ABSTRACT
The actin microfilaments are well known dynamic structures that support and organize the cell membrane and functions associated with the membrane such as ion channels and transporters. In addition, many aspects of cellular physiology seem to be actively modulated by changes in actin cytoskeleton dynamics, which involve reorganization and restructuring of the filaments. For both of these reasons, the actin cytoskeleton has attracted special attention since the early days of cell volume regulation research. Mechanisms controlling the actin equilibrium in response to external stimuli were studied and the signaling cascades leading to the regulation of actin cytoskeleton dynamics have been partially elucidated. They include: a) activation of specific actin binding proteins that regulate actin polymerization dynamics, b) activation of protein kinases or phosphatases regulating phosphorylation of specific cytoskeletal proteins and c) activation of signal transduction pathways leading from membrane receptor activation to actin reorganization involving small GTPases of the Rho and Rac families. These intracellular signal transducers are activated by extracellular stimuli that include hormones, growth factors, cytokines, or ions, many of them in turn are partially known to participate in cell volume regulation. These findings provide strong evidence that the actin cytoskeleton is involved in cell volume regulation by sensing and mediating extracellular signals.
Subject(s)
Actins/metabolism , Cell Size , Cytoskeleton/metabolism , Signal TransductionABSTRACT
Glomerular permeability for macromolecules depends partially on proper attachment of the glomerular epithelial cells (GEC) to the glomerular basement membrane (GBM). The latter requires integrity of the actin cytoskeleton, which in turn is regulated by specific actin-associated proteins. Since several glomerulopathies characterized by heavy proteinuria are associated with increased glomerular tumor necrosis factor alpha (TNF-alpha) expression, we studied the interaction of TNF-alpha with the actin cytoskeleton of cultured rat GEC. Incubation of GEC with 10 ng/ml TNF-alpha for variable time periods ranging from 15 min to 24 hr demonstrated a marked accentuation and redistribution of actin microfilaments, as shown by direct fluorescence analysis and confocal laser scanning microscopy. Quantitative biochemical determination of the G/total-actin ratio confirmed the above observations. Indeed, this ratio was significantly reduced, indicating substantial polymerization of G-actin and formation of F-actin. Concurrently, TNF-alpha rapidly induced tyrosine phosphorylation of both paxillin and focal adhesion kinase, without affecting the expression levels of these two proteins. In addition, tyrosine phosphorylation of vinculin became evident, indicating involvement of this focal adhesion marker in the observed actin reorganization. Inhibition of tyrosine phosphorylation by genistein prevented the reorganization of the actin cytoskeleton by TNF-alpha. We conclude that TNF-alpha induces substantial reorganization of actin cytoskeleton and focal adhesions. These effects occur simultaneously, with a prompt TNF-alpha-induced tyrosine phosphorylation of paxillin and focal adhesion kinase, indicating that these proteins, known to regulate actin polymerization and formation of focal adhesions, may be directly involved in the mechanism controlling the observed actin redistribution. These findings suggest that the observed TNF-alpha-actin cytoskeleton interactions may relate to the pathogenesis of glomerulopathies with heavy proteinuria, in which increased glomerular expression of TNF-alpha is associated with disturbances in the attachment of podocytes to the GBM.
