摘　要植物细胞骨架是主要由微丝和微管构成的动态的三维网络体系。微丝骨架和微管骨架在细胞内参与许多重要的生理过程，如细胞分裂、细胞生长、分化、细胞运动和信号转导等。虽然微丝和微管有许多各自不同的功能，但越来越多证据表明它们常常以相互协调的方式在细胞中发挥重要作用。Formin蛋白传统上认为是一个微丝调控因子，促进不分支的微丝索或者微丝束的形成。最近在动物和酵母中研究表明，Formin也参与了间期和有丝分裂期微管的动态组装。然而有关植物Formin在体内与微管骨架的相互作用还鲜有报道。在本研究中，我们将AtFH14全长与GFP的融合蛋白在烟草BY-2细胞中进行了表达，通过观察细胞系中的荧光定位，我们发现AtFH14在有丝分裂期定位在早前期微管带、纺锤体和成膜体上，利用β-tubulin抗体进行间接免疫荧光实验证实AtFH14-GFP在有丝分裂细胞中与微管共定位。同时在表达AtFH14的细胞中标记微丝，发现微丝被募集到纺锤体和成膜体上，且与AtFH14蛋白共定位。通过药理学实验，我们发现当微管解聚时，AtFH14定位也呈弥散分布；当微丝解聚时AtFH14仍特异地分布在有丝分裂器上，这说明AtFH14的定位更依赖于微管而不是微丝。进一步，对表达AtFH14细胞系进行动态观察，我们发现细胞有丝分裂器转换受到了抑制，细胞被延缓或阻滞在有丝分裂期。通过药理学实验证明，表达AtFH14的细胞有增强抵抗微管解聚药和增强抵抗微丝解聚药的作用。对AtFH14结构域进行分析，Pten和FH1FH2结构域在细胞分裂周期中都定位在有丝分裂器上，AtFH14全长的功能可能是由Pten结构域和FH1FH2结构域共同决定的。我们将AtFH14的不同结构域在洋葱表皮细胞中瞬时表达，结果显示AtFH14在细胞中呈点线状分布，Pten结构域呈点状分布，而FH1FH2结构域则定位在周质微管上，暗示AtFH14在不同类型的细胞中具有不同的作用。总之，本研究结果表明AtFH14可能在细胞周期中发挥微丝、微管交联因子的作用，其通过影响细胞分裂骨架装置的转变而参与细胞有丝分裂和胞质分裂的进程。关键词：Formin， 微丝， 微管， 微丝微管交联因子， BY-2细胞， 有丝分裂

ABSTRACTMicrotubules and microfilaments are two major cytoskeletal networks found in plant cells. Both of them play important roles in many aspects of fundamental processes. Although they both constitute separate cytoskeletal systems and fulfill many distinct functions, strong evidences are growing for functional interactions between microtubules and microfilaments in some cellular processes. Formins are known as microfilament regulators. Recently, it has been noted that Formins behave as prominent regulators of the microtubule cytoskeleton in interphase and mitosis. However, little is known about the function of plant Formin in regulating microtubules. In our research, full-length AtFH14 construct fused with GFP was transformed into wildtype BY-2 cells. The signal of AtFH14-GFP was concentrated on the preprophase band, spindle and phragmoplast, which co-localized with microtubules. By using rhodamine-phalloidin, we found that microfilaments co-distributed with AtFH14 on spindle and phragmoplast in overexpressing AtFH14 cells. Pharmacological experiment showed the fact that with the depolymerized microtubules, AtFH14-GFP signal became dispersed; but the depolymerized microfilaments had no appreciable effect on AtFH14 localization—illustrating that the observed AtFH14 localization relied on microtubules rather than microfilaments. Furthermore, overexpression of AtFH14 affected the cell division progression by inhibiting the turnover of the mitotic apparatus. We also found that overexpressing AtFH14 cells increased the resistance of cytoskeleton drug Oryzalin treatment and Lat B treatment. Expression of different truncated proteins of AtFH14 revealed that both Pten domain and FH1FH2 domain could target to PPB, spindle and phragmoplast, indicating that the location and function of AtFH14 was determined by both Pten and FH1FH2 domains. In onion epidermal cells, AtFH14 revealed dot-line structure, Pten domain showed dot array, while FH1FH2 was found to bind to cortical microtubule arrays and revealed mobile dynamics consistent with microtubule studies.These results demonstrate that AtFH14 is a unique plant Formin that crosslinks microtubules and microfilaments, which may play important roles in the process of cell division.KEY WORDS：Formin, microtubules, microfilaments, microtubule-microfilament cross-linker, BY-2 cell, mitosis