-I, Kinectin, and mDia2 can bind the wild type forms of both RhoA and Cdc42 in a GTP-dependent manner in vitro. Comparison of the ROCK-I and Kinectin sequences revealed a short region of sequence homology that is both required for interaction in the two-hybrid assay and sufficient for weak interaction in vitro. Sequences related to the ROCK-I/Kinectin sequence homology are present in heterotrimeric G protein β subunits and in theSaccharomyces cerevisiae Skn7 protein. We show that β2 and Skn7 can interact with mammalian RhoA and Cdc42 and yeast Rho1, both in vivo and in vitro. Functional assays in yeast suggest that the Skn7 ROCK-I/Kinectin homology region is required for its function in vivo.