Polymorphonuclear leukocytes (PMN) and macrophages, also called phagocytes, must regulate actin filament assembly in order to change shape, ingest particles, release granules and crawl to the sites of infection. A critical control point in the regulation of actin assembly in motile cells is the capping and uncapping of the barbed or (+) ends of actin filaments. Two barbed end capping protein likely to play key roles in the control of actin assembly during phagocyte movement will be investigated: 1. Macrophage capping protein (MCP_: This 38 kDa, Ca2+-sensitive protein caps the barbed ends of actin filaments, but does not sever them. MCP is the most abundant actin-binding protein in macrophages, representing 1% of the total cytoplasmic protein. Using immunofluorescence and confocal microscopy as well as immunogold electron microscopy, MCP's location in """"""""resting' and stimulated macrophages will be examined. In vivo Ca2+- sensitivity will be examined by studying MCP localization before and after intracellular Ca2+ is lowered by EGTA/AM treatment. To examine the in vivo effects of MCP, human MCP cDNA will be permanently transfected into monocyte cell liens, u937 and J774, using the beta-actin promoter driven lK4444 vector. The ability of transfected cells to crawl, degranulate and phagocytose will then be studied. Genomic MCP DNA has been cloned from a human placenta genomic library and is presently being sequenced. The MCP gene is being localized to a specific chromosomal site. Sequence analysis of human MCP cDNA reveals that MCP is a member of the gelsolin/villin protein family. Mutations in MCP cDNA based on primary structural comparisons to gelsolin and villin are being introduced by PCR. A mutant MCP protein has been expressed in E. Coli which has a new function, being capable of severing as well as capping. This accomplishment is unprecedented in the actin-binding protein field and will allow exploration of the interrelationships between monomer binding, capping and severing using the fluorescent probe, pyrenyl actin. X-ray crystallographic analysis of recombinant MCP and the severing mutant will complement our functional studies by allowing assessment of tertiary structure and actin contact sites. II. PMN actin polymerization inhibitor (Annexin VI). This 65kDa protein binds membrane lipids and also caps the barbed ends of actin filaments. Annexin VI represents 3-4% of the total protein in PMN and is likely to play an important role in agonist mediated membrane-actin interactions. The ability of this protein to sequester actin monomers and to sever actin filaments will be studied using pyrenyl actin. Confocal immunofluorescence will be used to localize this protein in PMN before and after stimulation. Annexin VI phosphorylation will be studied using immunoprecipitation and p32 labeling. In addition to providing a better understanding of phagocyte motility, these studies promise to provide new insights into macrophage development, the behavior of metastatic cancer cells, control of inflammation nd host defense.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI023262-10
Application #
2062113
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1985-08-01
Project End
1998-12-31
Budget Start
1995-01-01
Budget End
1995-12-31
Support Year
10
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Florida
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
During, Russell L; Gibson, Bruce G; Li, Wei et al. (2007) Anthrax lethal toxin paralyzes actin-based motility by blocking Hsp27 phosphorylation. EMBO J 26:2240-50
Zhang, Y; Vorobiev, Sergey M; Gibson, Bruce G et al. (2006) A CapG gain-of-function mutant reveals critical structural and functional determinants for actin filament severing. EMBO J 25:4458-67
Sidhu, Gurjit; Li, Wei; Laryngakis, Nicholas et al. (2005) Phosphoinositide 3-kinase is required for intracellular Listeria monocytogenes actin-based motility and filopod formation. J Biol Chem 280:11379-86
Larson, Laura; Arnaudeau, Serge; Gibson, Bruce et al. (2005) Gelsolin mediates calcium-dependent disassembly of Listeria actin tails. Proc Natl Acad Sci U S A 102:1921-6
During, Russell L; Li, Wei; Hao, Binghua et al. (2005) Anthrax lethal toxin paralyzes neutrophil actin-based motility. J Infect Dis 192:837-45
Parikh, Shefal S; Litherland, Sally A; Clare-Salzler, Michael J et al. (2003) CapG(-/-) mice have specific host defense defects that render them more susceptible than CapG(+/+) mice to Listeria monocytogenes infection but not to Salmonella enterica serovar Typhimurium infection. Infect Immun 71:6582-90
Southwick, Frederick S; Li, Wei; Zhang, Fangliang et al. (2003) Actin-based endosome and phagosome rocketing in macrophages: activation by the secretagogue antagonists lanthanum and zinc. Cell Motil Cytoskeleton 54:41-55
Bubb, Michael R; Yarmola, Elena G; Gibson, Bruce G et al. (2003) Depolymerization of actin filaments by profilin. Effects of profilin on capping protein function. J Biol Chem 278:24629-35
Zhang, Fangliang; Southwick, Frederick S; Purich, Daniel L (2002) Actin-based phagosome motility. Cell Motil Cytoskeleton 53:81-8
Witke, W; Li, W; Kwiatkowski, D J et al. (2001) Comparisons of CapG and gelsolin-null macrophages: demonstration of a unique role for CapG in receptor-mediated ruffling, phagocytosis, and vesicle rocketing. J Cell Biol 154:775-84

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