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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036507-03
Application #
3290603
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1986-12-01
Project End
1989-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
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
Hartwig, J H; Chambers, K A; Hopcia, K L et al. (1989) Association of profilin with filament-free regions of human leukocyte and platelet membranes and reversible membrane binding during platelet activation. J Cell Biol 109:1571-9
Hartwig, J H; Chambers, K A; Stossel, T P (1989) Association of gelsolin with actin filaments and cell membranes of macrophages and platelets. J Cell Biol 108:467-79
Hartwig, J H; Janmey, P A (1989) Stimulation of a calcium-dependent actin nucleation activity by phorbol 12-myristate 13-acetate in rabbit macrophage cytoskeletons. Biochim Biophys Acta 1010:64-71
Hartwig, J H; Yin, H L (1988) The organization and regulation of the macrophage actin skeleton. Cell Motil Cytoskeleton 10:117-25
Ezzell, R M; Kenney, D M; Egan, S et al. (1988) Localization of the domain of actin-binding protein that binds to membrane glycoprotein Ib and actin in human platelets. J Biol Chem 263:13303-9
Yin, H L; Hartwig, J H (1988) The structure of the macrophage actin skeleton. J Cell Sci Suppl 9:169-84