Polymorphonuclear leukocytes (PMNs) and macrophages required a contractile apparatus for shape changes and motility. Both purified actin-regulatory proteins and intact phagocytes will be studied to understand of how these cells regulate the dynamic changes in actin filament concentration associated with their movements. I) Actin-regulatory proteins purified from macrophages: A) The 41K protein is a Ca2+-sensitive monomer sequestering and capping protein which may be phosphorylated in the cell. The affinities of 41K protein for actin monomers and actin filament ends will be determined using the fluorescent probe pyrenyl-actin. The effects of changes in ionized calcium on end-blocking and monomer sequestering will be examined using Ca/EGTA solutions as well as a calcium electrode. The 41K protein's affinity for Ca2+ will be determined by equilibrium dialysis with Ca45. In vivo phosphorylation of 41K will be examined using P32 labeled macrophages and immunoprecipitation as well as immunoblots of two dimensional electrophoretic gels. The effects of in vitro phosphorylation on 41K function will also be examined; b) Acumentin, a very labile end blocking protein, will be studied using pyrenyl-actin depolymerization and elongation assays to determine if this protein blocks barbed filament ends; c) Profilin, an actin monomer sequestering protein which may also directly interact with filament, will be combined with actin monomers and barbed-end capped filaments to study profilin's effects on pointed end assembly. Limulus acrosomal actin bundles will be used to measure how effectively this protein can block actin assembly at the barbed filament end. Profilin also exists as a high affinity complex with actin. Ion exchange and gel filtration chromatography will be used to attempt to purify a cofactor or cofactors which may regulate profilin-actin conversion from a moderate to a high affinity complex. II) Actin filament assembly and profilin function in intact PMN Triton-soluble extracts in combination with poly-L-proline and DNAase I-conjugated sephadex beads as well as pyrenyl actin will be used to assess the relative importance of the various actin-modulating protein in controlling the large actin monomer pool found in PMNs. The concentrations of high affinity profilin-actin complex as well as actin sequestering activity will be measured before and after chemotactic stimulation. Recent evidence suggest profilin may no account for the high actin monomer content in PMNs. We will look for other proteins which may also sequester actin monomers and attempt to purify them using HPLC ion exchange chromatography. III) Finally the severe PMN motility disorder associated with defective actin assembly, called neutrophil actin dysfunction, will be reinvestigated. The possibility that this disorder is caused by a point mutation in the beta acting gene will be studied by cloning and sequencing techniques. Understanding how actin functions in phagocytes may result in a better understanding of motile processes in other cells, in particular platelets, endothelial and invasive neoplastic cells. This knowledge may also lead to the development of new methods for altering leukocyte movement and thereby improvement ability to enhance host defense and control inflammation.
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