Assembly dynamics of actin in adherent human neutrophils.
We have extended our previous studies of adherent neutrophils and compared 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 treatment effectively inhibited the depolymerization of F-actin fragments following cell lysis. Formaldehyde and phalloidin treatment reduced G-actin 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 fixed, there were highly significant differences in G-actin levels between the suspended and adherent cells as compared with unfixed cells. Adhesion to both laminin and fibronectin initiated a rapid rise in G-actin with a corresponding decrease in F-actin. However, the changes were more pronounced in cells adherent to laminin. The peak of depolymerization 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 [Ca2+]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 cells at 3 min (P < 0.02). Pertussis toxin, H-7, and staurosporin treatments did not alter the dynamic changes of actin in adherent cells, suggesting that these metabolic events are transduced by a G-protein and Protein Kinase C independent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Wang
J S
JS
William B. Castle Hematology Research Laboratory, Boston City Hospital, Massachusetts.
Pavlotsky
N
N
Tauber
A I
AI
Zaner
K S
KS
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000199:Actins / Q000378:metabolism*; D000470:Alkaloids / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007797:Laminin / Q000378:metabolism*; D009504:Neutrophils / Q000502:physiology*; D037342:Pertussis Toxin; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D013050:Spectrometry, Fluorescence; D019311:Staurosporine; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000199:Actins; D000470:Alkaloids; D004791:Enzyme Inhibitors; D005353:Fibronectins; D007546:Isoquinolines; D007797:Laminin; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D010566:Virulence Factors, Bordetella; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D037342:Pertussis Toxin; D003850:Deoxyribonuclease I; D019311:Staurosporine; D002118:Calcium