Golgi Myosin in Higher Plants

真核细胞的分泌系统将大分子转运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其合成位点到其作用位点的物理运动。在大多数物种中，这些载体是小泡状或管状运输容器。植物细胞在真核生物中是独特的，因为它们显示出整个高尔基体的快速的、基于肌动球蛋白的运动。这种运动提出了一种可能性，即植物高尔基体，分泌系统的一个主要细胞器，也提供长距离运输功能，除了其在合成，修饰和分泌产物的分选中的作用。该项目追求两个中心目标。首先是建立高尔基体运动的机制基础。第二个是测试的假设，快速的高尔基体堆栈运动是必要的适当功能的分泌系统的植物细胞和间接发挥作用的生长和发育。实验方法可以分为四个部分。(1)利用荧光蛋白和同种型特异性抗体融合鉴定定位于高尔基体的肌球蛋白马达蛋白。(2)通过反向遗传学，即T-DNA插入和RNA沉默，分离缺乏高尔基体运动的肌球蛋白突变体。(3)高尔基体肌球蛋白基因突变体的表型分析，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、测试候选Rab G蛋白和使用免疫共沉淀或酵母双杂交系统的一般筛选来鉴定介导高尔基体肌球蛋白的货物附着的蛋白质。总之，这个项目将确定植物细胞中高尔基体堆叠运动的分子基础，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.