Membrane Polarization and Endothelial Cell Motility

膜极化和内皮细胞运动

• 批准号: 6859334
• 负责人: PAUL L FOX
• 金额: $ 38.25万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6859334
• 关键词: MCF7 cell; P glycoprotein; Sf9 cell line; actins; affinity chromatography; atomic force microscopy; autoradiography; caveolins; cell membrane; cell motility; cholesterol; fluorescence microscopy; fluorescence recovery after photobleaching; green fluorescent proteins; growth factor; immunoprecipitation; membrane activity; phosphorylation; polymerase chain reaction; steroid biosynthesis; vascular endothelium; viscosity; western blottings

DESCRIPTION (provided by applicant): Endothelial cell (EC) movement is initiated by angiogenic growth factors, which trigger a sequence of spatially polarized intracellular events including the activation of motility-regulating small GTPases and the assembly of actin-dependent, force-generating systems at the cell anterior. Our primary interest is the role of the plasma membrane in cell movement. We have shown that membrane microviscosity is a key determinant of motility, and that basic fibroblast growth factor increases EC plasma membrane microviscosity as measured by fluorescence recovery after photobleaching (FRAP). Spatial analysis shows a highly polarized gradient of microviscosity in plasma membranes of rapidly migrating EC, with a leading edge that is substantially more viscous than the trailing edge. An important role of cholesterol in generation of this membrane microviscosity gradient is suggested by an increase in cholesterol content of the membrane, by gradient reversal upon cholesterol removal, and by relocalization of a fluorescent cholesterol analog, NBD-cholesterol, to the front of moving ECs. In studies of the mechanism that drives membrane polarization we have observed that caveolin-1, an intracellular cholesterol transport protein, is also highly polarized and accumulates in the rear of migrating ECs. In studies of the mechanism by which membrane physical properties regulate motility, we have found that increased membrane microviscosity increases the binding of Racl to plasma membranes in the front of moving ECs. We have also found that the ability of actin to deform large unitamellar vesicles is decreased when microviscosity is high, i.e., at an elevated cholesterol-to-phospholipid ratio. From these data we propose as a hypothesis that angiogenic growth factors alter cholesterol synthesis and trafficking to increase the membrane microviscosity at the leading edge of the moving cell. We further propose that increased microviscosity increases cell movement by increasing Racl-binding to the plasma membrane and by altering the barrier properties to improve the efficiency by which actin filaments propel the cell forward. We will test this hypothesis in three Specific Aims: (1) Determine the molecular mechanism regulating polarization of membrane microviscosity during EC movement, (2) determine the mechanism by which microviscosity regulates Racl binding to membranes and (3) determine the role of membrane microviscosity in regulation of actin filament formation and function. The experiments will make use of cultured cells expressing GFP-tagged These studies will provide basic information on mechanisms regulating cell motility and may lead, in the long-term, to molecular strategies to inhibit or enhance cell migration. Pharmacological agents based on these results may be useful for inhibition of tumor angiogenesis or to enhance collateral blood vessel formation and the healing of synthetic vascular grafts.

描述（由申请人提供）： 内皮细胞（EC）运动是由血管生成生长因子启动的，其触发一系列空间极化的细胞内事件，包括运动调节小GTP酶的激活和肌动蛋白依赖性的力产生系统在细胞前部的组装。我们的主要兴趣是质膜在细胞运动中的作用。我们已经表明，膜微粘度是一个关键的决定因素的运动，和碱性成纤维细胞生长因子增加EC质膜微粘度测量荧光恢复后光漂白（FRAP）。空间分析显示了一个高度极化梯度的微粘度在质膜的快速迁移EC，与前缘，这是实质上更粘比后缘。胆固醇在产生这种膜微粘度梯度中的重要作用是由膜的胆固醇含量的增加，胆固醇去除后的梯度逆转，以及荧光胆固醇类似物NBD-胆固醇的重新定位到移动的EC的前面而提出的。在驱动膜极化的机制的研究中，我们已经观察到，小窝蛋白-1，细胞内胆固醇转运蛋白，也是高度极化的，并积累在迁移EC的后方。在膜物理性质调节运动性的机制的研究中，我们已经发现，增加的膜微粘度增加了Racl与移动EC前面的质膜的结合。我们还发现，当微粘度高时，肌动蛋白使大的单层囊泡变形的能力降低，即，胆固醇与磷脂的比例升高从这些数据中，我们提出了一个假设，即血管生成生长因子改变胆固醇的合成和运输，以增加膜微粘度在移动的细胞的前沿。我们进一步提出，增加微粘度增加细胞运动，通过增加Racl结合到质膜，并通过改变屏障特性，以提高效率，其中肌动蛋白丝推动细胞前进。我们将在三个具体目标中检验这一假设：（1）确定EC运动过程中调节膜微粘度极化的分子机制，（2）确定微粘度调节Racl与膜结合的机制，（3）确定膜微粘度在调节肌动蛋白丝形成和功能中的作用。这些实验将利用表达GFP标记的培养细胞。这些研究将提供有关调节细胞运动机制的基本信息，并可能导致长期抑制或增强细胞迁移的分子策略。基于这些结果的药理学试剂可能用于抑制肿瘤血管生成或增强侧支血管形成和合成血管移植物的愈合。