MYOSIN VI FUNCTION IN INTRACELLULAR TRANSPORT/LOCALIZATI

关于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，每个成员在不同的亚细胞分布。它们对细胞组织、动力学和功能的相对贡献仍不清楚。 在我们以前的研究中，我们已经确定了第一个肌球蛋白的VI类（果蝇95 F肌球蛋白），并已表明，它是一个肌动蛋白为基础的细胞质转运蛋白参与膜重塑在合胞胚盘发育阶段。 随后，我们已经确定了肌球蛋白VI突变体，影响精子发生。 这些突变体的表型研究表明，肌球蛋白VI是一种转运蛋白在膜重塑在这个分化过程中，以及。 肌球蛋白VI也可能是重要的促进肌动蛋白和微管细胞骨架之间的相互作用。 我们发现，肌球蛋白VI和CLIP（细胞质连接蛋白-190），微管结合蛋白，是相关的，判断生化和免疫定位标准。 这两种蛋白质都参与细胞内转运。 这两种蛋白质在几个过程中，肌动蛋白和微管为基础的过程被认为是重要的。 其中两个是早期胚胎中RNP的神经元运输和锚定。 我们推测，肌球蛋白VI-CLIP相互作用促进运输组件从一个细胞骨架系统到其他的运动。 为了验证我们关于肌球蛋白VI功能的假设，我们的研究将集中在肌球蛋白VI在三种细胞类型中的膜重塑和转运作用：精子细胞，我们将主要使用遗传技术来识别相互作用的蛋白质;神经细胞，我们将使用生物化学分离和体内运动成像;和早期胚胎，在那里我们将研究相互作用的重要RNP本地化使用显性干扰分子和抗体抑制的方法。 CLIP在肌球蛋白VI介导的过程中的作用将使用体内表达的显性干扰分子进行分析。 我们将研究肌球蛋白VI参与这些过程的生化机制，使用体外实验，映射与其他蛋白质相互作用的重要领域，并定义与含肌球蛋白VI的复合物相关的生化活性。 由于它被认为是非常规的肌球蛋白在其他生物体中发挥的作用类似于我们提出的果蝇肌球蛋白VI，我们的实验将揭示肌球蛋白的功能和协会，应该普遍适用的重要信息。

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.