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.
White
Peter
P
Department of Natural Sciences, Colby-Sawyer College, New London, NH, USA.
Gu
Luo
L
Chen
Jing
J
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D047630:Depsipeptides; D005260:Female; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008387:Marine Toxins / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D010456:Peptides, Cyclic / Q000494:pharmacology; D011108:Polymers / Q000378:metabolism; D011817:Rabbits; D012995:Solubility; D013552:Swine; D013844:Thiazoles / Q000494:pharmacology*; D053778:Thiazolidines
D000199:Actins; D000970:Antineoplastic Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; D047630:Depsipeptides; D008387:Marine Toxins; D010456:Peptides, Cyclic; D011108:Polymers; D013844:Thiazoles; D053778:Thiazolidines; C057531:jasplakinolide; D000255:Adenosine Triphosphate; C086235:swinholide A; C037067:latrunculin A