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At FH16, an Arabidopsis Type II formin, Binds and Bundles both Microflaments and Microtubules, and Preferentially Binds to Microtubules

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Journal of Integrative Plant Biology 2013年第11期55卷 1002-1015页

作者： Jiaojiao Wang Yi Zhang Jing Wu Lei Meng Haiyun Ren The Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education College of Life Science Beijing Normal University

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.

关键词： Actin filament Arabidopsis cytoskeleton formin microtubule.

AtFH8 Is Involved in Root Development under Effect of Low-Dose Latrunculin B in Dividing Cells

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Molecular Plant 2011年第2期4卷 264-278页

作者： Xiu-Hua Xue Chun-Qing Guo Fei Du Quan-Long Lu Chuan-Mao Zhang Hai-Yun Ren Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education College of Life Sciencer Beijing Normal Universityl Beijing 100875China The Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education and the State Key Laboratory of Bio-Membrane and Membrane Bio-Engineering College of Life Science Peking University Beijing 100871 China

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.

关键词： Actin binding protein formin cell division root meristem lateral root initiation actin nucleator and bundler.

结直肠癌组织中formin2蛋白的表达及意义

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中华消化外科杂志 2015年第4期14卷 335-338页

作者：陈文平施海周毅李晓华初大可刘朝旭赵青川李纪鹏第四军医大学西京消化病医院消化三科西安710032 天津市人民医院肛肠外科 300121

目的探讨结直肠癌组织中formin 2（FMN2）蛋白的表达及其与结直肠癌患者临床病理因素和预后的关系.方法回顾性分析2008年1月至2011年12月第四军医大学西京消化病医院收治的335例结直肠癌患者的临床病理资料.收集患者手术切除的结直肠... 详细信息

目的探讨结直肠癌组织中formin 2（FMN2）蛋白的表达及其与结直肠癌患者临床病理因素和预后的关系.方法回顾性分析2008年1月至2011年12月第四军医大学西京消化病医院收治的335例结直肠癌患者的临床病理资料.收集患者手术切除的结直肠标本进行研究.采用免疫组织化学染色检测结直肠癌患者癌组织及癌旁组织中FMN2蛋白的表达情况,分析其与结直肠癌患者临床病理因素和预后的关系.采用电话或信件方式进行随访,随访时间截至2014年6月.率的比较以及FMN2表达与临床病理因素的关系分析采用χ^2检验.采用Kaplan-Meier法绘制生存曲线,Log-rank检验进行生存分析.结果 FMN2蛋白的表达位于结直肠癌组织细胞的细胞质.结直肠癌组织中FMN2蛋白的阳性表达率为53.73％（180/335）,癌旁组织中为80.90％（271/335）,两者比较,差异有统计学意义（χ^2=46.792,P＜0.05）.结直肠癌组织中FMN2蛋白的表达在结直肠癌患者的肿瘤分化程度、TNM分期及淋巴结转移方面比较,差异有统计学意义（χ^2=7.707,36.472,40.635,P＜0.05）.322例患者获得术后随访,随访率为96.12％（322/335）.随访时间为3～36个月,中位随访时间为26个月.其中173例FMN2蛋白阴性表达的结直肠癌患者中位生存时间为30个月,3年累积生存率为48.6％;149例FMN2蛋白阳性表达的结直肠癌患者中位生存时间为36个月,3年累积生存率为68.5％,两者生存情况比较,差异有统计学意义（χ^2=16.283,P＜0.05）.结论 FMN2蛋白在结直肠癌组织中表达下调,与结直肠癌的发生、发展密切相关,可作为判断患者预后的指标.

关键词：结直肠肿瘤 formin 2 预后免疫组织化学染色

Profilin Regulates Apical Actin Polymerization to Control Polarized Pollen Tube Growth

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Molecular Plant 2015年第12期8卷 1694-1709页

作者： Xiaonan Liu Xiaolu Qu Yuxiang Jiang Ming Chang Ruihui Zhang Youjun Wu Ying Fu Shanjin Huang State Key Laboratory of Plant Physiology and Biochemistry College of Biological Sciences China Agricultural University Beijing 100193 China Key Laboratory of Plant Molecular Physiology Institute of Botany Chinese Academy of Sciences Beijing 100093 China Center for Plant Biology School of Life Sciences Tsinghua University Beijing 100084 China Tsinghua-Peking Joint Center for Life Sciences Beijing 100084 China National Center for Plant Gene Research Beijing 100101 China present address: Department of Biological Sciences Dartmouth College Hanover NH 03755 USA

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.

关键词： pollen tube actin actin polymerization profilin formin

Actin Polymerization Mediated by AtFH5 Directs the Polarity Establishment and Vesicle Trafficking for Pollen Germination in Arabidopsis

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Molecular Plant 2018年第11期11卷 1389-1399页

作者： Chang Liu Yi Zhang Haiyun Ren Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education College of Life ScienceBeijing Normal UniversityBeijing 100875China

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.

关键词： Chinese cabbage Pollen germination Polarity establishment Vesicle trafficking Actin formin

拟南芥formin家族蛋白AFH14基因的克隆及功能结构域分析

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山西大同大学学报（自然科学版） 2011年第3期27卷 48-50页

作者：李艳花山西大同大学脑科学研究所山西大同037009

应用RT-PCR的方法从拟南芥cDNA中克隆了AFH14的全序列,并对其功能结构域进行分析。发现所克隆的目的基因编码区全长3 102 bp,与基因组序列比对发现该基因包含18个内含子和18个外显子,编码1 033个氨基酸残基。通过与其它formin成员的序... 详细信息

应用RT-PCR的方法从拟南芥cDNA中克隆了AFH14的全序列,并对其功能结构域进行分析。发现所克隆的目的基因编码区全长3 102 bp,与基因组序列比对发现该基因包含18个内含子和18个外显子,编码1 033个氨基酸残基。通过与其它formin成员的序列比对分析,发现AFH14是一个典型的拟南芥II型formin,包括PTEN结构域。

关键词： AFH14 formin PTEN FH1 FH2

Arabidopsis formin3 directs the formation of actin cables and polarized growth in pollen tubes

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Arabidopsis formin3 directs the formation of actin cables an...

AtFH7和AtFH20在拟南芥根系发育中的功能初探

中国植物学会植物细胞生物学2010年学术年会

作者： Jianrong Ye~(a,b,1),Yiyan Zheng~(c,1),An Yan~(b,d),Naizhi Chen~c,Zhangkui Wang~(a,b), Shanjin Huang~(c,2),and Zhenbiao Yang~(b,d,2*) 1 College of Biological Science,China Agricultural University,Beijing 100193,China 2 China Agricultural University-University of California-Riverside Joint Center for Biological Sciences and Biotechnology,China Agricultural University,Beijing 100193,China 3 Key Laboratory of Photosynthesis and Environmental Molecular Physiology,Institute of Botany,Chinese Academy of Sciences,Beijing 100093,China 4 Department of Botany and Plant Sciences,Center for Plant Cell Biology,Institute of Integrative Genome Biology,University of California,Riverside,California 92521

Cytoplasmic actin cables are the most prominent actin structures in plant cells,but the molecular mechanism underlying their formation is unknown.The function of these actin cables,which are proposed to modulate cytoplasmic streaming and intracellular movement of many organelles in plants,has not been studied by genetic means.Here,we show that Arabidopsis thaliana formin3(AFH3) is an actin nucleation factor responsible for the formation of longitudinal actin cables in pollen tubes. The Arabidopsis AFH3 gene encodes a 785-amino acid polypeptide,which contains a formin homology 1(FH1) and a FH2 domain.In vitro analysis revealed that the AFH3 FH1FH2 domains interact with the barbed end of actin filaments and have actin nucleation activity in the presence of G-actin or G actin-profilin.Overexpression of AFH3 in tobacco(Nicotiana tabacum) pollen tubes induced excessive actin cables,which extended into the tubes’apices.Specific downregulation of AFH3 eliminated actin cables in Arabidopsis pollen tubes and reduced the level of actin polymers in pollen grains.This led to the disruption of the reverse fountain streaming pattern in pollen tubes,confirming a role for actin cables in the regulation of cytoplasmic streaming.Furthermore,these tubes became wide and short and swelled at their tips,suggesting that actin cables may regulate growth polarity in pollen tubes.Thus,AFH3 regulates the formation of actin cables,which are important for cytoplasmic streaming and polarized growth in pollen tubes.

关键词： actin formin pollen tube

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AtFH7和AtFH20在拟南芥根系发育中的功能初探

作者：李应超西北大学

学位级别：硕士

植物细胞内存在着活跃的胞质环流,伴随着胞质环流,各种亚细胞成分高度动态,研究表明这种动态的性质依赖于细胞骨架,尤其是微丝的持续重塑,以响应发育和环境信号。肌动蛋白丝的形成与排列受到多种肌动蛋白结合蛋白的调节。formin蛋白作... 详细信息

植物细胞内存在着活跃的胞质环流,伴随着胞质环流,各种亚细胞成分高度动态,研究表明这种动态的性质依赖于细胞骨架,尤其是微丝的持续重塑,以响应发育和环境信号。肌动蛋白丝的形成与排列受到多种肌动蛋白结合蛋白的调节。formin蛋白作为肌动蛋白成核因子之一,广泛存在于真核细胞中,现已发现formin能够调控肌动蛋白的成核、成束,并且部分成员还能协调微丝与微管的协同作用,通过对骨架的调控,formin在细胞生长和形态建成等过程中发挥着关键性作用。与真菌和动物差异较大,植物中formin蛋白家族经过进化形成了特有的蛋白结构域组成,其结构域主要包括FH1和FH2结构域,而不包含DIA、DAD以及结合Rho GTP酶的GBD/FH3结构域。根据高度保守的结构域FH2对植物formin蛋白家族进行分析,分为两类亚家族蛋白,即Ⅰ类formin蛋白和Ⅱ类formin蛋白。由于植物中formin蛋白家族数目庞大,功能冗余现象明显,人们对植物中的formin蛋白研究仍然比较有限。根据对拟南芥中formin蛋白的定位和含量的生信数据分析,选择了在根毛中表达量较高的且有可能定位在细胞核的Ⅰ类formin蛋白AtFH7和Ⅱ类formin蛋白AtFH20进行功能的初步探究,筛选获得了AtFH7和AtFH20的T-DNA插入纯合突变体,并通过杂交得到红色荧光标记细胞核和绿色荧光标记微丝的atfh7和atfh20纯合突变体。对根的根长、分生区、根毛及其细胞核进行观察和统计分析,得到的主要研究成果如下:1.细胞核在根毛细胞发生程序性死亡后,会回迁至根毛基部,并进一步被降解。2.atfh7的根毛显著性变密变长,atfh20的根毛显著性变稀变短,AtFH7和AtFH20的双突formin7-1/20-1中根毛的长度和密度与野生型对照无明显差异。证明了AtFH7和AtFH20对根毛的起始和伸长过程可能具有相反调控功能,AtFH7对根毛的生长有抑制作用,AtFH20对根毛的生长有促进作用。3.atfh7、atfh20和formin7-1/20-1根毛中细胞核的形状和迁移趋势均无显著性变化。AtFH7和AtFH20作为肌动蛋白结合蛋白可能并不参与细胞核的形态调控,对细胞核的前移和回迁无决定性影响。4.atfh7的根和分生区都显著性变长,atfh20的根和分生区都显著性变短,formin7-1/20-1的根长与野生型对照无显著性差异。证明AtFH7和AtFH20对细胞的分裂可能具有相反的调控功能,AtFH7抑制细胞分裂,AtFH20促进细胞分裂。综上所述,Ⅰ类formin蛋白AtFH7和Ⅱ类formin蛋白AtFH20对细胞的分裂和极性生长均具有调控作用。

关键词：拟南芥根根毛细胞核 formin

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一种水稻微丝结合蛋白OsFH1的功能研究

一种水稻微丝结合蛋白OsFH1的功能研究

作者：张小清上海师范大学

学位级别：硕士

在植物体内，微丝细胞骨架在胞内运输、细胞生长、细胞极性和形态建成中都起到一个基础性的作用。在真核生物中，formin在微丝聚合和一些基于微丝的生理活动中起到中心调节者的作用。我们选择了水稻formin家族中的OsFH1基因对其进行体... 详细信息

在植物体内，微丝细胞骨架在胞内运输、细胞生长、细胞极性和形态建成中都起到一个基础性的作用。在真核生物中，formin在微丝聚合和一些基于微丝的生理活动中起到中心调节者的作用。我们选择了水稻formin家族中的OsFH1基因对其进行体外生化特性和体内生物学功能进行分析。近年来已报道的微丝结合蛋白中，formin家族引起更多的关注，在植物中，根据它们的FH2结构域的序列同源性，formin被分为两大类， I型和II型。目前关于formin的报道I型居多，OsFH1是一个典型的I类formin，在结构中含有信号肽、跨膜结构域、结合profilin/actin的FH1结构域和非常保守的FH2结构域。为了研究OsFH1的生物学功能，我们选取烟草花粉管作为我们研究的模式工具，在烟草花粉管中，过量表达OsFH1基因能够促使花粉管生长受阻、顶端膨大，同时用荧光标记的鬼笔环肽染色花粉管，在波长568nm的荧光下观察，可清晰见到在花粉管的顶端聚集大量的微丝。为了验证它在体外和微丝的关系，我们构建了FH2结构域和FH1FH2结构域的截短蛋白原核表达载体，并纯化得到纯度大于95%的融合蛋白。利用Pyrene标记微丝和全内反射荧光显微镜的方法，我们发现FH2融合蛋白和FH1FH2融合蛋白都能和微丝结合促进微丝成核而且可以结合在微丝的正端起到封端作用，FH1FH2融合蛋白能使微丝成束而FH2融合蛋白没有此成束活性。这些结果表明，OsFH1可以通过调节微丝聚合、成束等行为来调节花粉管的生长，这对我们了解和认识细胞骨架的形成和植物花粉管极性的生长有重要意义。

关键词：微丝花粉管顶端生长 formin 细胞骨架结构域