CYTOSKELETAL STRUCTURE IN THE INTESTINAL BRUSH BORDER

建议的研究将继续我们的努力，以表征肌动蛋白- 肠上皮细胞及其顶刷的基本细胞骨架 边界（BB）。 这些研究将以双重目标进行， 提供了对非肌肉细胞的基于肌动蛋白的细胞骨架的一般见解， 细胞，并剖析肌动蛋白的作用及其相关的结合 蛋白质在肠上皮细胞的生理功能。 我们的主要重点 在此期间，研究将在非传统的家庭 肌球蛋白--即，不同于常规的、双头的和 尾BB肌球蛋白，其在BB的末端网结构域中表达。 这将包括继续对鸟类BB进行生物化学和功能研究 肌球蛋白I 这种单头肌球蛋白，其轻链（lc）， 多种钙调素（CM），形成横向连接的桥梁， 微绒毛（MV）肌动蛋白核心的膜。 三组实验是 提出了 一个将调查CM相互作用的分子基础， BB肌球蛋白重链（hc），以及CM调节 这种酶的酶促、机械化学和膜结合活性 肌球蛋白 第二个目标将是阐明一个基因的调控功能， 最近发现的激酶磷酸化BB肌球蛋白的尾部结构域 我知道。 最后，我们计划识别和表征MV膜 作为BB肌球蛋白I受体的蛋白质。 第二大项目 是基于从人类肠道中提取的亚克隆 细胞系Caco-2可能表达多种非常规肌球蛋白， 到BB肌球蛋白I。 这些肌球蛋白的克隆策略进行了概述。 一旦克隆成功，我们计划通过以下方法来探测这些肌球蛋白在体内的功能： 截短（无头）肌球蛋白在转染的Caco-2细胞中的表达。 最后，我们概述了一项试点研究，我们希望在其中建立 应用遗传学和分子遗传学方法的可行性 剖析BB细胞骨架蛋白功能。 为此，我们建议 形成细胞骨架的蛋白质的分子克隆 果蝇中肠BB--从MV核心蛋白绒毛开始。

The studies proposed will continue our efforts to characterize the actin- based cytoskeleton of the intestinal epithelial cell and its apical brush border (BB). These studies will be pursued with the dual goals of providing general insights into the actin based cytoskeleton of nonmuscle cells, and also to dissect the roles of actin and its associated binding proteins in the physiologic functions of the enterocyte. Our primary focus of study during this grant period will be on the family of unconventional myosins--that is, myosins other than the conventional, two-headed and tailed BB myosin which is expressed in the terminal web domain of the BB. This will include continued biochemical and functional studies on avian BB myosin I. This single-headed myosin, that has as its light chains (lc), multiple calmodulins (CM), forms the bridges which laterally tether the microvillar (MV) actin core to the membrane. Three sets of experiments are proposed. One will investigate the molecular basis for CM interaction with BB myosin heavy chain (hc), as well as the mechanism by which CM regulates the enzymatic, mechanochemical and membrane binding activities of this myosin. A second goal will be to elucidate the regulatory functions of a recently discovered kinase that phosphorylates the tail domain of BB myosin I hc. Finally, we plan to identify and characterize the MV membrane protein that serves as a receptor for BB myosin I. A second major project is based on the discovery that subclones derived from the human intestinal cell line, Caco-2, may express multiple unconventional myosins in addition to BB myosin I. Strategies for the cloning of these myosins are outlined. Once cloned, we plan to probe the function of these myosins in vivo through expression of truncate (headless) myosins in transfected Caco-2 cells. Finally, we outline a pilot study in which we hope to establish the feasibility of applying genetic and molecular genetic approaches to dissecting BB cytoskeletal protein function. To this end we propose the molecular cloning of the proteins which form the cytoskeleton of the Drosophila midgut BB--beginning with the MV core protein villin.