Actin and villin compartmentation during ATP depletion and recovery in renal cultured cells.
ATP-depletion in renal cultured cells has been used as a model for studying various cytoskeletal and functional alterations induced by renal ischemia. This communication explores the reversibility of these effects utilizing a novel method [1] that depleted ATP (ATP-D) to 2% of control within 30 minutes and caused complete recovery (REC) of ATP in one hour. Under confocal microscopy, ATP-D (30 min) caused thinning of F-actin from the microvilli, cortical region, and basal stress fibers, with the concurrent appearance of intracellular F-actin patches. These changes were more pronounced after 60 minutes of ATP-D. One hour of REC following 30 minutes of ATP-D produced complete recovery of F-actin in each region of the cell. However, after 60 minutes of ATP-D, a heterogeneous F-actin recovery pattern was observed: almost complete recovery of the apical ring and microvilli, thinned cortical actin with occasional breaks along the basolateral membrane, and a dramatic reduction in basal stress fiber density. The time course of cortical actin and actin ring disruption and recovery coincided with a drop recovery in the transepithelial resistance and the cytoskeletal dissociation and reassociation of the Na,K-ATPase. Additionally, the microvilli retracted into the cells during ATP-D, a process that was reversed during REC. Triton extraction and confocal microscopy demonstrated that villin remained closely associated with microvillar actin during both ATP-D and REC. These distinctive regional differences in the responses of F-actin to ATP depletion and repletion in cultured renal epithelial cells may help to clarify some of the differential tubular responses to ischemia and reperfusion in the kidney.
Golenhofen
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Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA.
Doctor
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Bacallao
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Mandel
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LJ
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000704:Analysis of Variance; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008840:Microfilament Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013552:Swine
D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; C026615:villin; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase