Actin‐based centripetal flow: Phosphatase inhibition by calyculin‐A alters flow pattern, actin organization, and actomyosin distribution

Abstract

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.

Actomyosin contractility spatiotemporally regulates actin network dynamics in migrating cells

“…Moreover, the correlative distributions of F-actin and myosin II suggest that actomyosin contractility is important for the structural organization and integrity of F-actin network and SFs, in agreement with previous reports (Lin et al, 1996;Henson et al, 2003;Schaub et al, 2007).…”

“…Calyculin A is known to induce actomyosin contractility by inhibiting the activity of myosin light chain phosphatases (Ishihara et al, 1989;Henson et al, 2003), and blebbistatin is a well characterized specific inhibitor of myosin II activity (Straight et al, 2003). These concentrations were chosen to achieve a partial perturbation of actomyosin contractility without adversely affecting cell versatility.…”

E-cadherin junction formation involves an active kinetic nucleation process

“…4B). In contrast, treatment of cells with calyculin A (63), a phosphatase inhibitor that increases the cellular actomyosin contractility (64), resulted in an increase in the frequency of successful junction formation (Fig. 4B).…”

Actomyosin-generated tension controls the molecular kinetics of focal adhesions

“…These values are about 260, 20 and 220 times less than the corresponding half times for exit of these proteins from FAs following blebbistatin treatment. Performing the opposite experiment, using the serine/threonine phosphatase inhibitor calyculin A (5 nM) for 10 minutes to increase actomyosin contractility (Ishihara et al, 1989;Henson et al, 2003), resulted in a gradual increase in FA area, without influencing the intensity of paxillin-YFP ( Fig. 2A).…”