Regulation of Protozoan Myosin-I Function

小行星9514248 Zot实验室发现了第一个结合非丝状肌球蛋白的非肌动蛋白。 这种来自阿米巴的蛋白Acan 125通过src同源结构域SH 3与肌球蛋白-I结合。 另一种蛋白Acan 47与Acan 125共纯化，也可能是肌球蛋白结合蛋白。 本项目的目标是确定Acan 125和Acan 47的功能，以确定肌球蛋白-I的细胞作用。 具体目标是：1、通过克隆和测序cDNA确定Acan 125的结构及其SH 3结合位点; 2、确定Acan 47的结构和肌球蛋白-I结合特性; 3、使用单特异性高滴度抗体通过免疫荧光定位Acan 125并通过显微注射抑制其在活细胞中的功能，确定Acan 125在阿米巴中的作用。 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性、细胞内囊泡转运和空泡收缩。 此外，Acan 125和肌球蛋白-I将通过双标记免疫荧光同时定位，包括肌球蛋白-I的同种型特异性抗体，以检测肌球蛋白-I和Acan 125的特定同种型之间的关联模式的变化。 本项目将探讨不仅是肌球蛋白-I的功能问题，但也SH 3功能的更广泛的问题。 SH 3介导的协会之间的不同功能的蛋白质细胞膜表面上的瞬时形成。 细胞运动需要膜和细胞骨架系统的协调。 由于其脂质结合特性，肌动蛋白为基础的运动蛋白肌球蛋白-I已牵连在许多膜介导的运动过程。 SH 3结合伙伴的特性将可能揭示肌球蛋白-I和微丝系统的新作用。 总的目标是了解所有细胞中共同的基于肌动蛋白的细胞运动所涉及的过程。 ***

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. ***