ADF/cofilin mediates actin cytoskeletal alterations in LLC-PK cells during ATP depletion

Sharon L. Ashworth, Erica L. Southgate, Ruben M. Sandoval, Peter J. Meberg, James R. Bamburg, Bruce A. Molitoris

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44 Scopus citations

Abstract

Ischemic injury induces actin cytoskeleton disruption and aggregation, but mechanisms affecting these changes remain unclear. To determine the role of actin-depolymerizing factor (ADF)/ cofilin participation in ischemic-induced actin cytoskeletal breakdown, we utilized porcine kidney cultured cells, LLC-PKA4.8, and adenovirus containing wild-type (wt), constitutively active, and inactive Xenopus ADF/cofilin linked to green fluorescence protein [XAC(wt)-GFP] in an ATP depletion model. High adenoviral infectivity (70%) in LLC-PKA4.8 cells resulted in linearly increasing XAC(wt)-GFP and phosphorylated (p)XAC(wt)-GFP (inactive) expression. ATP depletion rapidly induced dephosphorylation, and, therefore, activation, of endogenous pcofilin as well as pXAC(wt)-GFP in conjunction with the formation of fluorescent XAC(wt)-GFP/actin aggregates and rods. No significant actin cytoskeletal alterations occurred with short-term ATP depletion of LLC-PKA4.8 cells expressing GFP or the constitutively inactive mutant XAC(S3E)-GFP, but cells expressing the constitutively active mutant demonstrated nearly instantaneous actin disruption with aggregate and rod formation. Confocal image three-dimensional volume reconstructions of normal and ATP-depleted LLC-PKA4.8 cells demonstrated that 25 min of ATP depletion induced a rapid increase in XAC(wt)-GFP apical and basal signal in addition to XAC-GFP/actin aggregate formation. These data demonstrate XAC(wt)-GFP participates in ischemia-induced actin cytoskeletal alterations and determines the rate and extent of these ATP depletion-induced cellular alterations.

Original languageEnglish (US)
Pages (from-to)F852-F862
JournalAmerican Journal of Physiology - Renal Physiology
Volume284
Issue number4 53-4
StatePublished - Apr 1 2003

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Keywords

  • Actin-depolymerizing factor
  • Ischemia
  • Microvilli
  • XAC-GFP

ASJC Scopus subject areas

  • Physiology

Cite this

Ashworth, S. L., Southgate, E. L., Sandoval, R. M., Meberg, P. J., Bamburg, J. R., & Molitoris, B. A. (2003). ADF/cofilin mediates actin cytoskeletal alterations in LLC-PK cells during ATP depletion. American Journal of Physiology - Renal Physiology, 284(4 53-4), F852-F862.