Abstract
Cell volume recovery in response to swelling requires reorganization of the cytoskeleton and fluid efflux. We have previously shown that electrolyte and fluid efflux via K+ and Cl- channels is controlled by swelling-induced activation of phospholipase Cgamma (PLCgamma). Recently, integrin engagement has been suggested to trigger responses to swelling through activation of Rho family GTPases and Src kinases. Because both PLCgamma and Rho GTPases can be regulated by Src during integrin-mediated cytoskeletal reorganization, we sought to identify swelling-induced Src effectors. Upon hypotonic challenge, Src was rapidly activated in transient plasma membrane protrusions, where it colocalized with Vav, an activator of Rho GTPases. Inhibition of Src with PP2 attenuated phosphorylation of Vav. PP2 also attenuated phosphorylation of PLCgamma, and inhibited swelling-mediated activation of K+ and Cl- channels and cell volume recovery. These findings suggest that swelling-induced Src regulates cytoskeletal dynamics, through Vav, and fluid efflux, through PLCgamma, and thus can coordinate structural reorganization with fluid balance to maintain cellular integrity.
Highlights
In addition to the restoration of fluid balance, a swollen cell must reorganize its disrupted cytoskeleton
Because we previously had observed irregular cell morphologies in connection with swelling-induced plasma membrane PLC␥ localization in HTC hepatoma cells [3], we investigated whether hypotonic exposure of these cells would elicit plasma membrane protrusions analogous to those in fibroblasts, and how the formation of swelling-induced membrane protrusions temporally correlated with volume recovery
Membrane protrusions transiently formed along the sides of the cells. (A video of this experiment is available in supplemental materials.) The protrusions expanded and contracted over a 3-min period, and subsequently could not be detected
Summary
PLC␥, phospholipase C␥; PP2, 4-amino5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PP3, 4-amino7-phenylpyrazol[3,4-d]pyrimidine; SES, standard extracellular solution; %RVD, percent volume recovery; FAK, focal adhesion kinase; pY-PLC␥, activated PLC␥; pY-Src, activated Src; pY-Vav, activated Vav. Src tyrosine kinases have been shown to be activated upon cell swelling via a mechanism consistent with integrin dependence, and kinase activity appears to be required for volume recovery [5]. Cdc and Rac help to initiate cell migration via mediators that trigger the formation of an actin polymerization complex at the leading edge, where localized actin polymerization provides the force necessary to form a membrane protrusion (10 –12). Src controls both the activation of Cdc and Rac1 [13] and the inhibition of RhoA [14] at the leading edge. Our findings suggest that through the recruitment and activation of Vav and PLC␥, Src coordinates the reorganization of the actin cytoskeleton with activation of ion channels to promote cell volume recovery
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