The research objective is to elucidate the molecular mechanisms by which Ca2+- and polyphosphoinositide (PPI)-dependent changes in actin filament length regulate the defense functions of lung macrophages. Particular emphasis will be focused on gelsolin, a 80 kDa PPI- and Ca binding protein first identified in rabbit lung macrophages. Gelsolin fragments actin filaments in the presence of uM Ca2+, and its activity is inhibited by PPI. Through regulation of actin filament length and polymerization, cytoplasmic gelsolin can cause reorganization of the actin cytoskeleton. Since both Ca2+ and PPI levels change transiently in cells following agonist stimulation, gelsolin may be the key control point in many cytological events. First, we will continue to characterize the functional domains of gelsolin. We have identified by limited proteolysis three actin binding sites and distinct Ca2+ and PPI- regulatory sites as well. We will focus on a gelsolin domain (designated CT28N) which binds to the side of actin filaments causing distortions which may contribute to PPI-inhibitable severing. We will study the effect of CT28N on actin filament morphology, and identify its PPI- and actin binding sites by NTCB cleavage. We will identify interactive sites between actin and gelsolin's 3 actin binding domains by chemical crosslinking, and prepare gelsolin crystals for analysis by X-ray crystallography. Second, we will use recombinant DNA technology to obtain further information about the boundaries as well as the interactions between gelsolin domains. Initially, mutated gelsolins with end deletions will be expressed in eukaryotic cells by gene transfection. We will capitalize on the fact that a gelsolin variant is secreted, so that it can be analyzed in the culture medium directly. Subsequently, specific alterations will be made by site-directed mutagenesis. Third, we will over/underexpress cytoplasmic gelsolin to assess its role in vivo. Gelsolin sense and antisense DNA will be transfected into fibroblasts and macrophage-like cells, and changes in cell shape and motility determined. We will look for compensatory changes in actin and other cytoskeletal proteins, to determine if there are mechanisms for maintaining a proper balance between them. Fourth, we will determine if the expression of cytoplasmic gelsolin changes under physiological and pathological conditions to gain further insight about its role in the cytoplasm. Our data indicate that cytoplasmic gelsolin expression decreases during the S phase of cell cycle, and after A23187 or cycloheximide treatment. The molecular basis for these changes will be determined.
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