Macrophages are host defense cells that move to, ingest, and kill airborn pathogens. These motile functions may be mediated in part by actin interacting with a number of actin-associated proteins. The research objective is to elucidate the mechanism by which one of these proteins, macrophage actin-binding protein (ABP), crosslinks actin filaments. Actin is the most abundant component of macrophage cortical cytoplasm and is organized into isotropic filament networks of definite rigidity. The crosslinking of actin filaments by ABP alters the mechanical properties of actin filaments and may play a role in defining their ultrastructure in cells. ABP is a large, extended molecule that has binding sites for actin near each of its molecular ends. In vitro, it modifies the architecture of actin filaments in solution such that networks in which filaments connect at perpendicular angles are formed. These networks are very similar in structure to actin within the cortical cytoplasm of cells. Particular emphasis will be placed in this proposal on studying the mechanism by which ABP generates the perpendicular branching of actin filaments. To accomplish this, ABP's (1) native conformation in solution, (2) primary structure, and (3) regulation in vivo will be studied. The first part of this proposal concerns an analysis of the structure of ABP rapidly frozen in physiological solutions. The second part pertains to defining functional domains on ABP. ABP will be subject to limited proteolysis to identify the subunit domains involved in binding to actin and in self-association. Both purified ABP and its cleavage fragments will be sequenced from their amino-termini. A panel of monoclonal antibodies will be prepared against ABP and their binding location on ABP domains determined in the electron microscope. Lastly, ABP's interaction with actin will be studied in resting and activated cells to identify mechanism(s) of control in vivo.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Cytology Study Section (CTY)
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Massachusetts General Hospital
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Hartwig, J H; Kwiatkowski, D J (1991) Actin-binding proteins. Curr Opin Cell Biol 3:87-97
Hartwig, J H; DeSisto, M (1991) The cytoskeleton of the resting human blood platelet: structure of the membrane skeleton and its attachment to actin filaments. J Cell Biol 112:407-25
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Yin, H L; Hartwig, J H (1988) The structure of the macrophage actin skeleton. J Cell Sci Suppl 9:169-84