The actin cytoskeleton is an important component of eukaryotic cell cytoskeleton and is temporally and spatially controlled by a series of actin binding proteins (ABPs). Among ABPs, formin family proteins have attract...The actin cytoskeleton is an important component of eukaryotic cell cytoskeleton and is temporally and spatially controlled by a series of actin binding proteins (ABPs). Among ABPs, formin family proteins have attracted much attention as they can nucleate unbranched actin filament from the profilin bound actin pool in vivo. In recent years, a number of formin family members from different organisms have been reported, and their characteristics are known more clearly, although some questions are still to be clarified. Here, we summarize the structures, func-tions and nucleation mechanisms of different formin family proteins, intending to compare them and give some new clues to the study of formins.展开更多
Both the mechanosensitive actin cytoskeleton and caveolae contribute to active processes such as cell migration,morphogenesis,and vesicular trafficking.Although distinct actin components are well studied,how they cont...Both the mechanosensitive actin cytoskeleton and caveolae contribute to active processes such as cell migration,morphogenesis,and vesicular trafficking.Although distinct actin components are well studied,how they contribute to cytoplasmic caveolae,especially in the context of mechano-stress,has remained elusive.Here,we identify two actin-associated mobility stereotypes of caveolin-1(CAV-1)-marked intracellular vesicles,which are characterized as‘dwelling’and‘go and dwelling’.In order to exploit the reason for their distinct dynamics,elongated actin-associated formin functions are perturbed.We find drastically decreased density,increased clustering,and compromised motility of cytoplasmic CAV-1 vesicles resulting from lacking actin nucleator formins by both chemical treatment and RNA silencing of formin genes.Furthermore,hypo-osmosis-stimulated diminishing of CAV-1 is dramatically intensified upon blocking formins.The clustering of CAV-1 vesicles when cells are cultured on soft substrate is also aggravated under formin inhibition condition.Together,we reveal that actin-associated formins are essential for maintaining the dynamic organization of cytoplasmic CAV-1 and importantly its sensitivity upon mechanical challenge.We conclude that tension-controlled actin formins act as a safety valve dampening excessive tension on CAV-1 and safeguarding CAV-1 against mechanical damage.展开更多
Formin是一种保守的肌动蛋白成核因子,其中只有少数Formin没有肌动蛋白成核能力。Formin家族包括DAAM(dishevelled-associated activator of morphogenesis)和INF(inverted formin)等成员。DAAM1表达上调会导致机体功能紊乱,进而导致特...Formin是一种保守的肌动蛋白成核因子,其中只有少数Formin没有肌动蛋白成核能力。Formin家族包括DAAM(dishevelled-associated activator of morphogenesis)和INF(inverted formin)等成员。DAAM1表达上调会导致机体功能紊乱,进而导致特发性肺动脉高压、卵巢癌、乳腺癌、胃癌的发生。DAAM2在机体内功能失调会导致髓鞘结构异常、类固醇耐药性肾病综合征、肾细胞癌、胰腺癌等疾病的发生。INF2表达上调可以诱导线粒体的分裂,促进细胞的增殖迁移,促进局灶节段性肾小球硬化、子宫内膜癌、甲状腺癌等疾病的发展。Formin的功能与人类疾病的发生和发展密切相关。本文综述了近年来Formin的研究进展及其与人类疾病之间的联系,为Formin相关疾病的深入研究和治疗提供参考和依据。展开更多
AIM To investigate whether promoter methylation is re-sponsible for the silencing of formin 2 （ FMN2） in colorectal cancer （CRC） and to analyze the association between FMN2 methylation and CRC.METHODSWe frst ident...AIM To investigate whether promoter methylation is re-sponsible for the silencing of formin 2 （ FMN2） in colorectal cancer （CRC） and to analyze the association between FMN2 methylation and CRC.METHODSWe frst identifed the expression levels and methylation levels of FMN2 in large-scale human CRC expression data-sets, including GEO and TCGA, and analyzed the relation-ship between the expression and methylation levels. Then, the methylation levels in four CpG regions adjacent to the FMN2 promoter were assessed by MethylTarget？ assays in CRC cells and in paired colorectal tumor samples and adjacent nontumor tissue samples. Furthermore, we inhibited DNA methylation in CRC cells with 5-Aza-2’-deoxycytidine and assessed the expression of FMN2 by qRT-PCR. Last, the association between FMN2 methylation patterns and clinical indicators was analyzed.RESULTSA statistically significant downregulation of FMN2 ex-pression in large-scale human CRC expression datasets was found. Subsequent analysis showed that a high frequency of hypermethylation occurred in the FMN2 gene promoter in CRC tissues; operating characteristic curve analysis revealed that FMN2 gene methylation had a good capability for discriminating between CRC and nontumor tissue samples （AUC = 0.8432, P 〈 0.0001）. MethylTarget？ assays showed that CRC cells and tissues displayed higher methylation of these CpG regions than nontumor tissue samples. Correlation analysis showed a strong inverse correlation between methylation and FMN2 expression, and the inhibition of DNA methylation with 5-Aza significantly increased endogenous FMN2 expression. Analysis of the association between FMN2 methylation patterns and clinical indicators showed that FMN2 methylation was signifcantly associated with age, N stage, lymphovascular invasion, and pathologic tumor stage. Notably, the highest methylation of FMN2 occurred in tissues from cases of early-stage CRC, including cases with no regional lymph node metastasis （N0）, cases in stages Ⅰ and Ⅱ, and cases with no lymphovascular inva-sion, but the methylation level began to decrease with tumor progression. Additionally, FMN2 promoter hyper-methylation was more common in patients 〉 60 years old and in colon cancer tissue.CONCLUSIONFMN2 promoter hypermethylation may be an important early event in CRC, most likely playing a critical role in cancer initiation, and can serve as an ideal diagnostic biomarker in elderly patients with early-stage colon cancer.展开更多
It is well established that guidance of axons during neuronal development is regulated by a variety of extracellular signals,governing cytoskeletal dynamics in growth cones.The actin and microtubule（MT）cytoskeleton ...It is well established that guidance of axons during neuronal development is regulated by a variety of extracellular signals,governing cytoskeletal dynamics in growth cones.The actin and microtubule（MT）cytoskeleton have both been shown to play important roles.However,a growing body of work suggests that a critical issue is the proper coordination of changes within these two major cytoskeletal systems（reviewed in Cammara-ta et al., 2016）.展开更多
The process of pollen germination is crucial for flowering plant reproduction,but the mechanisms through which pollen grains establish polarity and select germination sites are not well understood.In this study,we rep...The process of pollen germination is crucial for flowering plant reproduction,but the mechanisms through which pollen grains establish polarity and select germination sites are not well understood.In this study,we report that a formin family protein,AtFH5,is localized to the vesicles and rotates ahead of Lifeact-mEGFPlabeled actin filaments during pollen germination.The translocation of AtFH5 to the plasma membrane initiates the assembly of a collar-like actin structure at the prospective germination site prior to germination. Genetic and pharmacological evidence further revealed an interdependent relationship between the mobility of AtFH5-1 abeled vesicles and the polymerization of actin filaments:vesicle-localized AtFH5 promotes actin assembly,and the polymerization and elongation of actin filaments,in turn,is essential for the mobility of AtFH5-1 abeled vesicles in pollen grains.Taken together,our work revealed a molecular mechanism underlying the polarity establishment and vesicle mobility during pollen germination.展开更多
Pollen tube growth is an essential step during flowering plant reproduction, whose growth depends on a population of dynamic apical actin filaments. Apical actin filaments were thought to be involved in the regu- lati...Pollen tube growth is an essential step during flowering plant reproduction, whose growth depends on a population of dynamic apical actin filaments. Apical actin filaments were thought to be involved in the regu- lation of vesicle fusion and targeting in the pollen tube. However, the molecular mechanisms that regulate the construction of apical actin structures in the pollen tube remain largely unclear. Here, we identify profilin as an important player in the regulation of actin polymerization at the apical membrane in the pollen tube. Downregulation of profilin decreased the amount of filamentous actin and induced disorganization of apical actin filaments, and reduced tip-directed vesicle transport and accumulation in the pollen tube. Direct visualization of actin dynamics revealed that the elongation of actin filaments originating at the apical membrane decreased in profilin mutant pollen tubes. Mutant profilin that is defective in binding poly-L-proline only partially rescues the actin polymerization defect in profilin mutant pollen tubes, although it fully rescues the actin turnover phenotype. We propose that profilin controls the construction of actin structures at the pollen tube tip, presumably by favoring formin-mediated actin polymerization at the apical membrane.展开更多
Formins are well-known regulators that participate in the organization of the actin cytoskeleton in organisms. The Arabidopsis thaliana L. genome encodes 21 formins, which can be divided into two distinct subfamilies....Formins are well-known regulators that participate in the organization of the actin cytoskeleton in organisms. The Arabidopsis thaliana L. genome encodes 21 formins, which can be divided into two distinct subfamilies. However, type II formins have to date been less well characterized. Here, we cloned a type II formin, AtFH16, and characterized its biochemical activities on actin and microtubule dynamics. The results show that the FH1 FH2 structure of AtFH16 cannot nucleate actin polymerization efficiently, but can bind and bundle microfilaments. AtFH16 FHIFH2 is also able to bind and bundle microtubules, and preferentially binds microtubules over microfilaments in vitro, in addition, AtFH16 FHIFH2 co-localizes with microtubules in onion epidermal cells, indicating a higher binding affinity of AtFH16 FHIFH2 for microtubules rather than microfilaments in vivo. In conclusion, AtFH16 is able to interact with both microfilaments and microtubules, suggesting that AtFH16 probably functions as a bifunctional protein, and may thus participate in plant cellular processes.展开更多
Formin is a major protein responsible for regulating the nucleation of actin filaments, and as such, it permits the cell to control where and when to assemble actin arrays. It is encoded by a multigene family comprisi...Formin is a major protein responsible for regulating the nucleation of actin filaments, and as such, it permits the cell to control where and when to assemble actin arrays. It is encoded by a multigene family comprising 21 members in Arabidopsis thaliana. The Arabidopsis formins can be separated into two phylogenetically-distinct classes： there are 11 class I formins and 10 class II formins. Significant questions remain unanswered regarding the molecular mechanism of actin nucleation and elongation stimulated by each formin isovariant, and how the different isovariants coordinate to regulate actin dynamics in cells. Here, we characterize a class II formin, AtFH19, biochemically. We found that AtFH19 retains all general properties of the formin family, including nucleation and barbed end capping activity. It can also generate actin filaments from a pool of actin monomers bound to profilin. However, both the nucleation and barbed end capping activities of AtFH19 are less efficient compared to those of another well-characterized formin, AtFHI. Interestingly, AtFH19 FH1FH2 competes with AtFH1 FHIFH2 in binding actin filament barbed ends, and inhibits the effect of AtFH1 FHIFH2 on actin. We thus propose a mechanism in which two quantitatively different formins coordinate to regulate actin dynamics by competing for actin filament barbed ends.展开更多
Formins have been paid much attention for their potent nucleating activity. However, the connection between the in vivo functions of AtFHs （Arabidopsis thaliana formin homologs） and their effects on actin organizati...Formins have been paid much attention for their potent nucleating activity. However, the connection between the in vivo functions of AtFHs （Arabidopsis thaliana formin homologs） and their effects on actin organization is poorly understood, in this study, we characterized the bundling activity of AtFH8 in vitro and in vivo. Biochemical analysis showed that AtFH8（FH1FH2） could form dimers and bundle preformed actin filaments or induce stellar structures during actin polymerization. Expression of truncated forms of AtFH8 and immunolocalization analysis showed that AtFH8 localized primarily to nuclear envelope in interphase and to the new cell wall after cytokinesis, depending primarily on its N-terminal transmembrane domain. GUS histochemical staining showed AtFH8 was predominantly expressed in Arabidopsis root meristem, vasculature, and outgrowth points of lateral roots. The primary root growth and lateral root initiation of atfh8 could be decreased by latrunculin B （LatB）. Analysis of the number of dividing cells in Arabidopsis root tips showed that much fewer dividing cells in Lat B-treated atfh8 plants than wild-type plants, which indicates that AtFH8 was involved in cell division. Actin cytoskeleton in root meristem of atfh8-1 was more sensitive to LatB treatment than that of wild-type. Altogether, our results indicate that AtFH8 is an actin filament nucleator and bundler that functions in cell division and root development.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
基金This work was supported by the National Natural Science Foundation for Distinguished Young Scholars （Grant No. 30325005）the National Natural Science Foundation of China （Grant No. 30470176）the National Basic Research Program of China （Grant No. 2006CB 100100）.
文摘The actin cytoskeleton is an important component of eukaryotic cell cytoskeleton and is temporally and spatially controlled by a series of actin binding proteins (ABPs). Among ABPs, formin family proteins have attracted much attention as they can nucleate unbranched actin filament from the profilin bound actin pool in vivo. In recent years, a number of formin family members from different organisms have been reported, and their characteristics are known more clearly, although some questions are still to be clarified. Here, we summarize the structures, func-tions and nucleation mechanisms of different formin family proteins, intending to compare them and give some new clues to the study of formins.
基金This study was supported by grants from the National Natural Science Foundation of China(92054104 and 31970660)CAS–VPST Silk Road Science Fund 2021(GJHZ2021138)+2 种基金Shanghai Municipal Science and Technology Major Project(2019SHZDZX02)Natural Science Foundation of Shanghai(19ZR1463000)‘100 Talents Program’from the Chinese Academy of Sciences.
文摘Both the mechanosensitive actin cytoskeleton and caveolae contribute to active processes such as cell migration,morphogenesis,and vesicular trafficking.Although distinct actin components are well studied,how they contribute to cytoplasmic caveolae,especially in the context of mechano-stress,has remained elusive.Here,we identify two actin-associated mobility stereotypes of caveolin-1(CAV-1)-marked intracellular vesicles,which are characterized as‘dwelling’and‘go and dwelling’.In order to exploit the reason for their distinct dynamics,elongated actin-associated formin functions are perturbed.We find drastically decreased density,increased clustering,and compromised motility of cytoplasmic CAV-1 vesicles resulting from lacking actin nucleator formins by both chemical treatment and RNA silencing of formin genes.Furthermore,hypo-osmosis-stimulated diminishing of CAV-1 is dramatically intensified upon blocking formins.The clustering of CAV-1 vesicles when cells are cultured on soft substrate is also aggravated under formin inhibition condition.Together,we reveal that actin-associated formins are essential for maintaining the dynamic organization of cytoplasmic CAV-1 and importantly its sensitivity upon mechanical challenge.We conclude that tension-controlled actin formins act as a safety valve dampening excessive tension on CAV-1 and safeguarding CAV-1 against mechanical damage.
文摘Formin是一种保守的肌动蛋白成核因子,其中只有少数Formin没有肌动蛋白成核能力。Formin家族包括DAAM(dishevelled-associated activator of morphogenesis)和INF(inverted formin)等成员。DAAM1表达上调会导致机体功能紊乱,进而导致特发性肺动脉高压、卵巢癌、乳腺癌、胃癌的发生。DAAM2在机体内功能失调会导致髓鞘结构异常、类固醇耐药性肾病综合征、肾细胞癌、胰腺癌等疾病的发生。INF2表达上调可以诱导线粒体的分裂,促进细胞的增殖迁移,促进局灶节段性肾小球硬化、子宫内膜癌、甲状腺癌等疾病的发展。Formin的功能与人类疾病的发生和发展密切相关。本文综述了近年来Formin的研究进展及其与人类疾病之间的联系,为Formin相关疾病的深入研究和治疗提供参考和依据。
基金Supported by the National Nature Science Foundation of China,No.81773130the Fundamental Research Funds for the Central Universities of Central South University(the Key Projects of Postgraduate Independent Exploration and Innovation of Central South University,No.2018zzts050)the New Xiangya Talent Projects of the Third Xiangya Hospital of Central South University,No.JY201508
文摘AIM To investigate whether promoter methylation is re-sponsible for the silencing of formin 2 （ FMN2） in colorectal cancer （CRC） and to analyze the association between FMN2 methylation and CRC.METHODSWe frst identifed the expression levels and methylation levels of FMN2 in large-scale human CRC expression data-sets, including GEO and TCGA, and analyzed the relation-ship between the expression and methylation levels. Then, the methylation levels in four CpG regions adjacent to the FMN2 promoter were assessed by MethylTarget？ assays in CRC cells and in paired colorectal tumor samples and adjacent nontumor tissue samples. Furthermore, we inhibited DNA methylation in CRC cells with 5-Aza-2’-deoxycytidine and assessed the expression of FMN2 by qRT-PCR. Last, the association between FMN2 methylation patterns and clinical indicators was analyzed.RESULTSA statistically significant downregulation of FMN2 ex-pression in large-scale human CRC expression datasets was found. Subsequent analysis showed that a high frequency of hypermethylation occurred in the FMN2 gene promoter in CRC tissues; operating characteristic curve analysis revealed that FMN2 gene methylation had a good capability for discriminating between CRC and nontumor tissue samples （AUC = 0.8432, P 〈 0.0001）. MethylTarget？ assays showed that CRC cells and tissues displayed higher methylation of these CpG regions than nontumor tissue samples. Correlation analysis showed a strong inverse correlation between methylation and FMN2 expression, and the inhibition of DNA methylation with 5-Aza significantly increased endogenous FMN2 expression. Analysis of the association between FMN2 methylation patterns and clinical indicators showed that FMN2 methylation was signifcantly associated with age, N stage, lymphovascular invasion, and pathologic tumor stage. Notably, the highest methylation of FMN2 occurred in tissues from cases of early-stage CRC, including cases with no regional lymph node metastasis （N0）, cases in stages Ⅰ and Ⅱ, and cases with no lymphovascular inva-sion, but the methylation level began to decrease with tumor progression. Additionally, FMN2 promoter hyper-methylation was more common in patients 〉 60 years old and in colon cancer tissue.CONCLUSIONFMN2 promoter hypermethylation may be an important early event in CRC, most likely playing a critical role in cancer initiation, and can serve as an ideal diagnostic biomarker in elderly patients with early-stage colon cancer.
基金supported by the Hungarian Science Foundation(OTKA)(K109330 to JM)the Hungarian Brain Research Program(KTIA_NAP_13-2-2014-0007 to JM)+1 种基金the National Research,Development and Innovation Office(GINOP-2.3.2-15-2016-00001 and GINOP-2.3.2-15-2016-00032 to JM)by an MTA Postdoctoral Fellowship(to IF)
文摘It is well established that guidance of axons during neuronal development is regulated by a variety of extracellular signals,governing cytoskeletal dynamics in growth cones.The actin and microtubule（MT）cytoskeleton have both been shown to play important roles.However,a growing body of work suggests that a critical issue is the proper coordination of changes within these two major cytoskeletal systems（reviewed in Cammara-ta et al., 2016）.
基金grants from the National Natural Science Foundation of China (31570323 and 31770206).
文摘The process of pollen germination is crucial for flowering plant reproduction,but the mechanisms through which pollen grains establish polarity and select germination sites are not well understood.In this study,we report that a formin family protein,AtFH5,is localized to the vesicles and rotates ahead of Lifeact-mEGFPlabeled actin filaments during pollen germination.The translocation of AtFH5 to the plasma membrane initiates the assembly of a collar-like actin structure at the prospective germination site prior to germination. Genetic and pharmacological evidence further revealed an interdependent relationship between the mobility of AtFH5-1 abeled vesicles and the polymerization of actin filaments:vesicle-localized AtFH5 promotes actin assembly,and the polymerization and elongation of actin filaments,in turn,is essential for the mobility of AtFH5-1 abeled vesicles in pollen grains.Taken together,our work revealed a molecular mechanism underlying the polarity establishment and vesicle mobility during pollen germination.
文摘Pollen tube growth is an essential step during flowering plant reproduction, whose growth depends on a population of dynamic apical actin filaments. Apical actin filaments were thought to be involved in the regu- lation of vesicle fusion and targeting in the pollen tube. However, the molecular mechanisms that regulate the construction of apical actin structures in the pollen tube remain largely unclear. Here, we identify profilin as an important player in the regulation of actin polymerization at the apical membrane in the pollen tube. Downregulation of profilin decreased the amount of filamentous actin and induced disorganization of apical actin filaments, and reduced tip-directed vesicle transport and accumulation in the pollen tube. Direct visualization of actin dynamics revealed that the elongation of actin filaments originating at the apical membrane decreased in profilin mutant pollen tubes. Mutant profilin that is defective in binding poly-L-proline only partially rescues the actin polymerization defect in profilin mutant pollen tubes, although it fully rescues the actin turnover phenotype. We propose that profilin controls the construction of actin structures at the pollen tube tip, presumably by favoring formin-mediated actin polymerization at the apical membrane.
基金supported by the National Natural Science Foundation of China (31130005)the National Basic Research Program of China (2013CB126902) to H. R.
文摘Formins are well-known regulators that participate in the organization of the actin cytoskeleton in organisms. The Arabidopsis thaliana L. genome encodes 21 formins, which can be divided into two distinct subfamilies. However, type II formins have to date been less well characterized. Here, we cloned a type II formin, AtFH16, and characterized its biochemical activities on actin and microtubule dynamics. The results show that the FH1 FH2 structure of AtFH16 cannot nucleate actin polymerization efficiently, but can bind and bundle microfilaments. AtFH16 FHIFH2 is also able to bind and bundle microtubules, and preferentially binds microtubules over microfilaments in vitro, in addition, AtFH16 FHIFH2 co-localizes with microtubules in onion epidermal cells, indicating a higher binding affinity of AtFH16 FHIFH2 for microtubules rather than microfilaments in vivo. In conclusion, AtFH16 is able to interact with both microfilaments and microtubules, suggesting that AtFH16 probably functions as a bifunctional protein, and may thus participate in plant cellular processes.
基金supported by the China National Fund for Distinguished Young Scholars(31125004)partially supported by the CAS/SAFEA International Partnership Program for Creative Research Teams and SRF for ROCS,SEM
文摘Formin is a major protein responsible for regulating the nucleation of actin filaments, and as such, it permits the cell to control where and when to assemble actin arrays. It is encoded by a multigene family comprising 21 members in Arabidopsis thaliana. The Arabidopsis formins can be separated into two phylogenetically-distinct classes： there are 11 class I formins and 10 class II formins. Significant questions remain unanswered regarding the molecular mechanism of actin nucleation and elongation stimulated by each formin isovariant, and how the different isovariants coordinate to regulate actin dynamics in cells. Here, we characterize a class II formin, AtFH19, biochemically. We found that AtFH19 retains all general properties of the formin family, including nucleation and barbed end capping activity. It can also generate actin filaments from a pool of actin monomers bound to profilin. However, both the nucleation and barbed end capping activities of AtFH19 are less efficient compared to those of another well-characterized formin, AtFHI. Interestingly, AtFH19 FH1FH2 competes with AtFH1 FHIFH2 in binding actin filament barbed ends, and inhibits the effect of AtFH1 FHIFH2 on actin. We thus propose a mechanism in which two quantitatively different formins coordinate to regulate actin dynamics by competing for actin filament barbed ends.
文摘Formins have been paid much attention for their potent nucleating activity. However, the connection between the in vivo functions of AtFHs （Arabidopsis thaliana formin homologs） and their effects on actin organization is poorly understood, in this study, we characterized the bundling activity of AtFH8 in vitro and in vivo. Biochemical analysis showed that AtFH8（FH1FH2） could form dimers and bundle preformed actin filaments or induce stellar structures during actin polymerization. Expression of truncated forms of AtFH8 and immunolocalization analysis showed that AtFH8 localized primarily to nuclear envelope in interphase and to the new cell wall after cytokinesis, depending primarily on its N-terminal transmembrane domain. GUS histochemical staining showed AtFH8 was predominantly expressed in Arabidopsis root meristem, vasculature, and outgrowth points of lateral roots. The primary root growth and lateral root initiation of atfh8 could be decreased by latrunculin B （LatB）. Analysis of the number of dividing cells in Arabidopsis root tips showed that much fewer dividing cells in Lat B-treated atfh8 plants than wild-type plants, which indicates that AtFH8 was involved in cell division. Actin cytoskeleton in root meristem of atfh8-1 was more sensitive to LatB treatment than that of wild-type. Altogether, our results indicate that AtFH8 is an actin filament nucleator and bundler that functions in cell division and root development.
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.
