The human neutrophil is an essential component of the body's host defense and immune surveillance network. Motile functions such as chemotaxis, secretion, phagocytosis and diapedesis are necessary for the neutrophil to perform its host defense role and require cell shape change. The cellular cytoskeleton in general and the microfilamentous cytoskeleton in particular is thought to provide the force/structure which changes/supports cell shape. Elucidation of the basic biochemical mechanisms which regulate cell shape is thus important to an understanding of neutrophil function and dysfunction as well as the processes of cell division, embryogenesis and tumor metastasis. Our long range goal is to elucidate the role and describe the mechanisms whereby the microfilamentous cytoskeleton determines neutrophil shape. Preliminary studies suggest that actin polymerization to an extent > 35% above basal levels is required for full neutrophil shape change and that lesser amounts of polymerization result in intermediate shapes; that actin distribution within distinct cellular regions determines shape; that two F-actin pools exist in neutrophils (a labile and a stable pool), and that the two F-actin pools and/or their regulatory proteins may be important determinants of neutrophil functions including cell shape.
Our specific aims are: 1) to define and localize the labile and stable pools of F-actin in basal neutrophils and in neutrophils activated by chemotactic factor or adherence; 2) to determine the nature of the labile F-actin pool as oligomer or dilutionally depolymerizable polymer; 3) to define the interactions between the labile and stable F-actin pools and gelsolin and actin binding protein (ABP). Cell shape is characterized by scanning electron microscopy and quantitated by a video enhanced image analysis system; F-actin content is analyzed by FACS analysis of NBDphallacidin stained cells and by quantitative scanning of Coomasie blue stained SDS-PAGE gels; F-actin distribution is assessed by single cell morphometry and computerized videoimage analysis; quantitative differential high speed centrifugation is utilized to determine the nature of the labile F-actin pool; immunoblots with monoclonal anti-gelsolin and anti-ABP allow definition of interactions between the actin pools and regulatory proteins.
|Watts, R G (1996) Dephosphorylation of a 34kd triton-insoluble F-actin pool protein is associated with phorbol ester-induced actin polymerization in human polymorphonuclear leukocytes. Hematopathol Mol Hematol 10:69-84|
|Watts, R G (1996) Adherence of human phagocytes results in characteristic reorganization and redistribution of distinct F-actin pools. Hematopathol Mol Hematol 10:223-32|
|Watts, R G; Deaton, J D; Howard, T H (1995) Dynamics of triton-insoluble and triton-soluble F-actin pools in calcium-activated human polymorphonuclear leukocytes: evidence for regulation by gelsolin. Cell Motil Cytoskeleton 30:136-45|
|Watts, R G (1995) Role of gelsolin in the formation and organization of triton-soluble F-actin during myeloid differentiation of HL-60 cells. Blood 85:2212-21|
|Watts, R G (1994) Membranoproliferative glomerulonephritis and micronodular cirrhosis associated with hepatitis C virus infection in a chronically transfused sickle cell patient. Am J Hematol 46:162-3|
|Howard, T H; Watts, R G (1994) Actin polymerization and leukocyte function. Curr Opin Hematol 1:61-8|
|Watts, R G; Howard, T H (1994) Role of tropomyosin, alpha-actinin, and actin binding protein 280 in stabilizing Triton insoluble F-actin in basal and chemotactic factor activated neutrophils. Cell Motil Cytoskeleton 28:155-64|
|Watts, R G; Gray, B M; Patterson, H S et al. (1994) A clinically applicable technique to study cytoskeletal dynamics in normal and abnormal polymorphonuclear leukocytes isolated from small volume blood samples. Am J Med Sci 308:313-21|
|Watts, R G; Howard, T H (1993) Mechanisms for actin reorganization in chemotactic factor-activated polymorphonuclear leukocytes. Blood 81:2750-7|
|Watts, R G; Howard, T H (1992) Evidence for a gelsolin-rich, labile F-actin pool in human polymorphonuclear leukocytes. Cell Motil Cytoskeleton 21:25-37|
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