Actin-based centripetal flow: phosphatase inhibition by calyculin-A alters flow pattern, actin organization, and actomyosin distribution.
Previous studies have suggested that the actin-based centripetal flow process in sea urchin coelomocytes is the result of a two-part mechanism, actin polymerization at the cell edge coupled with actomyosin contraction at the cell center. In the present study, we have extended the testing of this two-part model by attempting to stimulate actomyosin contraction via treatment of coelomocytes with the phosphatase inhibitor Calyculin A (CalyA). The effects of this drug were studied using digitally-enhanced video microscopy of living cells combined with immunofluorescent localization and scanning electron microscopy. Under the influence of CalyA, the coelomocyte actin cytoskeleton undergoes a radical reorganization from a dense network to one displaying an array of tangential arcs and radial rivulets in which actin and the Arp2/3 complex concentrate. In addition, the structure and dynamics of the cell center are transformed due to the accumulation of actin and membrane in this region and the constriction of the central actomyosin ring. Physiological evidence of an increase in actomyosin-based contractility following CalyA treatment was demonstrated in experiments in which cells generated tears in their cell centers in response to the drug. Western blotting and immunofluorescent localization with antibodies against the phosphorylated form of the myosin regulatory light chain (MRLC) suggested that the demonstrated constriction of actomyosin distribution was the result of CalyA-induced phosphorylation of MRLC. Overall, the results suggest that there is significant cross talk between the two underlying mechanisms of actin polymerization and actomyosin contraction, and indicate that changes in actomyosin tension may be translated into alterations in the structural organization of the actin cytoskeleton.
Henson
John H
JH
Department of Biology, Dickinson College, Carlisle, Pennsylvania 17013, USA. henson@dickinson.edu
Kolnik
Sarah E
SE
Fried
Christopher A
CA
Nazarian
Ronniel
R
McGreevy
Jeanine
J
Schulberg
Katrina L
KL
Detweiler
Megan
M
Trabosh
Valerie A
VA
D000199:Actins / Q000378:metabolism*; D000205:Actomyosin / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008387:Marine Toxins; D020409:Molecular Motor Proteins / Q000378:metabolism; D010080:Oxazoles / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012617:Sea Urchins / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
D000199:Actins; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D020409:Molecular Motor Proteins; D010080:Oxazoles; C059041:calyculin A; D000205:Actomyosin; D010749:Phosphoprotein Phosphatases