Golgi Myosin in Higher Plants

真核细胞的分泌系统将大分子运输到细胞表面和细胞的裂解空间。这种运输需要膜结合载体中的分泌产物从它们的合成部位到作用部位的物理运动。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一无二的，因为它们显示出整个高尔基体堆叠的快速、基于肌动球蛋白的运动。这一运动提出了这样一种可能性，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、修饰和分类中起着公认的作用外，还具有长途运输功能。该项目追求两个核心目标。第一个是建立高尔基堆叠运动的机械基础。第二个是验证这样一个假设，即快速的高尔基体堆叠运动对于植物细胞分泌系统的正常运作是必要的，并间接地在生长和发育中发挥作用。实验方法可以分为四个部分。(1)利用与荧光蛋白和异构体特异性抗体的融合，鉴定定位于高尔基体堆叠的肌球蛋白马达蛋白。(2)分离缺乏由反向遗传学引起的高尔基运动的肌球蛋白突变体，即T-DNA插入和RNA沉默。(3)对高尔基体肌球蛋白基因(S)突变体的表型分析，特别是对标记蛋白在分泌系统中的转运效率进行了分析。(4)通过靶向方法，测试候选Rab G蛋白，以及使用免疫共沉淀或酵母双杂交系统的一般筛选，鉴定介导高尔基肌球蛋白附着的蛋白质。综上所述，该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.