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.
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