MYOSIN VI FUNCTION IN INTRACELLULAR TRANSPORT/LOCALIZATI

对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凝蛋白家族成员，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尚不清楚。在我们之前的研究中，我们已经确定了第一个VI类肌球蛋白（果蝇95F肌球蛋白），并表明它是一种基于动作蛋白的细胞质转运蛋白，参与合胞囊胚发育阶段的膜重塑。随后，我们确定了影响精子发生的肌球蛋白VI突变体。这些突变体的表型研究表明，肌球蛋白VI在这种分化过程中也是膜重塑的转运体。肌凝蛋白VI对于促进肌动蛋白和微管细胞骨架之间的相互作用也很重要。根据生化和免疫定位标准，我们发现肌球蛋白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运输有关。这两种蛋白在几个过程中相关，其中基于肌动蛋白和微管的过程被认为是重要的。其中两个是早期胚胎中的神经元转运和RNPs的锚定。我们假设肌凝蛋白VI-CLIP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运动。为了验证我们关于肌凝蛋白VI功能的假设，我们的研究将集中在肌凝蛋白VI在三种细胞类型中的膜重塑和运输作用：精子，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成像的运动在体内；在早期胚胎中，我们将使用显性干扰分子和抗体抑制方法研究对RNP定位重要的相互作用。CLIP在肌球蛋白VI介导的过程中的作用将通过在体内表达的显性干扰分子进行分析。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的复合物相关的生化活性。由于人们认为其他生物中的非常规肌球蛋白发挥的作用与我们在果蝇中提出的肌球蛋白VI类似，我们的实验将揭示肌球蛋白功能和关联的重要信息，这应该是普遍适用的。

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.