MECHANISM AND REGULATION OF ACTIN-BASED RETROGRADE FLOW

基于肌动蛋白的逆行流的机制和调控

• 批准号: 6082677
• 负责人: JOHN H HENSON
• 金额: $ 13.18万
• 依托单位: DICKINSON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6082677
• 关键词: actin binding protein; actins; cell motility; cytoskeleton; electron microscopy; enzyme activity; guanine nucleotide binding protein; microfilaments; micromanipulator; microtubules; myosins; protein kinase; sea urchins; second messengers; wound healing

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.

细胞运动的各个方面在细胞循环，分裂，迁移，膜运输和信号转导中起着基本作用，所有这些都对正常的细胞生长，发育和分化至关重要。 细胞运动是由统称为“细胞骨架”的许多结构和运动蛋白介导的。 逆行流是由肌动蛋白蛋白细胞骨架介导的细胞运动的一种形式，在真核细胞中广泛。 流动过程涉及层状质膜，膜蛋白和基础肌动蛋白细胞骨架的持续运动，从细胞周围向细胞中心。 该现象已被假定是在细胞易位，细胞迁移靶向以及形态发生方面的功能。 逆行流量仅在几种细胞类型（神经元，鱼角膜细胞和哺乳动物成纤维细胞）中进行了广泛的研究，尽管如此，尽管如此，这种基本过程的确切机制和调节仍然很大程度上是未知的。 该建议概述了一系列旨在使用独特的实验模型，海胆核细胞来阐明逆行流量的机理和调节的一系列实验。 这些细胞表现出高度夸张的流动形式，并具有许多特性，使它们非常适合本研究，包括其光学特性，细胞骨架蛋白的免疫学探针的可用性以及可以启动并停止流动的准备和细胞骨骼组织的准备就绪。 光和电子显微镜方法，结合药理学和微量渗透方法将用于解决以下特定目的：1。确定肌动蛋白丝之间的结构和功能关系，肌动蛋白丝与肌动蛋白结合和运动蛋白之间的结构和功能关系，以及肌动蛋白丝和肌动蛋白丝之间的肌动蛋白和运动蛋白之间的结构和功能关系。 2。确定细胞内第二信使的调节作用，蛋白激酶/磷酸酶活性以及单体GTPase蛋白的Rho家族在逆行流动过程中。 3。分析逆行流量和细胞骨架动力学在细胞伤口愈合中的作用。