title: Decreased actin solubility observed during ATP-depletion is mimicked by severing agents but not depolymerizing agents in isolated and cultured proximal tubular cells.
abstract: 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.
authors.affiliation: Department of Natural Sciences, Colby-Sawyer College, New London, NH, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: White
authors.forename: Luo
authors.initials: L
authors.lastname: Gu
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
mesh_terms: 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
chemical_list: 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