Abstract

Polymorphonuclear leukocytes (PMNs) and macrophages require a contractile apparatus for shape changes, movement, and phagocytosis. The major component of this motor is actin. Actin can exist as monomers or as filaments of varying lengths. Changes in actin filament concentration and length underlie the ability of these cells to move. The present studies should begin to clarify how phagocytes control actin filament assembly in their cytoplasm. (1) Studies of acumentin will be continued. This 65,000 dalton protein purified from macrophages and human granulocytes controls actin assembly by blocking actin monomer exchange at the low affinity end of actin filaments. Conditions which enhance and inhibit this activity will be assessed by viscometry. Acumentin's binding affinity for actin will be measured using a radiolabel sedimentation assay. Its presence in other cells and tissues will be demonstrated by nitrocellulose immunoblot. Acumentin's localization in phagocytes will be visualized by immunofluorescence (2) Actin assembly in various combinations of macrophage actin-modulating proteins (acumentin, profilin, gelsolin and any newly purifed proteins) will be studied by light acattering, pyrenyl-actin, DNAse I inhibition, and viscosity. (3) To see if recombination studies mimic whole extracts, PMN and macrophage extracts will be studied using a new fluorescent probe, pyrenyl-actin. Preliminary studies suggest the presence of a new calcium sensitive actin-modulating protein whose function is different than gelsolin's. (4) This new calcium sensitive regulatory protein will be purified by ion exchange and gel filtration chromatography. (5) A granulocyte actin polymerization defect discovered in two male children will be studied. These children also had a severe ultimately fatal PMN chemotactic defect. Actin will be purified from the granulocytes of one child's parents and subjected to thin layer and high pressure liquid chromatography peptide mapping. Understanding how actin works in phagocytes may lead to new methods for altering leukocyte movement and thereby improve our ability to enhance host defence and control inflammation.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI023262-03
Application #
3135160
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1985-08-01
Project End
1988-07-31
Budget Start
1987-08-01
Budget End
1988-07-31
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost

  Institution

Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104

  Publications

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

Showing the most recent 10 out of 26 publications