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.
