拟南芥EXO70C1调控胞质分裂的分子机理

Tethering, which mediates the first contact between vesicles and their target membranes, plays an important role in the regulation of SNAREs mediating membrane fusion. Exocyst, a tethering factor, is a highly conserved octameric complex that plays critical roles in tethering of Golgi-derived vesicles to target membranes. As a component of exocyst complex, EXO70 exhibits key roles in assembly of exocyst at the target membrane. During plant cytokinesis, the partitioning of the mother cell depends on targeted membrane trafficking to generate new cell wall between two daughter cells. Though the function of several SNAREs had been determined in membrane fusion, the regulatory mechanism that tethering of vesicles to target membranes is unknown. We found previously that Arabidopsis exo70c1 mutant plants show a wide array of abnormalities, including swelling cells at cotyledons, hypocotyls and roots. Further investigations revealed that cell wall stubs and binucleate cells were often observed with these cells, suggesting that exo70c1 is defective in cytokinesis. To characterize the function of EXO70C1 during plant cytokinesis, the sub-cellular localization of EXO70C1 and its dynamics will be investigated using electron microscopy and fluorescent-protein fusions, and the interacting proteins will be identified by using split-ubiquitin based yeast two-hybrid technology. The knowledge from this project will help to understand the mechanism that EXO70 mediated vesicles tethering regulates plant cytokinesis.

囊泡拴系过程介导囊泡与目的膜之间最初的接触，在时空上调控下游的膜融合事件。一类保守的蛋白复合体-胞泌复合体（Exocyst）-参与了运输囊泡在目的膜的拴系。该复合体由8个蛋白亚基组成，其中EXO70是调控复合体在靶膜组装的关键蛋白。植物细胞胞质分裂过程中，细胞板的形成是通过由SNARE介导膜融合实现的，但拴系机制调控这一过程的机理目前了解很少。前期我们发现拟南芥EXO70C1突变导致幼苗子叶、下胚轴及根部细胞不规则肿大，细胞学观察显示这些异常细胞呈现典型的胞质分裂缺陷表型，即细胞壁闭合不全和一胞多核，说明EXO70C1在植物胞质分裂过程中发挥重要作用。本项目将①通过GFP标记确定EXO70C1在胞质分裂过程中的亚细胞定位及动态行为特点；②通过鉴定EXO70C1互作蛋白了解EXO70C1参与胞质分裂的调控网络。项目通过对植物EXO70功能解析，将有助于理解囊泡拴系调控细胞板发育的分子机理。

囊泡运输是存在于所有真核生物的一种非常重要的生物学过程，直接参与了细胞生长、细胞壁形成、细胞分泌和细胞分裂等多项细胞学过程。EXO70是介导运输囊泡拴系到靶膜、启动膜融合事件的关键蛋白，在动物和酵母中由单基因编码，而在陆生植物基因组中存在大量拷贝，譬如拟南芥基因组有23个EXO70，EXO70大量扩增的生物学意义尚不明确。本项目综合利用细胞学、遗传学和分子生物学等方法，对EXO70C1及其同源基因EXO70A1的功能进行了初步分析，研究结果如下：(1)EXO70C1分布在花粉粒和花粉管细胞质中，在花粉萌发和花粉管极性生长中发挥重要作用。(2)EXO70C1与SNARE蛋白复合体成员SYP124/SYP125有互作，暗示胞泌复合体和SNARE复合体协同调控囊泡运输过程。(3)EXO70A1分布在下胚轴表皮细胞质膜及胞质中，参与了下胚轴细胞伸长所必需的物质运输。(4)EXO70A1在下胚轴表皮细胞质膜上的排布与皮层微管相关，而向质膜的运输和在质膜的上的分布依赖于微丝骨架。(5)筛选到一个胞质分裂异常突变体，其胚性细胞和花粉粒细胞胞质分裂异常，通过全基因组测序分析此表型可能是由于CDC20.4基因缺失造成的。项目研究结果为阐述EXO70介导的囊泡运输参与细胞极性生长和细胞伸长的分子机制奠定了基础，同时也有助于人们理解植物EXO70家族扩增的生物学意义。