Golgi Myosin in Higher Plants

真核细胞的分泌系统将大分子输送到细胞表面和细胞的裂解区室。这种转运需要分泌产物在膜结合载体中从合成位点到作用位点的物理运动。在大多数种类中，这些载体是小的囊状或管状运输容器。植物细胞在真核生物中是独一无二的，因为它们表现出快速的、基于肌动球蛋白的整个高尔基堆运动。这种运动提出了一种可能性，即植物高尔基体作为分泌系统的主要细胞器，除了在分泌产物的合成、修饰和分类中发挥公认的作用外，还具有远距离运输功能。这个项目追求两个中心目标。首先是建立高尔基堆运动的机制基础。二是验证高尔基体快速运动是植物细胞分泌系统正常运作所必需的假设，并间接在生长发育中发挥作用。实验方法可分为四个部分。(1)利用荧光蛋白和异构体特异性抗体的融合物鉴定高尔基体上的肌球蛋白运动蛋白。(2)通过T-DNA插入和RNA沉默等反向遗传方法分离出缺乏高尔基运动的肌球蛋白突变体。(3)分析高尔基肌球蛋白基因突变体的表型，特别是通过以下标记蛋白在分泌系统内的转运效率。(4)通过靶向方法鉴定介导高尔基肌球蛋白货物附着的蛋白质，测试候选Rab g蛋白，以及使用共免疫沉淀或酵母- 2杂交系统进行一般筛选。综上所述，该项目将确定植物细胞中高尔基体运动的分子基础，并测试其功能相关性。

The secretory system of eukaryotic cells transports macromolecules to the cell surface and lytic compartments of the cell. This transport requires the physical movement of secretory products in membrane-bounded carriers from their site of synthesis to their site of action. In most species, these carriers are small vesicular or tubular transport containers. Plants cells are unique among eukaryotes in that they display rapid, actomyosin-based movements of entire Golgi stacks. This movement raises the possibility that the plant Golgi, a major organelle of the secretory system, also provides long-distance transport functions, in addition to its well-established roles in synthesis, modification, and sorting of secretory products. This project pursues two central goals. The first is to establish the mechanistic basis underlying Golgi stack movements. The second is to test the hypothesis that rapid Golgi stack movements are necessary for proper functioning of the secretory system of plant cells and indirectly play a role in growth and development. The experimental approaches can be broken down into four parts. (1) Identification of myosin motor proteins that localize to Golgi stacks using fusions to fluorescent proteins and isoform-specific antibodies. (2) Isolation of myosin mutants that lack Golgi movements created by reverse genetics, i.e. T-DNA insertion and RNA silencing. (3) Analysis of the phenotype of mutants in Golgi myosin gene(s), particularly the transport efficiency within the secretory system by following marker proteins. (4) Identification of the proteins that mediate cargo attachment for Golgi myosin by both a targeted approach, testing candidate Rab G-proteins, and a general screen using co-immunoprecipitation or the yeast-two hybrid system. Taken together, this project will identify the molecular basis for Golgi stack movement in plant cells as well as test its functional relevance.