Proteins involved in the attachment of actin to cell membranes, particularly plasma membranes, will be investigated. Two types of actin-membrane association will be analyzed: (1) that which may involve a class of proteins found in many cell types that we and others have shown to be closely related to erythrocyte spectrin, and (2) that which occurs in specialized regions of membranes, such as in fibroblast adhesion plaques, where bundles of actin filaments terminate. A strategy of sequential analysis will be used to identify and characterize the components that link actin to the membrane in these different locations. During the investigation of non-erythrocyte spectrins we will attempt to identify proteins involved in linking spectrin itself to membranes. A variety of biochemical, immunological and electron microscopic approaches will be used to define the relationship between spectrin and cell surface and membrane components in non-erythroid cells. The role of spectrin in regulating the lateral mobility and distribution of cell surface components will be studied using fluorescence recovery after photobleaching and other techniques. We will try to dissociate spectrin from the plasma membrane by microinjecting live cells with antibodies against spectrin or against proteins to which it binds. Regarding the proteins involved in attachment of actin microfilament bundles to the plasma membrane, we will concentrate on the properties of vinculin and 215K, a new protein that we have recently identified infibroblast adhesion plaques. New proteins interacting with vinculin and 215K will be identified using affinity chromatography and chemical cross-linking reagents. In an attempt to develop a new model system for examining """"""""end-on"""""""" attachment of actin filaments to membranes, we will isolate thefascia adherens from cardiac muscle. This structrure is responsible for the membrane anchorage of actin in cardiac myofibrils. Again a strategy of sequential analysis will be used to define the components involved in attaching the actin filaments to this membrane.
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