Plastins are a family of actin binding proteins （ABPs） known to cross-link actin microfilaments in mammalian cells, creating actin microfilament bundles necessary to confer cell polarity and cell shape. Plastins als...Plastins are a family of actin binding proteins （ABPs） known to cross-link actin microfilaments in mammalian cells, creating actin microfilament bundles necessary to confer cell polarity and cell shape. Plastins also support cell movement in response to changes in environment, involved in cell/tissue growth and development. They also confer plasticity to cells and tissues in response to infection or other pathological conditions （e.g., inflammation）. In the testis, the cell-cell anchoring junction unique to the testis that is found at the Sertoli cell-cell interface at the blood-testis barrier （BTB） and at the Sertoli-spermatid （e.g., 8-19 spermatids in the rat testis） is the basal and the apical ectoplasmic specialization （ES）, respectively. The ES is an F-actin-rich anchoring junction constituted most notably by actin microfilament bundles. A recent report using RNAi that specifically knocks down plastin 3 has yielded some insightful information regarding the mechanism by which plastin 3 regulates the status of actin microfilament bundles at the ES via its intrinsic actin filament bundling activity. Herein, we provide a brief review on the role of plastins in the testis in light of this report, which together with recent findings in the field, we propose a likely model by which plastins regulate ES function during the epithelial cycle of sDermatogenesis via their intrinsic activity on actin microfilament organization in the rat testis.展开更多
Dynamics of the actin cytoskeleton are essential for pollen germination and pollen tube growth. ACTIN- DEPOLYMERIZlNG FACTORs （ADFs） typically contribute to actin turnover by severing/depolymerizing actin filaments....Dynamics of the actin cytoskeleton are essential for pollen germination and pollen tube growth. ACTIN- DEPOLYMERIZlNG FACTORs （ADFs） typically contribute to actin turnover by severing/depolymerizing actin filaments. Recently, we demonstrated that Arabidopsis subclass III ADFs （ADF5 and ADF9） evolved F-actin-bundling function from conserved F-actin-depolymerizing function. However, little is known about the physiological function, the evolutional significance, and the actin-bundling mechanism of these neo- functionalized ADFs. Here, we report that loss of ADF5 function caused delayed pollen germination, retarded pollen tube growth, and increased sensitive to latrunculin B （LatB） treatment by affecting the generation and maintenance of actin bundles. Examination of actin filament dynamics in living cells revealed that the bundling frequency was significantly decreased in adf5 pollen tubes, consistent with its biochem- ical functions. Further biochemical and genetic complementation analyses demonstrated that both the N- and C-terminal actin-binding domains of ADF5 are required for its physiological and biochemical functions. Interestingly, while both are atypical actin-bundling ADFs, ADF5, but not ADF9, plays an important role in mature pollen physiological activities. Taken together, our results suggest that ADF5 has evolved the function of bundling actin filaments and plays an important role in the formation, organization, and maintenance of actin bundles during pollen germination and pollen tube growth.展开更多
Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been impli- cated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin d...Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been impli- cated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin dynamics in plants is still not well understood. Using in vitro single-molecule imaging technology, we directly observed that the FH1-FH2 domain of an Arabidopsis thaliana formin, AtFH14, processively at- taches to the barbed end of actin filaments as a dimer and slows their elongation rate by 90%. The attach- ment persistence of FH1-FH2 is concentration dependent. Furthermore, by use of the triple-color total internal reflection fluorescence microscopy, we found that ABP29, a barbed-end capping protein, com- petes with FH1-FH2 at the filament barbed end, where its binding is mutually exclusive with AtFH14. In the presence of different plant profilin isoforms, FH1-FH2 enhances filament elongation rates from about 10 to 42 times. Filaments buckle when FH1-FH2 is anchored specifically to cover slides, further indicating that AtFH 14 moves processively on the elongating barbed end. At high concentration, AtFH 14 bundles actin filaments randomly into antiparallel or parallel spindle-like structures; however, the FH1-FH2-mediated bundles become thinner and longer in the presence of plant profilins. This is the direct demonstration of a processive formin from plants. Our results also illuminate the molecular mechanism of AtFH14 in regulating actin dynamics via association with profilin.展开更多
Regulation of actin dynamics is a central theme in cell biology that is important for different aspects of cell physiology. Villin, a member of the villin/gelsolin/fragmin superfamily of proteins, is an important regu...Regulation of actin dynamics is a central theme in cell biology that is important for different aspects of cell physiology. Villin, a member of the villin/gelsolin/fragmin superfamily of proteins, is an important regulator of actin. Villins contain six gelsolin homology domains （G1-G6） and an extra headpiece domain. In contrast to their mammalian counterparts, plant villins are expressed widely, implying that plant villins play a more general role in regulating actin dynamics. Some plant villins have a defined role in modifying actin dynamics in the pollentube; most of their in vivo activities remain to be ascertained. Recently, our understanding of the functions and mechanisms of action for plant villins has progressed rapidly, primarily due to the advent of Arabidopsis thaliana genetic approaches and imaging capabilities that can visualize actin dynamics at the single filament level in vitro and in living plant cells. In this review, we focus on discussing the biochemical activities and modes of regulation of plant villins. Here, we present current understand- ing of the functions of plant villins. Finally, we highlight some of the key unanswered questions regarding the functions and regulation of plant villins for future research.展开更多
Background:Living cells need to undergo subtle shape adaptations in response to the topography of their substrates.These shape changes are mainly determined by reorganization of their internal cytoskeleton,with a majo...Background:Living cells need to undergo subtle shape adaptations in response to the topography of their substrates.These shape changes are mainly determined by reorganization of their internal cytoskeleton,with a major contribution from filamentous(F)actin.Bundles of F-actin play a major role in determining cell shape and their interaction with substrates,either as“stress fibers,”or as our newly discovered“Concave Actin Bundles”(CABs),which mainly occur while endothelial cells wrap micro-fibers in culture.Methods:To better understand the morphology and functions of these CABs,it is necessary to recognize and analyze as many of them as possible in complex cellular ensembles,which is a demanding and time-consuming task.In this study,we present a novel algorithm to automatically recognize CABs without further human intervention.We developed and employed a multilayer perceptron artificial neural network(“the recognizer”),which was trained to identify CABs.Results:The recognizer demonstrated high overall recognition rate and reliability in both randomized training,and in subsequent testing experiments.Conclusion:It would be an effective replacement for validation by visual detection which is both tedious and inherently prone to errors.展开更多
It has been proposed that cortical fine actin filaments are needed for the morphogenesis of pavement cells(PCs).However,the precise role and regulation mechanisms of actin filaments in PC morphogenesis are not well un...It has been proposed that cortical fine actin filaments are needed for the morphogenesis of pavement cells(PCs).However,the precise role and regulation mechanisms of actin filaments in PC morphogenesis are not well understood.Here,we found that Arabidopsis thaliana ACTIN DEPOLYMERIZING FACTOR9(ADF9) is required for the morphogenesis of PC,which is negatively regulated by the R2R3 MYELOBLASTOSIS(MYB) transcription factor MYB52.In adf9 mutants,the lobe number of cotyledon PCs was significantly reduced,while the average lobe length did not differ significantly compared to that of wild type(Col-0),except for the variations in cell area and circularity,whereas the PC shapes in ADF9 overexpression seedlings showed different results.ADF9 decorated actin filaments,and colocalized with plasma membrane.The extent of filament bundling and actin filament bundling activity in adf9 mutant decreased.In addition,MYB52 directly targeted the promoter of ADF9 and negatively regulated its expression.The myb52-2 mutant showed increased lobe number and cell area,reduced cell circularity of PCs,and the PC phenotypes were suppressed when ADF9 was knocked out.Taken together,our data demonstrate that actin filaments play an important role in the morphogenesis of PC and reveal a transcriptional mechanism underlying MYB52 regulation of ADF9-mediated actin filament bundling in PC morphogenesis.展开更多
Our previous study demonstrated that WLIMla has dual roles in fiber elongation and secondary cell wall synthesis in upland cotton, and the protein acts either as an actin-binding protein or as a transcription factor. ...Our previous study demonstrated that WLIMla has dual roles in fiber elongation and secondary cell wall synthesis in upland cotton, and the protein acts either as an actin-binding protein or as a transcription factor. Because WLIMla consists of two different LIM domains, it is possible that these elements contribute differentially to the dual functions of the protein. In this study, we dissected the two LIM domains and characterized their biochemical functions. By using red fluorescent protein （RFP） fusion, co-sedimentation, and DNA binding methods, we found that the two domains of WLIM 1 a, domain 1 （D 1） and domain2 （D2）, possessed different biochemical properties. While D1 contributed primarily to the actin filament-bundling activity of WLIMla, D2 contributed to the DNA-binding activity of the protein; both D1 and D2 relied on a linker sequence for their ac- tivities. In addition, we found that WLIMla and its two LIM domains form dimers in vitro. These results may lead to a better understanding of the molecular mechanisms of dual functions of WLIMla during cotton fiber development.展开更多
文摘Plastins are a family of actin binding proteins （ABPs） known to cross-link actin microfilaments in mammalian cells, creating actin microfilament bundles necessary to confer cell polarity and cell shape. Plastins also support cell movement in response to changes in environment, involved in cell/tissue growth and development. They also confer plasticity to cells and tissues in response to infection or other pathological conditions （e.g., inflammation）. In the testis, the cell-cell anchoring junction unique to the testis that is found at the Sertoli cell-cell interface at the blood-testis barrier （BTB） and at the Sertoli-spermatid （e.g., 8-19 spermatids in the rat testis） is the basal and the apical ectoplasmic specialization （ES）, respectively. The ES is an F-actin-rich anchoring junction constituted most notably by actin microfilament bundles. A recent report using RNAi that specifically knocks down plastin 3 has yielded some insightful information regarding the mechanism by which plastin 3 regulates the status of actin microfilament bundles at the ES via its intrinsic actin filament bundling activity. Herein, we provide a brief review on the role of plastins in the testis in light of this report, which together with recent findings in the field, we propose a likely model by which plastins regulate ES function during the epithelial cycle of sDermatogenesis via their intrinsic activity on actin microfilament organization in the rat testis.
基金This work was supported by the National Basic Research Program （2014CB954203）, the National Natural Science Foundation of China （31670180, 31470283）, and the Fundamental Research Funds for the Cen- tral Universities （Izujbky-2016-bt05） to Y.X.
文摘Dynamics of the actin cytoskeleton are essential for pollen germination and pollen tube growth. ACTIN- DEPOLYMERIZlNG FACTORs （ADFs） typically contribute to actin turnover by severing/depolymerizing actin filaments. Recently, we demonstrated that Arabidopsis subclass III ADFs （ADF5 and ADF9） evolved F-actin-bundling function from conserved F-actin-depolymerizing function. However, little is known about the physiological function, the evolutional significance, and the actin-bundling mechanism of these neo- functionalized ADFs. Here, we report that loss of ADF5 function caused delayed pollen germination, retarded pollen tube growth, and increased sensitive to latrunculin B （LatB） treatment by affecting the generation and maintenance of actin bundles. Examination of actin filament dynamics in living cells revealed that the bundling frequency was significantly decreased in adf5 pollen tubes, consistent with its biochem- ical functions. Further biochemical and genetic complementation analyses demonstrated that both the N- and C-terminal actin-binding domains of ADF5 are required for its physiological and biochemical functions. Interestingly, while both are atypical actin-bundling ADFs, ADF5, but not ADF9, plays an important role in mature pollen physiological activities. Taken together, our results suggest that ADF5 has evolved the function of bundling actin filaments and plays an important role in the formation, organization, and maintenance of actin bundles during pollen germination and pollen tube growth.
文摘Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been impli- cated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin dynamics in plants is still not well understood. Using in vitro single-molecule imaging technology, we directly observed that the FH1-FH2 domain of an Arabidopsis thaliana formin, AtFH14, processively at- taches to the barbed end of actin filaments as a dimer and slows their elongation rate by 90%. The attach- ment persistence of FH1-FH2 is concentration dependent. Furthermore, by use of the triple-color total internal reflection fluorescence microscopy, we found that ABP29, a barbed-end capping protein, com- petes with FH1-FH2 at the filament barbed end, where its binding is mutually exclusive with AtFH14. In the presence of different plant profilin isoforms, FH1-FH2 enhances filament elongation rates from about 10 to 42 times. Filaments buckle when FH1-FH2 is anchored specifically to cover slides, further indicating that AtFH 14 moves processively on the elongating barbed end. At high concentration, AtFH 14 bundles actin filaments randomly into antiparallel or parallel spindle-like structures; however, the FH1-FH2-mediated bundles become thinner and longer in the presence of plant profilins. This is the direct demonstration of a processive formin from plants. Our results also illuminate the molecular mechanism of AtFH14 in regulating actin dynamics via association with profilin.
基金supported by grants from the National Natural Science Foundation of China (31125004 and 31071179)the Ministry of Science and Technology of China (2013CB945100 and 2011CB944600)
文摘Regulation of actin dynamics is a central theme in cell biology that is important for different aspects of cell physiology. Villin, a member of the villin/gelsolin/fragmin superfamily of proteins, is an important regulator of actin. Villins contain six gelsolin homology domains （G1-G6） and an extra headpiece domain. In contrast to their mammalian counterparts, plant villins are expressed widely, implying that plant villins play a more general role in regulating actin dynamics. Some plant villins have a defined role in modifying actin dynamics in the pollentube; most of their in vivo activities remain to be ascertained. Recently, our understanding of the functions and mechanisms of action for plant villins has progressed rapidly, primarily due to the advent of Arabidopsis thaliana genetic approaches and imaging capabilities that can visualize actin dynamics at the single filament level in vitro and in living plant cells. In this review, we focus on discussing the biochemical activities and modes of regulation of plant villins. Here, we present current understand- ing of the functions of plant villins. Finally, we highlight some of the key unanswered questions regarding the functions and regulation of plant villins for future research.
文摘Background:Living cells need to undergo subtle shape adaptations in response to the topography of their substrates.These shape changes are mainly determined by reorganization of their internal cytoskeleton,with a major contribution from filamentous(F)actin.Bundles of F-actin play a major role in determining cell shape and their interaction with substrates,either as“stress fibers,”or as our newly discovered“Concave Actin Bundles”(CABs),which mainly occur while endothelial cells wrap micro-fibers in culture.Methods:To better understand the morphology and functions of these CABs,it is necessary to recognize and analyze as many of them as possible in complex cellular ensembles,which is a demanding and time-consuming task.In this study,we present a novel algorithm to automatically recognize CABs without further human intervention.We developed and employed a multilayer perceptron artificial neural network(“the recognizer”),which was trained to identify CABs.Results:The recognizer demonstrated high overall recognition rate and reliability in both randomized training,and in subsequent testing experiments.Conclusion:It would be an effective replacement for validation by visual detection which is both tedious and inherently prone to errors.
基金supported by National Natural Science Foundation of China(31970661 and 32370736)Liaoning Education Foundation(JYTQN2024007)Young and Middle-aged Science and Technology Innovation Talent Support Program of Shenyang(RC220273)。
文摘It has been proposed that cortical fine actin filaments are needed for the morphogenesis of pavement cells(PCs).However,the precise role and regulation mechanisms of actin filaments in PC morphogenesis are not well understood.Here,we found that Arabidopsis thaliana ACTIN DEPOLYMERIZING FACTOR9(ADF9) is required for the morphogenesis of PC,which is negatively regulated by the R2R3 MYELOBLASTOSIS(MYB) transcription factor MYB52.In adf9 mutants,the lobe number of cotyledon PCs was significantly reduced,while the average lobe length did not differ significantly compared to that of wild type(Col-0),except for the variations in cell area and circularity,whereas the PC shapes in ADF9 overexpression seedlings showed different results.ADF9 decorated actin filaments,and colocalized with plasma membrane.The extent of filament bundling and actin filament bundling activity in adf9 mutant decreased.In addition,MYB52 directly targeted the promoter of ADF9 and negatively regulated its expression.The myb52-2 mutant showed increased lobe number and cell area,reduced cell circularity of PCs,and the PC phenotypes were suppressed when ADF9 was knocked out.Taken together,our data demonstrate that actin filaments play an important role in the morphogenesis of PC and reveal a transcriptional mechanism underlying MYB52 regulation of ADF9-mediated actin filament bundling in PC morphogenesis.
基金the National Basic Research Priorities Program (U1303281)the China Postdoctoral Science Foundation
文摘Our previous study demonstrated that WLIMla has dual roles in fiber elongation and secondary cell wall synthesis in upland cotton, and the protein acts either as an actin-binding protein or as a transcription factor. Because WLIMla consists of two different LIM domains, it is possible that these elements contribute differentially to the dual functions of the protein. In this study, we dissected the two LIM domains and characterized their biochemical functions. By using red fluorescent protein （RFP） fusion, co-sedimentation, and DNA binding methods, we found that the two domains of WLIM 1 a, domain 1 （D 1） and domain2 （D2）, possessed different biochemical properties. While D1 contributed primarily to the actin filament-bundling activity of WLIMla, D2 contributed to the DNA-binding activity of the protein; both D1 and D2 relied on a linker sequence for their ac- tivities. In addition, we found that WLIMla and its two LIM domains form dimers in vitro. These results may lead to a better understanding of the molecular mechanisms of dual functions of WLIMla during cotton fiber development.
