The research objective is to elucidate the molecular mechanism by which calcium-dependent changes in actin filament length regulates the defense functions of lung macrophages, and the clearance of actin filaments from the blood stream. Particular emphasis will be focused on a 90 kDa, Ca2+ binding protein first identified in rabbit lung macrophages, and subsequently extracellularly in blood plasma as a molecular variant. This protein, called gelsolin, fragments actin filaments in the presence of MuM Ca2+. Through regulation of actin filament length, cytoplasmic gelsolin can cause gel-sol transformation of the cytoplasm and is, therefore, likely to be a key control point for many cytological events. Extracellularly, through shortening of actin filaments and/or interaction with other plasma proteins, plasma gelsolin may contribute to maintenance of hemostasis by a novel mechanism. In the first aspect of this proposal, research will be directed at examining the relation between the structure and function of gelsolin. Gelsolin will be subjected to limited proteolysis to identify the part of the molecule involved in binding to actin, and to explore the basis for activation of gelsolin by Ca2+. The second aspect of this proposal will examine the role of gelsolin in regulating shape changes and movement of the phagocytes. Particular emphasis will be placed on comparing gelsolin-actin interaction in resting and activated cells, its reversibility and dependence on [Ca2+]i, and identifying the mechanisms for maintaining gelsolin homeostasis. The third aspect of the work will determine how synthesis and secretion of gelsolin can be regulated. The possibility of feedback regulation on synthesis of both cytoplasmic and plasma gelsolin, and the steps involved in processing of gelsolin for secretion will be studied to shed light on how the proteins are regulated in health and disease. The fourth aspect will examine the molecular biology of the gelsolin gene, to complement approaches from biochemical and cell biological perspectives. The fact that gelsolin is a unique example of a mammalian protein with molecular variants for cytoskeletal and secretory purposes makes study of how gelsolin variants are derived and regulated at the gen level particularly interesting, and our findings may have far reaching ramifications.
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