MEMBRANE CYTOSKELETON INTERACTIONS IN NEUTROPHILS

对中性粒细胞（PMNs）和其他细胞的大量研究表明， 细胞骨架是运动相关过程的中心， 和扩散，细胞迁移，趋化性，吞噬作用，内吞作用， 胞吐作用 更具体地说，这些过程似乎需要 表面膜和底层之间的联系 含肌动蛋白的细胞骨架。 然而，这些分子的细节 一般缺乏互动。 如果不详细了解 膜蛋白-细胞骨架蛋白相互作用，我们不能评估 这些相互作用在细胞对外界环境的反应中的重要性 环境 在拟议的研究中，我们将研究 膜蛋白与肌动蛋白为基础的细胞骨架在人类中性粒细胞。 我们有 选择的膜蛋白，可能直接或间接参与 细胞附着在基质上，因为这似乎需要 膜细胞骨架的相互作用，是一个必要的步骤， 迁移到炎症部位。 我们将重点关注两个 140 KD的糖蛋白（与PMN不同 粘附糖蛋白MO-1和p150，95），以及结合 细胞外基质蛋白血小板反应蛋白（TSP）。 的140 KD蛋白是我们特别感兴趣的，因为它是减少在中性粒细胞 分离自表现出白细胞粘附缺陷（MO-1）的患者 缺陷）。 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启动或修改在细胞 activation. 该提案的具体目标是：（1）确定和 使用细胞表面标记表征表面膜蛋白 技术，特异性凝集素结合，TSP亲和层析，和 用于细胞粘附、细胞铺展和O2产生的功能测定;（2） 评估表面膜蛋白和细胞膜蛋白之间的关系， 细胞骨架在稳态和配体激发（激活）的中性粒细胞使用 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 膜相关的细胞骨架在稳态和激活的中性粒细胞使用 免疫印迹、凝胶覆盖和免疫沉淀技术;（4） 从稳态和活化中分离特异性表面膜蛋白 使用亲和层析，凝胶过滤， 蔗糖梯度离心和免疫沉淀程序;和（5） 为了表征这些分离的蛋白质与F-肌动蛋白的相互作用， 直接可视化、共沉降、粘滞性和芘-肌动蛋白 测定。 我们的最终目标是确定这些膜细胞骨架 相互作用导致基于运动性的功能，如PMN粘附， 传播，以及这些相互作用的改变如何导致特定的 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.