INVOLVEMENT OF GTP-BINDING PROTEINS IN ACTIN POLYMERIZATION IN HUMAN NEUTROPHILS

The motility of human neutrophils, which is of vital importance for the role of these cells in host defense, is based on rapid and dynamic changes of the filamentous actin (F-actin) network. Consequently, to understand how neutrophils move and ingest particles, we need to know how polymerization and depolymerization of actin are regulated. Previous studies by several investigators have, based on indirect evidence obtained with pertussis toxin, suggested a role for GTP-binding protein(s) (G protein) in chemotaxis-induced, but not phagocytosis-induced, reorganization of the F-actin network. The aim of the present investigation was to study the effects of directly activated G proteins (i.e., without prior ligand-receptor complex formation) on the F-actin content in human neutrophils. AlF4- induced a pronounced and sustained increase in F-actin in intact neutrophils. This effect coincided with an increase in cytosolic free Ca2+, indicating that phospholipase C and the subsequent transduction mechanism were also activated. Inhibition of phospholipase C activity by extensive depression of the cytosolic free Ca2+level (< 20 nM) only marginally affected the AlF4--induced rise in F-actin content. The major part of the AlF4--induced rise in F-actin content was also resistant to pertussis toxin, suggesting that pertussis toxin-insensitive G proteins in neutrophils are also able to trigger actin polymerization. The specificity of AlF4- in activating G proteins was also tested in permeabilized cells. In this case the effect was more rapid and could be totally abolished by guanosine 5′-[β-thio]diphosphate. In analogy, in permeabilized cells guanosine 5′-[γ-thio]triphosphate mimicked the effect of AlF4- on actin polymerization, and the effect induced by this nonhydrolyzable GTP analogue could also be totally abolished by guanosine 5′-[β-thio]diphosphate. In summary, the present data support our previous hypothesis that G proteins are intimately linked to actin polymerization in human neutrophils.