Luo Gu | PubMed Health

PubMed2003

Calyculin A-induced actin phosphorylation and depolymerization in renal epithelial cells.

This study reports actin phosphorylation and coincident actin cytoskeleton alterations in renal epithelial cell line, LLC-PK1. Serine phosphorylation of actin was first observed in vitro after the cell lysate was incubated with phosphatase inhibitors and ATP. Both the phosphorylated actin and actin kinase activities were found in the cytoskeletal fraction. Actin phosphorylation was later detected in living LLC-PK1 cells after incubation with the phosphatase inhibitor calyculin A. Calyculin A-induced actin phosphorylation was associated with reorganization of the actin cytoskeleton, including net actin depolymerization, loss of cell-cell junction and stress fiber F-actin filaments, and redistribution of F-actin filaments in the periphery of the rounded cells. Actin phosphorylation was abolished by 3-h ATP depletion but not by the non-specific kinase inhibitor staurosporine. These results demonstrate that renal epithelial cells contain kinase/phosphatase activities and actin can be phosphorylated in LLC-PK1 cells. Actin phosphorylation may play an important role in regulating the organization of the actin cytoskeleton in renal epithelium.

Actin Cytoskeleton↗

PubMed2002

Decreased actin solubility observed during ATP-depletion is mimicked by severing agents but not depolymerizing agents in isolated and cultured proximal tubular cells.

The microvilli of the apical membrane of proximal tubule (PT) cells are supported by the underlying actin cytoskeleton. Ischaemic or anoxic ATP-depletion leads to the disruption of the actin cytoskeleton, resulting in microvillar retraction and loss of membrane polarity. Using isolated PT cells, we have previously demonstrated that actin filaments (F-actin) are likely severed during ATP-depletion. A sequential extraction protocol revealed a decrease in actin solubility, resulting in the sequestration of a distinct F-actin pool with the insoluble cellular complex in ATP-depleted PT cells. We demonstrate here that decreased actin solubility is not only a reliable end-marker of ATP-depletion induced injury in freshly isolated PT cells, but also serves as a biochemical marker in the cultured proximal tubular cell line LLC-PK1. In the present studies, we also investigated specific actin-binding drugs to determine if they mimic the effects observed during energy depletion. Jasplakinolide (JP), a compound which binds F-actin and prevents depolymerization, did not effect actin solubility during ATP-depletion. Furthermore, swinholide A (SA), an F-actin severing agent, resulted in decreased actin solubility, mimicking the effects of ATP-depletion. Interestingly, latrunculin A (LA), an agent which depolymerizes F-actin, did not reduce actin solubility, but rather resulted in an increase in digitonin-soluble actin. Taken collectively, our results support previous work and suggest that disruption of the actin cytoskeleton during ATP-depletion is mediated by F-actin severing/fragmentation and not depolymerization. The differential effects of F-actin disrupting agents and the consistencies observed in both models of ischaemic injury will provide a basis for a more detailed understanding of the pathological events of PT-cell dysfunction.

Actins↗