MYOSIN VI FUNCTION IN INTRACELLULAR TRANSPORT/LOCALIZATI

许多不同的非常规肌球蛋白在体内的功能知之甚少。大多数细胞表达许多肌球蛋白家族成员，每个成员都以不同的亚细胞分布。它们对细胞组织、动力学和功能的相对贡献尚不清楚。在我们以前的研究中，我们已经鉴定出第一个VI类肌球蛋白(Drodexila 95F Myosin)，并表明它是一种基于肌动蛋白的细胞质转运蛋白，参与了合胞胚层发育阶段的细胞膜重塑。随后，我们发现了影响精子发生的肌球蛋白VI突变体。对这些突变体的表型研究表明，肌球蛋白VI在分化过程中的膜重塑过程中也是一个转运蛋白。肌球蛋白VI对促进肌动蛋白和微管细胞骨架之间的相互作用也可能是重要的。我们发现，根据生化和免疫定位标准，肌球蛋白VI和微管结合蛋白CLIP(细胞质连接蛋白-190)是相关的。这两种蛋白都与细胞内转运有关。这两种蛋白质在几个过程中都有关联，其中基于肌动蛋白和基于微管的过程都被认为是重要的。其中两个是RNPs在早期胚胎中的神经元运输和锚定。我们假设，肌球蛋白VI-CLIP的相互作用促进了运输成分从一个细胞骨架系统到另一个细胞骨架系统的移动。为了检验我们关于肌球蛋白VI功能的假设，我们的研究将集中在三种细胞类型中肌球蛋白VI的膜重构和转运角色：精子细胞，在那里我们将主要使用基因技术来确定相互作用的蛋白质；神经元细胞，在那里我们将使用生化分离和活体运动成像；以及早期胚胎，在那里我们将使用主要干扰分子和抗体抑制方法来研究对于RNP定位非常重要的相互作用。将使用体内表达的主要干扰分子来分析CLIP在肌球蛋白VI介导的过程中的作用。我们将通过体外实验来研究肌球蛋白VI参与这些过程的生化机制，这些实验绘制了与其他蛋白质相互作用的重要结构域，并定义了与含有肌球蛋白VI的复合体相关的生化活性。由于人们认为其他生物中的非传统肌球蛋白发挥着与我们在果蝇中的肌球蛋白VI相似的作用，我们的实验将揭示关于肌球蛋白功能和联系的重要信息，这些信息应该是普遍适用的。

Little is known about the functions of the many different unconventional myosins in vivo. Most cells express many myosin family members, each in a distinct subcellular distribution. Their relative contributions to cellular organization, dynamics, and function remains unclear. In our previous studies we have identified the first myosin of class VI (Drosophila 95F myosin) and have shown that it is an actin-based cytoplasmic transporter involved in membrane remodeling during the syncytial blastoderm stage of development. Subsequently, we have identified myosin VI mutants that affect spermatogenesis. Phenotypic studies of these mutants suggest that myosin VI is a transporter during membrane remodeling in this differentiation process as well. Myosin VI may also be important for facilitating interactions between the actin and microtubule cytoskeletons. We discovered that myosin VI and CLIP (cytoplasmic linker protein-190), a microtubule- binding protein, are associated, as judged by biochemical and immunolocalization criteria. Both of these proteins have been implicated in intracellular transport. The two proteins are associated in several processes in which both actin-based and microtubule-based processes are thought to be important. Two of these are neuronal transport and anchoring of RNPs in early embryos. We hypothesize that the myosin VI-CLIP interaction facilitates movement of transported components from one cytoskeletal system to the other. To test our hypotheses about myosin VI function, our studies will focus on membrane remodeling and transport roles for myosin VI in three cell types: spermatids, where we will use primarily genetic techniques to identify interacting proteins; neuronal cells, where we will use biochemical fractionation and imaging of motility in vivo; and the early embryo, where we will investigate interactions important for RNP localization using dominant interfering molecules and antibody inhibition approaches. CLIP's role in myosin VI mediated-processes will be analyzed using dominant interfering molecules expressed in vivo. We will examine the biochemical mechanism of myosin VI's participation in these processes using experiments in vitro that map the domains important for interactions with other proteins and define the biochemical activities associated with complexes containing myosin VI. Since it is thought that unconventional myosins in other organisms play roles similar to those we propose for myosin VI in Drosophila, our experiments will reveal important information about myosin function and associations that should be generally applicable.