Cytoskeleton - Membrane Interactions

描述（由申请人提供）：肌动蛋白细胞骨架和富含胆固醇的液体有序膜结构域（也称为“脂筏”）均参与细胞粘附、运动、存活和膜运输过程中的信号转导。然而，潜在的分子机制尚不清楚。本申请提出了一种新型的膜骨架，首先描述和表征在当前的资助期，这可能是一个“缺失的环节”，在了解肌动蛋白和肌球蛋白参与脂筏信号的继续研究。由于脂筏组织蛋白（漂浮蛋白、气孔蛋白）和信号蛋白（Src家族激酶、异源三聚体Gi蛋白、基质金属蛋白酶）与丝状肌动蛋白、肌球蛋白I和II以及其他膜骨架蛋白（α-辅肌动蛋白、胞衬蛋白、超绒毛蛋白）的紧密结合，含有这种膜骨架的耐洗涤剂膜（DRM-H）表现出比其他富含胆固醇的膜组分更高的浮力密度。DRM-H相关的膜骨架存在于许多运动细胞中，并明显调节细胞粘附、收缩性和细胞外调节激酶（ERK 1/2）的信号传导。 我们提出DRM-H膜骨架通过促进脂筏相关蛋白的局部重排参与Src和/或ERK信号传导。超绒毛蛋白，一种近膜蛋白，也直接结合到肌动蛋白和肌球蛋白II，似乎是这种调节的关键控制点。为了验证这些假设，我们建议：（1）阐明肌动蛋白，肌球蛋白，超绒毛蛋白，和其他DRM-H蛋白的信号支架的形成和重组的作用;（2）探索DRM-H蛋白调节粘着斑功能和细胞收缩性的机制;（3）确定DRM-H膜骨架在膜运输过程中的作用。这些研究描述了一种新的机制，肌动蛋白和肌球蛋白II的附着富含胆固醇的膜结构域，并将提供深入了解这些领域的细胞骨架调节机制。这个项目的长期目标是了解肌动蛋白为基础的膜骨架如何在细胞运动，粘附和信号传导过程中发挥作用。这些信息将增加我们对正常细胞行为和与免疫功能障碍、癌细胞侵袭、肌营养不良、糖尿病和发育异常相关的病理状况的理解。

DESCRIPTION (provided by applicant): Both the actin cytoskeleton and cholesterol-rich liquid-ordered membrane domains, also called 'lipid rafts', are involved in signal transduction during cell adhesion, motility, survival, and membrane trafficking. However, the underlying molecular mechanisms are unclear. This application proposes the continued study of a new type of membrane skeleton, first described and characterized in the current funding period, that may represent a 'missing link' in understanding actin and myosin involvement in lipid raft signaling. Detergent-resistant membranes containing this membrane skeleton (DRM-H) exhibit a higher buoyant density than do other cholesterol-rich membrane fractions due to the tight association of lipid raft organizing proteins (flotillins, stomatin) and signaling proteins (Src family kinases, heterotrimeric Gi proteins, matrix metalloproteinase) with filamentous actin, myosins I and II, and other membrane skeleton proteins (alpha-actinin, fodrin, supervillin). DRM-H-related membrane skeletons are present in many motile cells and apparently regulate cell adhesion, contractility, and signaling to extracellularly regulated kinases (ERK1/2). We propose that the DRM-H membrane skeleton is involved in Src and/or ERK signaling by promoting local rearrangements of lipid raft-associated proteins. Supervillin, a membrane-proximal protein that also binds directly to actin and myosin II, appears to be a key control point for this regulation. To test these hypotheses, we propose to: (1) elucidate the roles of actin, myosin, supervillin, and other DRM-H proteins in the formation and reorganization of signaling scaffolds; (2) explore the mechanisms by which DRM-H proteins modulate focal adhesion function and cellular contractility; and (3) determine the role of the DRM-H membrane skeleton during membrane trafficking. These studies describe a novel mechanism for the attachment of actin and myosin II to cholesterol-rich membrane domains and will provide insight into cytoskeletal regulatory mechanisms in these domains. The long-range goal of this project is to understand how actin-based membrane skeletons function during cell motility, adhesion, and signaling. This information will increase our understanding of both normal cellular behaviors and pathological conditions associated with immune dysfunction, cancer cell invasion, muscular dystrophy, diabetes, and developmental abnormalities.