MEMBRANE CYTOSKELETON INTERACTIONS IN NEUTROPHILS

对中性粒细胞(PMN)和其他细胞的大量研究表明 细胞骨架是黏附等运动相关过程的核心。 和扩散，细胞迁移，趋化，吞噬，内吞，和 胞吐。更具体地说，这些进程似乎需要 表膜和下层膜之间的联系 含肌动蛋白的细胞骨架。然而，这些分子的细节 一般情况下，缺乏互动。在没有详细了解的情况下 膜蛋白-细胞骨架蛋白的相互作用，我们不能评估 这些相互作用在细胞对其外部环境的反应中的重要性 环境。在拟议的研究中，我们将研究 人中性粒细胞中以肌动蛋白为基础的细胞骨架的膜蛋白。我们有 可能直接或间接参与的选定的膜蛋白 细胞附着到底物上，因为这似乎需要 膜-细胞骨架相互作用是PMN的必要步骤 迁移到发炎的地方。我们的注意力集中在两个方面 膜蛋白的类型，一种140KD的糖蛋白(与PMN不同 粘附性糖蛋白MO-1和p150，95)和膜蛋白(S)结合 细胞外基质蛋白，血小板凝血酶敏感蛋白(TSP)。140岁的 KD蛋白是我们特别感兴趣的，因为它在PMN中被还原 分离自一例表现为白细胞黏附缺陷(MO-1)的患者 不足)。我们的目标是确定膜蛋白如何与 肌动蛋白，以及这种关联是如何在细胞期间启动或修改的 激活。该提案的具体目标是：(1)确定和 用细胞表面标记技术表征表膜蛋白 技术、特异性凝集素结合、TSP亲和层析和 细胞黏附、细胞铺展和产氧量的功能测定； 目的：探讨细胞表膜蛋白与细胞周期的关系。 细胞骨架在稳态和配体挑战(激活)的PMN中的应用 SDS-PAGE和放射自显影，双标记免疫荧光显微镜， 和肌动蛋白聚合试验；(3)鉴定 膜相关细胞骨架在稳态和激活的PMN中的应用 免疫印迹、凝胶覆盖和免疫沉淀技术； 从稳态和活化状态中分离特定表面膜蛋白 使用亲和层析、凝胶过滤、 蔗糖梯度离心法和免疫沉淀法；以及(5) 研究这些分离蛋白与F-肌动蛋白的相互作用 直接观察法、共沉淀法、粘度法和芘-肌动蛋白 化验。我们的最终目标是确定这些膜细胞骨架是如何 相互作用导致基于运动的功能，如PMN黏附和 传播，以及这些相互作用中的变化可能如何导致特定的 疾病状态。

Numerous studies on neutrophils (PMNs) and other cells suggest that the cytoskeleton is cental to motility associated processes such as adhesion and spreading, cell migration, chemotaxis, phagocytosis, endocytosis, and exocytosis. More specifically, these processes appear to require an association between the surface membrane and the underlying actin-containing cytoskeleton. However, the molecular details of these interactions are generally lacking. Without a detailed knowledge of membrane protein-cytoskeletal protein interactions, we cannot evaluate the importance of these interactions in the cell's response to its external environment. In the proposed study, we will examine the interaction of membrane proteins with the actin-based cytoskeleton in human PMNs. We have selected membrane proteins that may be directly or indirectly involved in cell attachment to a substrate, since this appears to require membrane-cytoskeleton interactions and is a requisite step for PMN migration to a site of inflammation. We will focus our attention on two types of membrane proteins, a 140KD glycoprotein (distinct form the PMN adhesive glycoproteins MO-1 and p150,95), and membrane protein(s) that bind the extracellular matrix protein, platelet thrombospondin (TSP). The 140 KD protein is of particular interest to us because it is reduced in PMNs isolated from a patient exhibiting leukocyte adhesion deficiency (MO-1 deficient). Our goal is to determine how membrane proteins interact with actin, and how this association is initiated or modified during cell activation. The specific aims of the proposal are: (1) to identify and characterize surface membrane proteins using cell surface labeling techniques, specific lectrin binding, TSP affinity chromatography, and functional assays for cell adhesion, cell spreading and O2 production; (2) to evaluate the relationship between surface membrane proteins and the cytoskeleton in steady-state and ligand-challenged (activated) PMNs using SDS-PAGE and autoradiography, double label immunofluorescence microscopy, and actin polymerization assays; (3) to identify components of the membrane-associated cytoskeleton in steady-state and activated PMNs using immunoblotting, gel overlay and immunoprecipitation techniques; (4) to isolate specific surface membrane proteins from steady-state and activated PMNs using a combination of affinity chromatography, gel filtration, sucrose gradient centrifugation and immunoprecipitation procedures; and (5) to characterize the interaction of these isolated proteins with F-actin by direct visualization, co-sedimentation, viscometry, and pyrene-actin assays. Our eventual goal is to determine how these membrane-cytoskeleton interactions lead to motility-based functions such as PMN adhesion and spreading, and how alterations in these interactions might lead to specific disease states.