微管和微丝是真核生物中普遍存在的蛋白纤维结构，它们与中间纤维共同构成细胞内一个相互联系的三维网络体系－细胞骨架。不同的骨架系统在不同的生命活动中行使不同的功能，然而大量的研究表明，在一些细胞生命活动中，微管和微丝间存在广泛的相互作用。Formin蛋白是存在于真核生物中一类重要的肌动蛋白动态调节因子，在细胞极性建立、细胞分裂和形态发生中起调控作用。近年来，formin的研究颇受关注，在动物和酵母细胞中，formin蛋白除了调控微丝骨架动态，也能稳定微管骨架。在本研究中，通过与其它formin成员的序列比对分析，发现AtFH14是一个典型的拟南芥II型formin，细胞生物学和生化实验显示AtFH14是微管与微丝的交联蛋白。首先，我们鉴定的AtFH14的两个T-DNA插入突变体，发现它们在小孢子发育过程中产生二分体、三分体、五分体以及一些形态不规则的四分体。为了探究AtFH14在四分体形成过程中的功能，我们对AtFH14的定位进行了观察。利用制备的AtFH14的单克隆抗体，通过间接免疫荧光实验证明AtFH14与减数分裂期花粉母细胞中期I、中期II纺锤体以及后期I、胞质分裂晚期成膜体微管共定位，而与所有时期的幅射状微管没有明显的共分布的现象。进一步用α-tubulin抗体标记减数分裂花粉母细胞的微管，发现突变体中有些中期I纺锤体比野生型的小；中期II，出现大量的两个纺锤体平行排列模式，而非正常的垂直排布方式；胞质分裂晚期，在野生细胞的胞质相连的部位有成膜体微管的明显聚集，而在突变体中却没有明显的成膜体微管的出现。这些结果说明AtFH14影响了花粉母细胞减数分裂时期的微管列阵。此外，我们还观察到在野生拟南芥药壁组织细胞的有丝分裂早前期微管带，纺锤体和成膜体微管也有定位，为了深入研究AtFH14对细胞有丝分裂的影响，我们以拟南芥悬浮细胞为材料，对AtFH14在细胞有丝分裂进程中的分布及功能进行了观察与分析。AtFH14单克隆抗体间接免疫荧光实验证明AtFH14与有丝分裂期的早前期微管带，纺锤体和成膜体微管共定位。通过双色标记方法观察了AtFH14表达量下降对有丝分裂器微管、微丝列阵的影响。在RNAi细胞群体中，表型的严重程度不同：一些细胞没有明显异常，微丝在纺锤体周围形成一个网状结构，成膜体微丝与成膜体微管共分布，但微丝比微管稍短；一些细胞纺锤体周围的网状微丝以及成膜体微丝消失；一些细胞有丝分裂器微管列阵部分解体，只留有少量微管残余，而微丝完全解聚；一些细胞微管、微丝列阵完全解聚。然而对照细胞微管、微丝列阵均正常分布。这些结果说明AtFH14在细胞分裂过程中可能是一个既结合微管又结合微丝的蛋白。为了验证AtFH14是否能直接作用于微管或/和微丝，我们利用体外系统对原核表达的AtFH14（FH1FH2）重组蛋白与微管或微丝的相互作用进行了研究。利用离心共沉淀方法的结果表明，AtFH14（FH1FH2）与聚合好的微管、微丝共孵育时，具有浓度依赖性的微管或微丝结合能力，并在一定浓度时可以达到饱和，说明AtFH14（FH1FH2）能特异性的直接结合微管或微丝。荧光标记和透射电镜实验证明AtFH14（FH1FH2）具有使微管或微丝成束的性质，并在体外与微管或微丝共分布。此外，在微管与微丝共同存在的情况下，AtFH14（FH1FH2）既能结合微管，同时也能结合微丝。体外荧光实验证明低浓度的AtFH14（FH1FH2）的微管结合成束能力强于与微丝结合成束的能力。在高浓度AtFH14（FH1FH2）存在的体系，诱导微管、微丝交联；并在单体肌动蛋白聚合过程中，电镜观察发现微管或微管束与微丝或微丝束平行排列、微管或微管束与微丝端部相粘连等现象，而在对照样品中更多的是观察到微丝横跨微管的现象。通过以上细胞生物学和生化实验充分证明AtFH14是一个微管或微丝的成束蛋白，同时亦是微管与微丝的交联蛋白。AtFH14很可能是通过连接微管与微丝骨架结构进而对细胞分裂装置起稳定作用，从而参与细胞有丝分裂和花粉母细胞减数分裂的调控。

Microtubules and microfilaments are two major cytoskeletal networks found in plant cells. Both of them play important roles in many aspects of fundamental processes of plant cells growth and development, such as cell division, cell expansion, and intracellular organization and motility. There is no question that they constitute separate cytoskeletal systems and fulfill many distinct functions. However, strong evidences are growing for functional interactions between microtubules and microfilaments in some cellular processes. Formins have long been known as microfilament regulators, but recently are implicated a role of microtubule interaction. In this study, AtFH14, a type II formin from Arabidopsis thaliana, was found to regulate both microtubule and microfilament arrays. To investigate the function of AtFH14 in the Arabidopsis, two T-DNA insertion mutants were identified with PCR, two mutants had a similar phenotype that the atfh14 lines produced dyads, triads, polyads, and abnormal tetrads with irregular distribution pattern of microspores. AtFH14 antibody and anti-α-tubulin antibody were used in staining Arabidopsis pollen mother cells, the AtFH14 co-localized with the spindle in metaphase I, II and the phragmoplast in anaphase I and advanced cytokinesis. Furthermore, immunofluorescence microscopy was performed with tubulin antibody. Compared to the wild type, irregular microtubule arrays were observed in atfh14 cells. Metaphase I spindle was smaller than that of the wild type. Metaphase II spindles were at right angle to each other. At the same stage, the spindles of the some atfh14 cells exhibited parallel arrangement. At advanced cytokinesis, there were apparently phragmoplast microtubules between the two nuclei, but in atfh14 cells, the phragmoplast microtubule systems did not obviously appear. To test if the AtFH14 was necessary during the cell division in Arabidopsis cells, AtFH14 antibody and anti-α-tubulin antibody were used in staining Arabidopsis suspension cells, the AtFH14 co-localized with proprephase band, spindle and phragmoplast. Compared the knockdown cells with the wild-type cells, we categorized the cell phenotypes as severe, strong, mild and normal. The microtubule and microfilament array are destroyed with different severity. These observations indicated that AtFH14 play important roles during the cell division as a microtubule and microfilament crosslinker.The direct binding activity of AtFH14(FH1FH2) to the two filaments was tested in vitro using co-sedimentation experiments, respectively. AtFH14(FH1FH2) protein directly co-sedimented with microtubules and microfilaments in a concentration-dependent manner. In vitro fluorescent and TEM experiments showed that AtFH14 bundled them and co-localized along microtubule or microfilament bundles. In the presence of both microtubules and microfilaments, AtFH14 preferentially bound to microtubules, and had higher affinity with the microtubule, but cross-linked microtubules and microfilaments when excessive AtFH14 was present. During the process of G-actin polymerization, TEM experiment showed that the microfilament or microfilament bundles elongated from the surface of the single microtubule or microtubule bundles.These results demonstrate that AtFH14 is a plant formin that cross-links microtubules and microfilaments, which is important in the process of plant cell division.