ADF/cofilin mediates actin cytoskeletal alterations in LLC-PK cells during ATP depletion.
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-PK(A4.8), 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-PK(A4.8) 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-PK(A4.8) 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-PK(A4.8) 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.
Ashworth
Sharon L
SL
Division of Nephrology, Department of Medicine, Indiana University, and Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 46202-5116, USA.
Southgate
Erica L
EL
Sandoval
Ruben M
RM
Meberg
Peter J
PJ
Bamburg
James R
JR
Molitoris
Bruce A
BA
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D051345:Destrin; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014981:Xenopus
D051339:Actin Depolymerizing Factors; D000199:Actins; D051345:Destrin; C495825:Dstn protein, mouse; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000968:Antimycin A; D000255:Adenosine Triphosphate