MECHANISM AND REGULATION OF ACTIN-BASED RETROGRADE FLOW

细胞运动在细胞周期、分裂、迁移、膜运输和信号转导等过程中起着重要作用，所有这些都是细胞正常生长、发育和分化所必需的。细胞的运动是由一系列结构和运动蛋白介导的，统称为“细胞骨架”。逆行流动是一种由肌动蛋白细胞骨架介导的细胞运动形式，广泛存在于真核细胞中。这一流动过程涉及膜脂质膜、膜蛋白和潜在的肌动蛋白细胞骨架从细胞外围向细胞中心的持续运动。这一现象被认为在细胞移位、细胞迁移靶向和形态发生等方面发挥作用。仅在少数细胞类型(神经元、鱼类角质形成细胞和哺乳动物成纤维细胞)中对逆行流动进行了广泛的研究，尽管有这些努力，但这一基本过程的确切机制和调控在很大程度上仍不清楚。这项建议概述了一系列实验，旨在利用独特的实验模型--海胆体腔细胞--阐明逆行流动的机制和调节。这些细胞表现出一种高度夸张的流动形式，并拥有许多使它们非常适合于这项研究的特性，包括它们的光学特性，细胞骨架蛋白的免疫探针的可用性，以及流动可以开始和停止以及细胞骨架组织可以改变的准备情况。光镜和电子显微镜结合药理学和显微操作的方法将被用来解决以下具体目标：1.确定细胞逆行流动过程中肌动蛋白细丝之间、肌动蛋白结合蛋白和运动蛋白之间以及肌动蛋白细丝和微管之间的结构和功能关系。2.确定细胞内第二信使、蛋白激酶/磷酸酶活性和Rho家族单体GTP酶蛋白在逆行血流过程中的调节作用。3.分析逆流和细胞骨架动力学在细胞创伤修复中的作用。

Aspects of cell motility play fundamental roles in cell cycling, division, migration, membrane transport and signal transduction, all of which are essential for normal cell growth, development and differentiation. Cell motility is mediated by a host of structural and motor proteins known collectively as the "cytoskeleton". Retrograde flow is a form of cell motility that is mediated by the actin protein cytoskeleton and is widespread in eukaryotic cells. The flow process involves the continual movement of the lamellipodial plasma membrane, membrane proteins and underlying actin cytoskeleton from the cell periphery towards the cell center. This phenomenon has been postulated as functioning in cellular translocation, in the targeting of cellular migrations, and in aspects of morphogenesis. Retrograde flow has been extensively studied in only a few cell types (neurons, fish keratocytes and mammalian fibroblasts) and, despite these efforts, the exact mechanism and regulation of this fundamental process is still largely unknown. This proposal outlines a series of experiments aimed at elucidating the mechanism and regulation of retrograde flow using a unique experimental model, the sea urchin coelomocyte. These cells display a highly exaggerated form of flow and possess a number of properties which make them well suited for this study, including their optical properties, the availability of immunological probes for cytoskeletal proteins, and the readiness with which flow can be started and stopped and the cytoskeletal organization can be altered. Light and electron microscopic methods, combined with pharmacological and micromanipulation approaches will be used to address the following Specific Aims: 1. To determine the structural and functional relationships between actin filaments, between actin filaments and actin-binding and motor proteins, and between actin filaments and microtubules in cells undergoing retrograde flow. 2. To determine the regulatory roles of intracellular second messengers, protein kinase/phosphatase activity, and the Rho family of monomeric GTPase proteins on the process of retrograde flow. 3. To analyze the role of retrograde flow and cytoskeletal dynamics in cell wound healing.