ASSEMBLY DYNAMICS OF ACTIN IN ADHERENT HUMAN NEUTROPHILS

We have extended our previous studies of adherent neutrophils and comp ared actin depolymerization and intracellular calcium changes induced by adherence to laminin and fibronectin. In order to accurately assess cellular actin changes, F-actin depolymerization in the cell lysates must be inhibited. We found that phalloidin or 3.7% formaldehyde treat ment effectively inhibited the depolymerization of F-actin fragments f ollowing cell lysis. Formaldehyde and phalloidin treatment reduced G-a ctin levels 75-80% in suspended cells, 35-73% in cells adherent for 1 min, and about 50% for cells adherent for 3 min. When the actin was fi xed, there were highly significant differences in G-actin levels betwe en the suspended and adherent cells as compared with unfixed cells. Ad hesion to both laminin and fibronectin initiated a rapid rise in G-act in with a corresponding decrease in F-actin. However, the changes were more pronounced in cells adherent to laminin. The peak of depolymeriz ation occurred by 1 min and, thereafter, G-actin decreased and F-actin increased reaching a steady state at 5 min. Adhesion to both laminin- and fibronectin-coated surfaces was accompanied by an increase of [Ca 2+](i) with a peak at 3 min, followed by a decrease from 3-5 min and a steady state attained between 5 and 10 min. The rise of [Ca2+](i) in laminin-adherent cells was about twice that in fibronectin-adherent ce lls at 3 min (P < 0.02). Pertussis toxin, H-7, and staurosporin treatm ents did not alter the dynamic changes of actin in adherent cells, sug gesting that these metabolic events are transduced by a G-protein and Protein Kinase C independent mechanism. The results support the hypoth esis that a transient mobilization of F-actin to a monomeric pool, whi ch then serves as a source for further repolymerization, is induced by adherence of neutrophils to extracellular matrix proteins. (C) 1993 W iley-Liss, Inc.