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.
Showing the most recent 10 out of 11 publications
