Regulation of Protozoan Myosin-I Function

9514248 Zot实验室发现了第一个与非丝状肌球蛋白结合的非肌动蛋白。这种来自棘阿米巴的蛋白质Acan125通过src同源结构域SH3与肌球蛋白-I结合。另一种蛋白质Acan47与Acan125结合，也可能是一种肌球蛋白结合蛋白。该项目的目标是确定Acan125和Acan47的功能，以便确定肌球蛋白-I的细胞角色。其具体目的是：1，通过克隆和测序确定Acan125的结构及其SH3结合部位；2，确定Acan47的结构和肌球蛋白I结合特性；3，利用单特异性高效价抗体确定Acan125在棘阿米巴中的作用，通过免疫荧光定位Acan125，并通过显微注射抑制其在活细胞中的功能。将监测注射抗体的细胞的肌球蛋白相关功能，如形状变化和细胞运动、细胞内囊泡运输和空泡收缩。此外，Acan125和肌球蛋白-I将通过包括肌球蛋白-I的同型特异性抗体在内的双标记免疫荧光同时定位，以检测特定肌球蛋白-I和Acan125之间联系模式的变化。这个项目不仅将探索肌球蛋白-I功能的问题，而且还将探索更广泛的SH3功能问题。SH3介导的结合在细胞膜表面具有不同功能的蛋白质之间瞬间形成。细胞运动需要膜和细胞骨架系统的协调。肌球蛋白-I是一种以肌动蛋白为基础的运动蛋白，由于其与脂质结合的特性，它参与了许多膜介导的运动过程。SH3结合伙伴的特征将可能揭示肌球蛋白-I和微丝系统的新作用。总体目标是了解基于肌动蛋白的细胞运动过程，这些过程在所有细胞中都是常见的。***

9514248 Zot The Zot laboratory has discovered the first non-actin protein that binds a nonfilamentous myosin. This protein, Acan125, from Acanthamoeba, binds to myosin-I through a src homology domain, SH3. Another protein, Acan47, copurifies with Acan125 and may also be a myosin-binding protein. The goals of this project are to determine the functions of Acan125 and Acan47 in order to determine cellular roles for myosin-I. The specific aims are to: 1, determine the structure of Acan125 and its SH3 binding site by cloning and sequencing the cDNA; 2, determine the structure and myosin-I binding characteristics of Acan47; and 3, establish a role for Acan125 in Acanthamoeba using monospecific high-titer antibodies to localize Acan125 by immunofluorescence and to inhibit its function in living cells by microinjection. Antibody-injected cells will be monitored for myosin-associated functions such as shape changes and cell motility, intracellular vesicular transport, and vacuolar contraction. In addition, Acan125 and myosin-I will be localized simultaneously by double-label immunofluorescence, including isoform-specific antibodies to myosin-I to detect changes in patterns of associations between specific isoforms of myosin-I and Acan125. This project will explore not only issues of myosin-I function, but also the broader issue of SH3 function. SH3-mediated associations form transiently between proteins of diverse functions on the surfaces of cellular membranes. Cellular movement requires the coordination of membrane and cytoskeletal systems. As a result of its lipid binding properties, the actin-based motor protein myosin-I has been implicated in many membrane-mediated motile processes. The characterization of SH3 binding partners will likely reveal novel roles for myosin-I and the microfilament system. the overall goal is to understand the processes involved in actin-based cell motility that are common among all cells. ***