ADF/cofilin mediates actin cytoskeletal alterations in LLC-PKcells during ATP d

ÇáÓð

×÷ÕߣºSharon L.Ashworth, Erica L.Southgate, Ruben M.Sandoval, Peter J.Meberg, James R.Bamburg,  Bruce A.Molitoris×÷Õßµ¥Î»£º1 Division of Nephrology, Department of Medicine,Indiana University, and Roudebush Veterans Affairs Medical Center,Indianapolis, Indiana 46202-5116; Department ofBiology, University of North Dakota, Grand Forks, NorthDakota 58201; and Department of Biochemistryand Molecular Biology, Colorado State U - B% B0 ~( Z% J4 F$ R. X" v( L
                  9 z5 Z6 ^' @( G1 `
                  ; ?- C) z6 @3 H9 r8 M$ X
         
# q7 s" K. P+ Y9 A4 |1 _                         + `5 ^0 p  `4 \4 ]
            + T. s4 h$ j; H, j4 |
            6 i4 z" k3 n' }3 K4 o( f  d
            ! g; s) {% q# x
            
  v$ R$ j0 b: L4 y# x5 c                        m2 U! n  f: s# ^: U) t7 j+ ^
        0 x- `% R+ J$ h. p
        
: C% N9 ~9 j' n4 J0 w        ) G, M$ s2 @  l4 q  a6 ]+ a; e1 i
          ¡¾ÕªÒª¡¿9 e& X$ c8 l: I& W1 f
      Ischemic injury induces actincytoskeleton disruption and aggregation, but mechanisms affecting thesechanges remain unclear. To determine the role of actin-depolymerizingfactor (ADF)/ cofilin participation in ischemic-induced actincytoskeletal breakdown, we utilized porcine kidney cultured cells,LLC-PK A4.8, and adenovirus containing wild-type (wt),constitutively active, and inactive Xenopus ADF/cofilinlinked to green fluorescence protein [XAC(wt)-GFP] in an ATPdepletion model. High adenoviral infectivity (70%) in LLC-PK A4.8 cells resulted in linearly increasingXAC(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 inconjunction with the formation of fluorescent XAC(wt)-GFP/actinaggregates and rods. No significant actin cytoskeletal alterationsoccurred with short-term ATP depletion of LLC-PK A4.8 cellsexpressing GFP or the constitutively inactive mutant XAC(S3E)-GFP, butcells expressing the constitutively active mutant demonstrated nearly instantaneous actin disruption with aggregate and rod formation. Confocal image three-dimensional volume reconstructions of normal andATP-depleted LLC-PK A4.8 cells demonstrated that 25 min of ATP depletion induced a rapid increase in XAC(wt)-GFP apical and basalsignal in addition to XAC-GFP/actin aggregate formation. These datademonstrate XAC(wt)-GFP participates in ischemia-induced actincytoskeletal alterations and determines the rate and extent of theseATP depletion-induced cellular alterations.
  a2 s5 l7 P; U1 O9 ~& h+ W          ¡¾¹Ø¼ü´Ê¡¿ ischemia microvilli actindepolymerizing factor XACGFP
; {( o% X+ b7 s3 T: Z                  INTRODUCTION
( k: q. ^1 Y# f) ~2 Z2 v
. N' E7 i1 b) D2 A% a; M( EISCHEMIA-INDUCED CELL INJURY of polarized proximal tubule cells results in severebiochemical, physiological, and morphological alterations ( 13, 20, 33 ). The extent of cellular injury is affected by the lengthand severity of the ischemic insult ( 14 ). Cellularchanges in surface membrane polarity, junctional complexes, and theactin cytoskeleton are among the earliest observed alterations( 14, 22, 23, 30 ). Within 5 min of ischemic injuryinduction, renal proximal tubule actin cytoskeletal alterations beginwith the apical microvilli showing signs of degeneration ( 13, 14, 20, 21 ). With increasing duration of ischemic injury,the apical microvilli suffer further damage with complete disintegration of their microfilament cores and overlying plasma membranes. Microvillar membranes fuse or coalesce to form enlarged structures, and membrane vesicles or blebs also form ( 28 ).These abnormal microvillar vesicles are internalized within theproximal tubule cytoplasm or lost into the proximal tubule lumen. Thecellular mechanisms responsible for the microfilament alterations arenot known. In addition to microvillar F-actin rearrangement in proximal tubule cells in response to ischemia, cytosolic F-actinredistributes with formation of F-actin aggregates ( 12, 15, 24 ).# k/ X' q& c+ H. S) H/ t) I$ X

7 g3 w( r( M: |Our previous in vivo data are consistent with a role for theactin-depolymerizing factor (ADF)/cofilin family of proteins inproximal tubule apical microvillar breakdown ( 3, 29 ). TheADF/cofilin family of proteins is necessary for eukaryotic cellsurvival, although the number and type of isoforms may vary betweencell types ( 32 ). These proteins are among the mostimportant cellular regulators of actin filament dynamics. They bindF-actin in a pH-dependent manner and have been shown to mediate F-actin severing and depolymerization ( 5 ). Under physiologicalconditions, ADF has a diffuse cytoplasmic distribution with little orno localization in the apical region of proximal tubule cells, but withinduction of ischemia, this distribution pattern changesdramatically. Within 15 min of ischemia, the phosphorylated orinactive form of the ADF protein ( 25 ) is rapidlydephosphorylated ( 29 ) and translocated from the cytoplasminto the terminal web and apical microvilli ( 3 ). Bothactin and ADF have been localized to luminal membrane vesicles thathave been lost from the apical surface during ischemic injury.Although these data are consistent with participation of ADF/cofilin indestruction of the F-actin core of microvilli in response toischemic injury of proximal tubule cells, direct proof for thisrole is lacking.' s& G8 C3 l7 {/ v4 M: \
$ v7 M4 P" Q. s2 Q( R, B% O, u
Therefore, the present studies were undertaken to directly evaluate therole ADF plays in F-actin destruction and reorganization duringischemic cell injury. To accomplish this goal, we utilized theproximal tubule cell line LLC-PK because several studies have demonstrated F-actin reorganization observed in rat proximal tubule cells in response to ischemic insult can be mimicked in LLC-PK cells by inducing ATP depletion through treatment with antimycin A insubstrate-depleted medium ( 4, 10, 15, 24 ). Recently, adenoviral constructs containing cDNAs of the wild-type (wt)ADF/cofilin isoform, Xenopus ADF/cofilin, XAC(wt)-greenfluorescent protein (GFP), the constitutively active mutant,XAC(S3A)-GFP, and the inactive mutant, XAC(S3E)-GFP, have becomeavailable ( 1, 18 ) and allowed for expression of theseproteins in LLC-PK cells. These unique tools have been successfullyused for expression of the wild-type ADF/cofilin isoform to directlydemonstrate XAC(wt)-GFP-mediated alterations in actin dynamics in cells( 6, 18 ). With the use of these probes, we manipulatedexpression of wild-type and mutant XAC-GFP isoforms and studied theeffects of their expression on the actin cytoskeleton in proximaltubule cultured cells under physiological and ATP-depleted conditions.Our data indicate a direct role for ADF/cofilin proteins in mediatingthe severe actin cytoskeletal alterations observed in response tocellular ATP depletion in addition to dramatically impacting the rateand extent of these cellular alterations.' C  y4 s7 j  v, o/ X2 e5 p
) ^* T" k5 v' D6 y9 b2 q0 ]
METHODS. C: K6 i5 i7 _4 b9 |

; E( H. L0 s# i& h( vCell culture. Cell culture experiments were performed on three proximal tubule celllines, two of which were porcine cell lines clonally derived fromLLC-PK(wt) (LLC-PK 10 and LLC-PK A4.8 ), and theS 1 mouse cell line (a kind gift from Dr. G. T. Nagami,Univ. of California at Los Angeles School of Medicine, Los Angeles,CA). The LLC-PK A4.8 cell line was maintained in alow-glucose (1 mg/ml glucose) DMEM (Sigma D-5523) containing 10% FBS,100 U/ml of penicillin, and 100 µg/ml of streptomycin, pH 7.4, at37°C in 5% CO 2 incubators. The LLC-PK 10 cellline was maintained and expanded on plastic tissue culture dishes inDMEM (JRH Biosciences, no. 56-498) containing 10% FBS, 100 U/ml ofpenicillin, and 100 µg/ml of streptomycin, pH 7.4. The S 1 cells were cultured in a 50:50 mixture of Ham's F-12MEM supplementedwith 2 mM L -glutamine, 10 mM sodium-HEPES, 2 mM sodiumpyruvate, insulin, sodium selenite, and sodium bicarbonate and 7þtal calf serum, penicillin, and streptomycin. For immunofluorescence studies, cells were grown on glass coverslips, whereas cells for protein extraction were grown on plastic dishes. Cells treated for ATPdepletion were incubated in 0.1 µM antimycin A diluted insubstrate-free DMEM (no glucose, pyruvate, serum, or amino acids), pH7.4, or in depletion buffer, 1× PBS containing 0.5 mMCaCl 2, and 1.0 mM MgCl 2, pH 7.4, for designatedtime intervals.; e6 y' Q$ d. s

7 P/ P5 }7 P( g) O1 R) SAdenoviral construction. XAC-GFP (wt, S3A mutant and S3E mutant) clones were constructed in theClontech phGFP-S65T vector by H. Abe, Chiba University, and generouslyshared with us. The 1,300-bp XAC-phGFP (wt, S3A and S3E) inserts wereremoved from the phGFP plasmid with Sac I and Xba I( Sac I site was blunt ended by degrading the 3' overhang withmung bean nuclease). The XAC-phGFP inserts (wt, S3A or S3E) were clonedinto the Xba I and blunt ended Kpn I site of theshuttle vector plasmid for adenovirus production by homologousrecombination in HEK-293 cells as previously described( 18 ). The fusion proteins were expressed under control ofthe immediate early promoter of the cytomegalovirus.
1 ~1 @6 a3 s% X* P4 a' h
' I2 a  K. [/ zAdenoviral infection. The cells were infected at 40-60% confluency with a viralmultiplicity of infection of 25 for 18 h with adenovirusexpressing GFP, XAC(wt)-GFP, the constitutively active mutantXAC(S3A)-GFP, or the inactive mutant form XAC(S3E)-GFP. Cellcultures were harvested at 18, 28, and 51 h postinfection withcell extracts prepared and examined by SDS-PAGE, followed by Westernblot analysis. By 24 h postinfection, 70-80% of the treatedcells were expressing XAC-GFP isoforms as observed by epifluorescencemicroscopy. All studies were done at 24 h postinfection unlessotherwise stated.) B( _) g1 }/ T

7 B- G5 C* |/ n; J: S4 f! cSDS-PAGE and Western analysis. LLC-PK or S 1 cellular proteins were extracted in a 2% SDSbuffer (2% SDS, 10 mM Tris, pH 7.6, 10 mM NaF, 5 mM DTT, 2 mM EGTA) and boiled. Protein concentration was determined by a filter paper dye-binding assay ( 19 ). Equal protein concentrations (5 µg of total extract protein) were loaded in each lane and separated by SDS-PAGE on 15% isocratic gels. For Western blot analysis, separated proteins were transferred to a polyvinylidene fluoride membrane, and the membrane was blocked with 5% nonfat dry milk or 10%newborn calf serum in 1× Tris-buffered saline with Tween. Forimmunodetection, the rabbit primary antibodies to XAC (1:10,000), tothe phosphopeptide epitope of phosphorylated ADF/cofilin[pADF/pcofilin (also recognizes pXAC)] (1:1,000), and to ADF(1:10,000) or mouse primary monoclonal antibody to cofilin (1:5), wereutilized and followed by horseradish peroxidase-conjugated goatanti-rabbit or goat anti-mouse secondary antibodies (1:30,000). Proteinbands were detected by enhanced chemiluminescence (Pierce, Rockford, IL) or stained with 4-chloro-1-napthol and quantified by densitometry.
+ @% v: ^; l6 B/ A& ?' P' H' k# U
2 x3 m& Y5 H. o: \Microscopy. LLC-PK A4.8 cells were fixed in 4% paraformaldehyde or3.7% formaldehyde for 1 h and permeabilized with 0.1% TritonX-100. F-actin was stained with rhodamine-phalloidin (Molecular Probes, Eugene, OR; 1:60 dilution) or Texas red-phalloidin (1:200; 1:10). Confocal images were acquired with an MRC-1024 laser-scanning confocalmicroscope (Bio-Rad, Hercules, CA) using a Nikon Diaphot 200 invertedmicroscope with a ×100, 1.4-numerical aperture (NA) oil-immersionobjective or a ×60, 1.2-NA water-immersion objective. Live cell imageswere captured with a Nikon Diaphot inverted microscope with a ×40,0.85-NA objective and a PXL cooled charge-coupled device camerawith a Kodak 1400 chip (Photometrics, Tucson, AZ). Metamorph software(Universal Imaging, West Chester, PA) was used to process the imagesand to reconstruct basal-to-apical three-dimensional reconstructions.* w5 x; y1 A+ ]; |  j7 z' K; o
7 q4 c/ D7 {9 E6 U) V: b5 [
RESULTS
5 \8 G5 [1 E( N) e' ?, R$ ?$ s  L' r
ATP depletion of LLC-PK A4.8 cells reduces pcofilinsignal. Initial studies were undertaken to determine the effect of ATPdepletion on the phosphorylation status of cofilin inLLC-PK A4.8 cells. ADF and cofilin are two highly conservedand related proteins, but differentially expressed proteins withsimilar, but distinct, actin-binding properties belonging to the samefamily of actin-associated proteins ( 5, 32 ). With the useof isoform-specific antibodies and an anti-phosphoepitope antibody thatrecognizes the phosphorylated form of each isoform, the cellularexpression of these proteins can be determined. With the use of theseprobes, we found that the endogenous expression of ADF and cofilinisoforms in porcine proximal tubule cell lines was notequivalent. The LLC-PK 4.8 cells had ample expression ofcofilin and little or no expression of ADF (Fig. 1 A ). The LLC-PK 10 cell line expressed ADF with no expression of cofilin, whereas theS 1 mouse cell line expressed both isoforms.
( Z- Y; ]9 X  U3 `# @, |% l2 d) u$ A, z5 N1 R
Fig. 1. ATP depletion activates cofilin in LLC-PK A4.8 cells. Cellular homogenate proteins (equal amounts) fromS 1, LLC-PK A4.8, and LLC-PK 10 cellswere probed using isoform-specific antibodies for actin-depolymerizingfactor (ADF), cofilin, or the phosphoepitope of phosphorylated (p)ADFand pcofilin, which also recognize pXAC(wt)-green fluorescence protein(GFP). Under physiological conditions, the cofilin isoform waspredominately expressed and phosphorylated in LLC-PK A4.8 cells, whereas ADF was very low or nonexistent ( A ). InLLC-PK 10 cells, the expression pattern was reversed, withADF primarily expressed and phosphorylated, and neither pcofilin norcofilin was detected. Both isoforms, ADF and cofilin, were expressedand detected in mouse proximal tubule S 1 cells. Westernblots of homogenates of LLC-PK A4.8 cells ATP depleted for0, 5, 15, and 30 min in depletion medium containing 0.1 µM antimycinA were probed with an antibody specific for the phosphoepitope ofADF/cofilin ( B ). Equal amounts of total protein (5 µg)were loaded in each lane with 3 replicates for each time point. Theconcentration of the inactive pcofilin isoform decreased withincreasing time of ATP depletion.3 d4 K% r  x8 t' u
! t. P$ K# \* V* n
As shown in Fig. 1 B, antimycin A-induced ATP depletionof LLC-PK 4.8 cells diminished the pcofilin signal in atime-dependent manner consistent with previously published in vivo ratkidney data that demonstrated ischemia induced atime-dependent dephosphorylation of phosphorylated ADF( 29 ). Induction of ATP depletion for 5 min had noeffect on cofilin phosphorylation, but within 15 min, a 60% decreasewas observed in pcofilin concentration. By 30 min of ATP depletion,pcofilin had been reduced to was no change in totalcellular cofilin (data not shown), these data imply that ATP depletioninduced a rapid duration-dependent dephosphorylation of cofilin.
7 ~. N, E$ W) u6 W9 m, ]0 J( B' L
" R1 {5 p8 {) bExpression of XAC-GFP through adenoviral infection ofLLC-PK A4.8 cells. To obtain direct evidence regarding the role of cofilin-mediatedcellular actin destruction and reorganization and microvillar F-actincore degeneration during ATP depletion, we utilized adenoviral vectorscontaining GFP, XAC(wt)-GFP, XAC(S3A)-GFP, or XAC(S3E)-GFP cDNA toexpress GFP or ADF/cofilin protein isoforms in LLC-PK A4.8 cells. In characterization studies, LLC-PK A4.8 cellsinfected with adenovirus containing the cDNA for XAC(wt)-GFPdemonstrated expression of the XAC(wt)-GFP fusion protein as early as18 h postinfection, as detected by GFP fluorescence (Fig. 2 A ) andWestern blotting techniques (Fig. 2 B ). The fraction ofGFP-expressing cells increased from ~70 to 90% over the 51-h periodpostinfection (Fig. 2 A ). The level of XAC(wt)-GFP expressionincreased linearly over the 51-h period postinfection (Fig. 2 B ). The level of phosphorylated XAC(wt)-GFP, as detected byWestern blot analysis, also increased linearly over the 51-h periodpostinfection (Fig. 2 B ). Therefore, as the wild-type XAC-GFPprotein was expressed, it was regulated through phosphorylation by acellular kinase. The level of endogenous cofilin, as detected byWestern blot analysis, remained constant during the first 18 hpostinfection of XAC(wt)-GFP, but decreased endogenous cofilin levelswere observed at 28 and 51 h postinfection.  U3 S" ^; Z- D. s" Q/ F

( b+ O  e" `5 M& d2 a  qFig. 2. Adenoviral Xenopus ADF/cofilin cDNAs(wild-type) linked to GFP [XAC(wt)-GFP] expression and itsphosphorylation increased from 18 to 51 h postinfection with adownregulation of endogenous cofilin. LLC-PK A4.8 cellcultures were infected with the adenovirus containing XAC(wt)-GFP witha multiplicity of infection of 25 for 18 h. A : 24 h postinfection, 70-90% of the cells showed varying levels of GFPfluorescence as observed by confocal microscopy. Cell cultures wereharvested at 18, 28, and 51 h postinfection, and the cell extractswere examined by SDS-PAGE and Western blot analysis loading of 5 µgof total extract protein per lane ( B ). The blots were probedwith rabbit anti-XAC, rabbit antibody to the phosphoepitope of ADF, andcofilin, which also recognized pXAC(wt)-GFP or mouse anti-cofilinprimary antibodies, followed by horseradish peroxidase-conjugated goatanti-rabbit or goat anti-mouse secondary antibodies. XAC(wt)-GFPexpression was observed by Western blot analysis as early as 18 hafter adenoviral infection and continued to increase for up to 51 h. An increase in the pXAC(wt)-GFP was also documented during this timeframe, suggesting XAC(wt)-GFP could be phosphorylated by a cellularkinase. A decrease in the concentration of endogenous cofilin was notedat 28 h postinfection and remained lower at 51 hpostinfection. In response to ATP depletion (Fig. 2 C ),pXAC(wt)-GFP was dephosphorylated, as demonstrated by Western blotanalysis of cellular homogenates of XAC(wt)-GFP expressing culturedcells probed with an antibody to pADF/pcofilin that alsorecognizes the phosphoepitope of 45-kDa pXAC(wt)-GFP.5 v3 X6 d' s# V2 }( y3 d

+ K+ U9 O; ]7 B9 y! w! AATP depletion resulted in rapid dephosphorylation of pXAC(wt)-GFP (Fig. 2 C ), as was seen for endogenous pcofilin (Fig. 1 B ). Compared with control levels, there was a 60% decreasein pXAC(wt)-GFP in response to 5 min of ATP depletion, which wasfurther reduced to 10% pXAC(wt)-GFP at 30 min of ATP depletion,showing dephosphorylation by an endogenous phosphatase. Together, thesedata indicate that ample expression, physiological phosphorylation, anddephosphorylation, in response to ATP depletion of XAC(wt)-GFP,occurred at the cellular level. Endogenous cofilin was alsodownregulated in response to XAC(wt)-GFP expression.
! a/ }, N7 R' r, T/ O2 L6 c: Q3 A. N
XAC-GFP expression did not alter F-actin under physiologicalconditions. To determine the effect of XAC(wt)-GFP expression on the F-actincytoskeleton of LLC-PK A4.8 cells, cells were stained with rhodamine or Texas red-phalloidin 24 h postinfection withadenovirus containing XAC(wt)-GFP. In Fig. 3, A-F, reconstructedbasal-to-apical images and single-plane basal images ofuninfected control cells ( A and B ) andXAC(wt)-GFP-expressing cells ( C-F ) are presented. InFig. 3, C-F, comparison of actin cytoskeletal stressfibers, microvillar microfilaments, and cortical actinnetwork can be drawn between high ( a ), medium( b ), and low ( c ) XAC(wt)-GFP-expressing cells andnonexpressing cells ( d ). These data demonstrate that adenoviral infection and XAC(wt)-GFP expression did not affect thedistribution or composition of the dense F-actin bundles that composebasal stress fibers, apical microvillar microfilament cores, or thecortical actin orientation of LLC-PK A4.8 cells, suggestingXAC(wt)-GFP expression does not alter cellular actin architecture underphysiological conditions, implying physiological regulation andfunction of the XAC(wt)-GFP proteins.* C  b- M8 D4 |$ v( y! _1 b
/ q# N3 v3 U: k9 r1 o
Fig. 3. Actin cytoarchitecture was preserved inadenoviral-infected LLC-PK A4.8 cells expressing XAC-GFPunder physiological conditions. Rhodamine-phalloidin staining ofF-actin in LLC-PK A4.8 cells expressing XAC(wt)-GFP( C and D ) did not demonstrate alterations in theF-actin structures compared with uninfected LLC-PK A4.8 cells ( A and B ) in three-dimensionalreconstructed and basal images. The rhodamine-phalloidin-stained,F-actin-bundled stress fibers in the control cells ( B ) weresimilar in size and frequency to those of the XAC(wt)-GFP-expressingLLC-PK A4.8 cells ( D ). In the reconstructedimages, microvillar actin present in noninfected cells ( A )was comparable in number, size, and intensity as inXAC(wt)-GFP-infected cells ( C ). To demonstrate differentlevels of XAC(wt)-GFP expression and its effect on actin organization,GFP fluorescent cells shown in images E and F (high expression, a; medium expression, b; lowexpression, c; and no expression, d ) can becompared with their corresponding rhodamine-phalloidin-stained F-actinimages in C and D. No notable difference in theactin stress fibers or apical microvillar actin could be discernedbetween higher XAC(wt)-GFP-expressing cells ( a ) comparedwith lower expressing cells ( b and c ) ornonexpressing cells ( d ).
% m+ S) Q- Y# k- }6 |4 h
; E4 r/ ~; `7 |; k7 }( C/ D1 {3 |XAC(wt)-GFP translocates to the surface membrane domain in responseto ATP depletion. Basal-to-apical reconstructions ( x-z axes images)demonstrate that, under physiological conditions (Fig. 4 A ), F-actin primarily locatedto basal and lateral aspects of the cell and in the microvilli at theapical surface. The expression of XAC(wt)-GFP under physiological conditions (Fig. 4 B ) was primarily detected in the cytoplasmof the LLC-PK A4.8 cells with little or no colocalization offluorescence with Texas red-phalloidin F-actin staining in the apical,basal, or lateral cellular regions. However, XAC(wt)-GFP-expressingcells that were ATP depleted for 25 min (Fig. 4 C ) hadintense XAC(wt)-GFP fluorescence, and colocalization of XAC(wt)-GFPwith F-actin staining in the apical and basal aspects of the cell.Also, F-actin and XAC(wt)-GFP were colocalized to dense aggregates(multiple orange/yellow areas) in the cytoplasm. These data are inagreement with and extend our previous observations showing rapidrelocalization of ADF to the apical domain of rat proximal tubule cellsduring ischemia ( 3 ).& P( Q# @# }! {- m% [  V
3 ^* N, n( G6 u/ o# S/ E; ~, Z
Fig. 4. ATP depletion induced surface membrane localization ofXAC-GFP. Untreated uninfected XAC(wt)-GFP-expressing and ATP-depletedXAC(wt)-GFP-expressing LLC-PK A4.8 were stained with Texasred-phalloidin, and through-focus images were taken. Metamorph softwarewas used to reconstruct basal-to-apical images. These three-dimensional( x-z or y-z axes) reconstructed vertical sliceimages of control and ATP-depleted LLC-PK A4.8 cellsdemonstrated that under physiological conditions, F-actin localized tothe cortical aspects of the cell and in the microvilli at the apicalsurface ( A ). Under physiological conditions, the fluorescentsignal for XAC(wt)-GFP was primarily detected in the cytoplasm ofcultured cells with little or no fluorescence in the apical aspects ofthe cell ( B ). XAC(wt)-GFP-expressing cells that were ATPdepleted for 25 min demonstrated a significant increase in thecytoplasmic XAC(wt)-GFP fluorescence colocalizing (yellow) with F-actinin the surface membrane regions and in aggregates in thecytosol ( C ).
" ?% O8 c* a* ^$ }! C. n3 p' S2 B: d: M8 H! ^% `3 q& ^, V
XAC mediates F-actin aggregation and rod formation during ATPdepletion. We next sought to determine the effect of XAC expression and ATPdepletion on the F-actin cytoskeleton. Cell monolayers infected withthe XAC(wt)-GFP adenovirus were ATP depleted with antimycin A indepletion media for 25 min and stained for F-actin using Texasred-phalloidin (1:10). In Fig. 5, A-C, both XAC(wt)-GFP-expressing and uninfected cells(arrows) were present in the same monolayer. Uninfected cells, ATPdepleted for 25 min (Fig. 5, A-C, arrows), werecharacterized by minimal disturbance in the fine-mesh cortical andstress fiber F-actin staining (Fig. 5 A, arrow). These data are similar to what we previously described under physiological orshort-term, ATP-depleted conditions ( 24 ). However, inXAC(wt)-GFP-expressing neighboring cells undergoing ATP depletion,intracellular F-actin disruption and aggregation were readily seen,with higher XAC(wt)-GFP-expressing cells being disrupted to a greaterextent than cells with lower expression levels. Colocalization ofXAC(wt)-GFP and F-actin, as demonstrated by intense yellow fluorescence(Fig. 5 C, open square), was apparent inXAC(wt)-GFP-expressing cells. The F-actin- and XAC-GFP-stainedaggregates had a much brighter GFP signal than the Texas red-phalloidinF-actin signal. We believe this difference in staining propertiesresults from the known competition between XAC-GFP and phalloidin forF-actin binding ( 5, 17 ).  X9 u6 f- F) s5 y2 e$ Y2 w' I* @

& I- U# ^1 E8 Z$ `$ t* [  Y$ Y, w' MFig. 5. ATP depletion induces formation of actin aggregates androds in XAC(wt)-GFP-expressing cells. Twenty-five minutes of ATPdepletion of LLC-PK A4.8 cells expressing XAC(wt)-GFP with0.1 µM antimycin A in depletion buffer induced rapid and extensiveformation of aggregates (small square in A - C )characterized by Texas red-phalloidin staining ( A ) andXAC(wt)-GFP fluorescence ( B ). Uninfected cells in the samemonolayer (arrow in A - C ) did not demonstrate anycomparable changes to their cytoskeletal structure, although theyunderwent the same ischemic insult. Merged images( C ) demonstrate that the aggregates contain both XAC(wt)-GFPand F-actin through the colocalization of XAC(wt)-GFP signalwith Texas red-phalloidin signal.
2 g1 K$ ?4 m' y5 j0 u9 [6 {! u' r( J" F1 n7 _4 u
To evaluate whether rod and aggregate formation, in response to ATPdepletion in XAC(wt)-GFP-expressing cells, was a direct result ofXAC(wt)-GFP-mediated actin alterations, we expressed either theconstitutively active mutant XAC(S3A)-GFP, the inactive mutantXAC(S3E)-GFP, or GFP in LLC-PK A4.8 cells. No rods oraggregates formed in the GFP (Fig. 6, A-C ) or S3Emutant-infected cells (Fig. 6, D-F ), even when ATPdepleted for 30 min. These data support the hypothesis that the activeADF/cofilin isoform directly mediates breakdown of the actincytoskeleton, leading to formation of ADF/cofilin aggregates and rods,whereas the inactive isoform cannot induce these events. These datafurther support a role for the dephosphorylated and activatedADF/cofilin proteins mediating the cellular actin changes observed withATP depletion during renal ischemia. Expression of theconstitutively active isoform led to spontaneous disruption of theactin cytoskeleton, with formation of rods and aggregates oftenresulting in detachment or cell death by 24 h postinfection (Fig. 6, G-I ). In addition, we observed a reduction incellular stress fibers in cells expressing the constitutively activemutant.
, s1 B" |& L  E$ c+ ]; `: i7 `$ P9 \8 F9 Z4 K% [/ r; z
Fig. 6. Aggregates and rods do not form in response to ATPdepletion of LLC-PK A4.8 cells expressing GFP orXAC(S3E)-GFP but spontaneously form in nondepletedXAC(S3A)-GFP-expressing cells. LLC-PK A4.8 cultured cellsinfected with adenovirus containing the cDNA for GFP( A-C ) and for the constitutively inactive mutantXAC(S3E)-GFP ( D-F ) were ATP depleted for 30 min andstained with Texas red-phalloidin to analyze their actin cytoskeleton.Close examination of the actin stress fibers and microvilli did notdemonstrate any notable differences between GFP- orXAC(S3E)-GFP-expressing cells and noninfected cells. However,LLC-PK A4.8 cultured cells infected with adenoviruscontaining the cDNA for the constitutively active XAC(S3A)-GFP isoform( G-I ) demonstrated spontaneous cytoskeletal changes.The actin stress fibers and microvilli were disrupted with formation ofaggregates and rods, with many infected cells breaking apart andlifting from the coverslip.( z, k; [! I& [" l& U5 P1 x
( \, t" Z" _. m6 o% S1 t3 ?
ATP depletion induces rapid formation of aggregates and rods incells expressing XAC(wt)-GFP. Next, we sought to determine the time course of F-actin alterations incontrol and XAC(wt)-GFP-expressing cells in response to ATP depletion.Rapid and extensive appearance of XAC-GFP/F-actin aggregates and rodswould directly indicate an important and early role for ADF/cofilinproteins in mediating F-actin disruption. To test this hypothesis, weundertook ATP depletion studies of cells infected with eitherXAC(wt)-GFP, GFP, or XAC(S3E)-GFP. In GFP- and XAC(S3E)-GFP-expressingcells, as well as in uninfected cells, we did not observe alterationsto the actin cytoskeleton comparable with the severe alterationsobserved in XAC(wt)-GFP-expressing cells in response to the same timeof ATP depletion (Figs. 5 and 6 ). XAC(wt)-GFP-, GFP-, andXAC(S3E)-GFP-expressing cells all demonstrated a high percentage of GFPsignal, indicating a similar level of infection and GFP proteinexpression. In addition, the wild-type XAC(wt)-GFP-expressing cellsappeared similar in morphology to uninfected cells or cells infectedwith GFP or the inactive S3E mutant. During ATP depletion, the GFPintensity and distribution at 2 min were comparable in theXAC(wt)-GFP-, GFP-, XAC(S3E)-GFP-expressing cells (Fig. 7, A, D, and G ). A homogenous cytosolicdistribution of GFP was observed, and nuclear localization was alsonoted. By 10 min of ATP depletion, localization of the GFP signal began to change in the XAC(wt)-GFP-expressing cells but not in the GFP- orXAC(S3E)-GFP-expressing cells (data not shown). By 20 min, cellsexpressing XAC(wt)-GFP had a reduction in the homogenous cytosolicXAC(wt)-GFP signal and an accumulation of cytoplasmic XAC(wt)-GFP-stained aggregates (Fig. 7 B, arrows). By 40 min,this effect was further enhanced in the XAC(wt)-GFP-infected cells (Fig. 7 C ), but the GFP- and XAC(S3E)-GFP-expressing cellsstill demonstrated no change in the diffuse GFP fluorescence (Fig. 7, F and I )." W- K: h8 U: I7 X- ^4 {
- b3 G' b: q9 S+ Y+ A4 ?
Fig. 7. ATP depletion of LLC-PK A4.8 cells expressingXAC(wt)-GFP rapidly induced formation of rods and aggregates ofXAC(wt)-GFP. LLC-PK A4.8 cells infected with adenoviruscontaining either XAC(wt)-GFP, GFP, or XAC(S3E)-GFP were ATP depletedin depletion buffer containing 0.1 µM antimycin A and observed for 40 min. During the first 10 min of ATP depletion, GFP-stained aggregatesformed in the XAC(wt)-GFP- ( A-C ) but not the GFP( D-F )- or XAC(S3E)-GFP ( G-I )-expressingcells. By 20 min of ATP depletion, the XAC(wt)-GFP signal was observedin large clumps (arrows) in most cells ( B ) expressingXAC(wt)-GFP, but not in the GFP ( E ) or S3E mutant-( H ) expressing cells ( E ). As XAC(wt)-GFP-stainedrod and aggregate (arrows) formation continued in theXAC(wt)-GFP-expressing cells, the diffuse cytoplasmic XAC(wt)-GFPstaining decreased ( B ). By 40 min, this effect was furtherenhanced in the XAC(wt)-GFP-expressing cells ( C ). Incontrast, depletion of the cells infected with GFP( D-F ) or the XAC(S3E)-GFP constitutively inactivemutant-expressing cells did not demonstrate any notable changesin the diffuse GFP staining observed under controlconditions.
/ x; d$ k6 M' @, J# r+ o9 C( \. r" z% p  X' B' j
DISCUSSION
7 q  q) J% V9 i
. g% L9 z. R0 l2 XThis is the first study to directly demonstrate that theADF/cofilin family of proteins mediates dramatic alterations to actin filament cytoarchitecture in response to ATP depletion. The ADF/cofilin family of proteins orchestrates actin dynamics primarily through accelerating the rate of pointed-end F-actin depolymerization and bysevering long F-actin filaments ( 5 ). To mediate cellular changes in actin dynamics, these stimulus/responsive proteins preferentially bind ADP-charged F-actin in a pH-dependent manner ( 7, 8, 11, 16 ). The ADF/cofilin proteins substantially increase the polymerization rate of actin, with ADP-actinpolymerization affected to a greater extent than ATP-actinpolymerization ( 11 ). The actin-binding properties of thisfamily of proteins are primarily regulated by phosphorylation anddephosphorylation. Also, ADF/cofilin proteins compete for F-actinbinding with other actin-binding proteins and phalloidin. Two kinasefamilies have been identified to specifically phosphorylate ADF/cofilinon serine-3, each with different upstream regulators. The Lim kinasefamily, the first identified ADF/cofilin-specific kinase, isphosphorylated, and its kinase activity is significantly increasedthrough downstream effects of the Rho family of small GTPases, Rac,Rho, and Cdc42. In turn, the activated Lim kinase phosphorylates andinactivates the ADF/cofilin protein family ( 2, 34 ). Thesecond family of ADF/cofilin-specific kinases, the testicular proteinkinase family (TESK1 or TESK2), includes serine/threonine kinasesstimulated through the integrin-mediated signaling pathway( 31 ). Phosphorylated ADF/cofilin proteins can no longerbind F- or G-actin to regulate actin dynamics ( 9, 25 ).Recently, the ADF/cofilin-specific phosphatase slingshot has been shownto dephosphorylate and activate ADF/cofilin at serine-3( 26 ).
# m  r/ l, q2 \. ~2 l* z% f- q; i% W
7 n6 ]0 u; m1 Z* O! M7 w) Y: ?Our previous studies suggested the ADF/cofilin family of proteinsplayed a significant role in ischemia-induced renal cell injuryof proximal tubule cells ( 3, 29 ). Acute renal failure mediates functional changes in the biochemical, physiological, andmorphological aspects of proximal tubule cells ( 30 ). The extent of these cellular alterations depends on the time and severity of the cellular injury, with apical membrane microvilli being extremelysensitive because they contain the majority of F-actin in these cells( 14, 15, 24 ). Clinical consequences resulting fromischemic injury include tubular obstruction from apicalmembrane blebbing, back-leak between cells that have loss theirjunctional complex integrity, reduced Na   reabsorption fromredistribution of ion pumps in the membrane, and abnormaltubuloglomerular feedback ( 30 ).% o; t! n) g: z5 b" T
/ I5 e" Y2 x9 l) }9 u2 B
Changes in the actin cytoarchitecture occur early and precede the otherobserved biochemical, functional, and structural alterations, suggesting actin changes are, in part, responsible for the subsequent destructive cellular changes. Within 5 min of renal artery clamping, weobserved dephosphorylation/activation of ADF, along with localization of this small protein into the apical microvillar region of the proximal tubule cell, where F-actin staining patterns show initial alterations ( 3 ). By 15 min of ischemia-inducedinjury, the apical membrane begins to coalesce and form luminal orcytoplasmic blebs or vesicles containing high concentrations of ADF andG-actin. In addition, microvillar microfilament destruction isconcurrent with increased G-actin concentration in the apical membraneregion. These events occur in a time frame to suggest that ADF locates to this region to participate through F-actin severing anddepolymerization in the breakdown of the microvillar microfilamentcore. In addition to microvillar microfilament changes, aggregates ofF-actin have been observed in the cytoplasm of injured proximal tubulecells ( 12, 15, 24 ).$ \; z4 R  c  x. ]) h8 X" j

, g. A% ~/ y% [1 C4 NAlthough our previous studies suggested dephosphorylation/activationand relocalization of ADF were coincident with microvillar microfilament core disintegration in response to ischemicinjury, we could not directly test the involvement of ADF in thisprocess. Therefore, to directly evaluate the role of the ADF/cofilinfamily of proteins in proximal tubule cell actin alterations, weexpressed the ADF/cofilin isoform XAC(wt)-GFP by adenoviral infectionin the proximal tubule cultured cell line LLC-PK A4.8. Inthese cells, endogenous cofilin expression is (data not shown). With expression of XAC(wt)-GFP,we observed a decrease in endogenous cofilin levels, suggesting thatendogenous cofilin played a minimal role in actin alterations inresponse to ATP depletion in XAC(wt)-GFPLLC-PK A4.8 -expressing cells. Although expression of GFP,XAC(S3E)-GFP, or XAC(wt)-GFP in these cells did not alter the integrityof their actin cytoskeleton, inducing ATP depletion in theXAC(wt)-GFP-expressing cells resulted in extremely rapid and extensivechanges in the actin cytoarchitecture (Figs. 3, 5, and 6 ) comparable tothe phenotype observed in uninfected cells that underwent a much longerischemic insult ( 12 ). XAC(wt)-GFP-containing aggregates and rods appeared within 10 min of ATP depletion and increased in number and size with depletion time. Actin aggregates 30 min of ATP depletion.These aggregates were primarily located in the cytoplasm, although rodswere also observed in the nucleus. As the number of XAC(wt)-GFP/actinaggregates increased, stress fibers and the fine meshwork of thecortical F-actin disappeared, suggesting XAC(wt)-GFP bound F-actin todepolymerize, sever, and redistribute the characteristic F-actinmeshwork into dense aggregates of F-actin bound by XAC(wt)-GFP. BecauseXAC(wt)-GFP competes with phalloidin for F-actin binding, increasedconcentrations of Texas red-phalloidin were utilized to insurephalloidin binding and, therefore, visualization of F-actin. Also, withATP depletion, the XAC(wt)-GFP relocalized into basal and apicalregions of the cells. Therefore, with ATP depletion, XAC(wt)-GFP signalsignificantly increased and rapidly moved from a diffuse cytoplasmicdistribution into aggregates along with F-actin. To achieve thisremodeling, XAC(wt)-GFP must be activated from its predepletion stateand relocalized to bind F-actin with subsequent F-actindepolymerization and severing activity, followed by localization ofXAC(wt)-GFP along with F-actin to new abnormal actin aggregate and rodstructures (Fig. 8 ). These data extendour kidney in vivo studies by providing direct evidence thatXAC(wt)-GFP relocalizes and participates in F-actin destruction andremodeling. Finally, in cells infected with the constitutively activeform of XAC(S3A)-GFP, spontaneously occurring aggregates and rods wereseen postinfection, and 24 h later, the entire actin cytoskeletonwas disrupted. This resulted in cell detachment and death (Fig. 6 ).These data, and the lack of F-actin disruption in response to ATPdepletion in GFP- and XAC(S3E)-GFP-expressing cells (Fig. 6 ), furtherdemonstrate that activation of ADF/cofilin is required to bring aboutthese cytoskeletal alterations.
0 P( j3 B  V) \! m8 m8 e) i" |5 j3 p
* m" E+ l- g- ~7 V0 O8 nFig. 8. Model for actin aggregate formation during ATP depletion.Under physiological conditions, F-actin and the proteins of theADF/cofilin family, in conjunction with other actin-binding proteins,interact in a regulated manner to maintain the F-actin architecture ofthe cell. Through actin subunit treadmilling and ATP hydrolysis, theactin filaments are polarized with ATP-actin subunits at the barbed endand ADP-actin subunits at the pointed end. Activity of the ADF/cofilinprotein family is regulated by phosphorylation. Under physiologicalconditions, both the active and inactive phosphorylated forms arepresent ( A ). Ischemia in vivo or ATP depletion invitro induces pADF/pcofilin dephosphorylation/activation, leading toADF/cofilin proteins cooperatively binding to the ADP-actin subunits ofF-actin ( B ). Once bound to F-actin, ADF/cofilin proteinssever long actin filaments and accelerate F-actin pointed-enddepolymerization, producing both ADF/cofilin:ADP-actin dimers andADF/cofilin proteins bound to F-actin fragments ( C ). Wepostulate that the lack of cellular ATP results in unregulatedADF/cofilin-mediated F-actin destruction followed by ADF/cofilin-actinaggregate formation ( D ).
- w' S" x" b% _2 ]5 z* w! V3 n& y2 N: r; g1 U. ]
The mechanism for formation of ADF/cofilin rods and aggregates isunknown, although recent studies by Pfannstiel and coworkers ( 27 ) suggest cofilin oligomers may induce actin bundlingactivity, leading to aggregate formation. At present, there are no data to support this in LLC-PK A4.8 cells that have been ATPdepleted. Although it is possible that XAC(wt)-GFP proteins may formoligomers in response to long-term ATP depletion in oxidizingconditions, short-term ATP depletion results in a drop in intracellularpH that is not consistent with reported conditions for cofilin oligomer formation ( 27 ).
8 c$ e, I3 q- I5 q
, M2 C8 h$ g+ ]6 P9 VIn summary, these studies strongly suggest ATP depletion induced ADFdephosphorylation/activation and relocalization to mediate F-actinalterations. By expressing the ADF/cofilin protein, and through its GFPfluorescent tag, we were able to follow its activity in response to ATPdepletion. With the use of this powerful tool, we demonstrated that ATPdepletion rapidly stimulated movement of the XAC(wt)-GFP signal from adiffuse cytoplasmic distribution to localize at sites of F-actin and tonewly formed actin aggregates and rod structures. These data stronglysuggest XAC(wt)-GFP bound, depolymerized, and severed F-actin toremodel actin into XAC(wt)-GFP-containing aggregates and rods. Thesedata further substantiate a mechanistic role for ADF/cofilin proteinsin mediating the rapid actin cytoskeletal remodeling that leads to thefunctional changes observed in the biochemical, physiological, andmorphological aspects of the proximal tubule cells in response toischemia-induced injury.3 p" _8 V0 e/ O/ \0 C0 U; v

' G9 ], J. A5 |( k, [ACKNOWLEDGEMENTS
+ C7 @( ?* A2 V
2 e- r: u2 k, M# Y, G( [1 {We thank Laurie Minamide and Melanie Hosford for technicalexpertise and helpful discussions.6 J9 p  L7 @- B+ @2 y  V
          ¡¾²Î¿¼ÎÄÏס¿, E0 P# M5 a9 ?- {1 l& j* n2 R
1. Abe, H,Obinata T,Minamide LS,andBamburg JR. Xenopus laevis actin-depolymerizing factor/cofilin: a phosphorylation-regulated protein essential for development. J Cell Biol 132:871-885,1996 .
: p! R2 Y1 z1 K9 ?/ A5 N
: i% t3 U+ T/ }) i- r) i+ N. O* z9 ~/ x: U1 x4 f& }/ I! g; |
4 N$ Y# S: i0 Q- u% p5 V+ ?9 O* c
2. Arber, S,Barbayannis FA,Hanser H,Schneider C,Stanyon CA,Bernard O,andCaroni P. Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393:805-809,1998 .  q# I4 u* Y% d
7 [" d/ x$ ^- `6 b1 s/ Y/ e
/ B5 a4 C* M, F" w
8 C7 Y4 u+ I6 f9 u# k  ]* P- E
3. Ashworth, SL,Sandoval RM,Hosford M,Bamburg JR,andMolitoris BA. Ischemic injury induces ADF relocalization to the apical domain of rat proximal tubule cells. Am J Physiol Renal Physiol 280:F886-F894,2001 .
1 T# F, j. q, k8 \
7 L* U) e/ q" E* C. U1 F' c1 U$ E( _0 {+ g0 C
- J& f* \0 b/ l; P1 m/ K" Q
4. Bacallao, R,Garfinkel A,Monke S,Zampighi G,andMandel LJ. ATP depletion: a novel method to study junctional properties in epithelial tissues. I. Rearrangement of the actin cytoskeleton. J Cell Sci 107:3301-3313,1994 .2 _! {9 u+ F2 W" S9 h& w. u) @. l, V

  D2 A1 c8 h& c& |  M1 @
2 e! v: n; n' F% g( R7 b% d. E* T+ l" \4 J& H
5. Bamburg, JR. Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu Rev Cell Dev Biol 15:185-230,1999  .
& J, E" @9 ]0 l4 a" I! S( B) q+ u: Q6 @( R6 U6 ?/ @8 F
2 B: d3 A& H9 b4 U# b

5 H" F# A4 x2 D6. Bernstein, BW,Painter WB,Chen H,Minamide LS,Abe H,andBamburg JR. Intracellular pH modulation of ADF/cofilin proteins. Cell Motil Cytoskeleton 47:319-336,2000  .
" p( m  S9 n" m5 y* Z2 L/ a  |, c9 w. z; J9 C7 M
3 B$ {2 }3 E( n4 e

% S% |( l' J9 {, K" e7. Blanchoin, L,andPollard TD. Interaction of actin monomers with Acanthamoeba actophorin (ADF/cofilin) and profilin. J Biol Chem 273:25106-25111,1998 .4 c# V' ^0 k: d' J# C
, C( ?/ y: e7 _6 ]/ E! }0 I& F) ^
3 T8 s# R- W, L" Y' P
9 [6 f4 }/ Y. j2 t# j, \7 V
8. Blanchoin, L,andPollard TD. Mechanism of interaction of Acanthamoeba actophorin (ADF/cofilin) with actin filaments. J Biol Chem 274:15538-15546,1999 ., D6 e; Q: P  L( C! u

% `" Z5 }5 j# a  U7 T- N. k$ S* ~* {) B+ y7 B
, V- v: b6 G& f% d, a. U4 b* O0 s
9. Blanchoin, L,Robinson RC,Choe S,andPollard TD. Phosphorylation of Acanthamoeba actophorin (ADF/cofilin) blocks interaction with actin without a change in atomic structure. J Mol Biol 295:203-211,2000  .5 a" c. h5 _5 t& X. r' Q% s3 v

* Y* e  N0 f. r5 E6 u) n9 S0 H7 h; L2 u/ @2 D

( c% D+ z5 f$ n% p10. Canfield, PE,Geerdes AM,andMolitoris BA. Effect of reversible ATP depletion on tight-junction integrity in LLC-PK 1 cells. Am J Physiol Renal Fluid Electrolyte Physiol 261:F1038-F1045,1991 .
; |- r1 _7 E/ o6 p+ D; c
. T  {+ D- b# x# U0 q+ G2 ^
( u  N7 C1 X7 r6 x3 ]6 B$ B0 @% v2 [3 [" O0 R
11. Carlier, MF,Laurent V,Santolini J,Melki R,Didry D,Xia GX,Hong Y,Chua NH,andPantaloni D. Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility. J Cell Biol 136:1307-1323,1997 .
- E8 J1 [- _) U/ a; ?1 p
/ ?  ]2 k1 F1 \5 N# y! t% E, }+ \, @4 M' C3 W! T$ R( j) \" K

) X, ^( ]; a# t% L  _4 s+ B12. Fish, EM,andMolitoris BA. Extracellular acidosis minimizes actin cytoskeletal alterations during ATP depletion. Am J Physiol Renal Fluid Electrolyte Physiol 267:F566-F572,1994 .
. I- W1 ]5 G1 {6 L
6 X% U9 u  g3 ~3 G( @+ ?% z7 C: G) d+ c/ c$ M2 E5 Y  y- ~4 w

# j# t  V* \+ N; k) F# K4 }13. Kellerman, PS,andBogusky RT. Microfilament disruption occurs very early in ischemic proximal tubule cell injury. Kidney Int 42:896-902,1992  .
* c) m. A4 ~) L+ F& h, I1 B) c  F8 t# f. O

% m. y. g0 R: i* W: z/ o
5 h% m: ^6 `' w+ a& u14. Kellerman, PS,Clark RAF,Hoilien CA,Linas SL,andMolitoris BA. Role of microfilaments in maintenance of proximal tubule structural and functional integrity. Am J Physiol Renal Fluid Electrolyte Physiol 259:F279-F285,1990 .5 h  B% P( {1 W  s

! U" t- l( x5 p- v1 R0 Q9 A2 F/ ?
: ~' g8 F) l: F$ a  U; Q# ]6 |1 k" F  G" j& e! w8 v1 l
15. Kroshian, VM,Sheridan AM,andLieberthal W. Functional and cytoskeletal changes induced by sublethal injury in proximal tubular epithelial cells. Am J Physiol Renal Fluid Electrolyte Physiol 266:F21-F30,1994 .
$ z/ C- C0 y) N& q% c+ _: I' \$ \+ k, ]  r2 d9 {

; ~. U9 Z2 M9 |
1 T/ ~, d! L' {" {9 m- a9 N16. Maciver, SK,andWeeds AG. Actophorin preferentially binds monomeric ADP-actin over ATP-bound actin: consequences for cell locomotion. FEBS Lett 347:251-256,1994  ./ p2 B0 V" G* Y+ U0 c3 V5 ~2 }

4 M! N8 T1 `4 x8 Q7 X* V3 Z. G
: q4 v. T1 m$ l( v, L4 ]4 J
/ N7 l( U6 _" B4 w; G8 C# N17. McGough, A,Pope B,Chiu W,andWeeds A. Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function. J Cell Biol 138:771-781,1997 .+ i( o3 r( F- f; y0 ~" m2 t- }

0 ~$ z9 Z# }2 r6 w  Y- y0 y1 h
4 Y1 z5 _, i# }2 z3 z2 I3 j) ^, s
: L' t5 n/ }/ Z* N18. Meberg, PJ,andBamburg JR. Increase in neurite outgrowth mediated by overexpression of actin depolymerizing factor. J Neurosci 20:2459-2469,2000 .
% m5 V% y4 f# A( h
; [- E; B* `8 K3 v; m# }
! \. \) v& A" V- x. K# s
4 v4 c* E" B- J+ Q6 C19. Minamide, LS,andBamburg JR. A filter paper dye-binding assay for quantitative determination of protein without interference from reducing agents or detergents. Anal Biochem 190:66-70,1990  .* h- B2 J& s( a3 t

  w7 a" s: T8 X, Z8 j; O- w* y4 o7 t8 H
1 D7 T( B3 y2 E. |3 w) E! D: Z+ z, J# |2 s+ l# W: o. ?1 X& p3 i
20. Molitoris, BA. New insights into the cell biology of ischemic acute renal failure. J Am Soc Nephrol 1:1263-1270,1991 .! J* U$ f1 R4 V4 @( S5 D. Z' t

* l& d: ?  ]. Y/ ~8 ?# F1 e4 Z( M0 O" z
  g3 W! o) R. X& M  O$ B
21. Molitoris, BA. Putting the actin cytoskeleton into perspective: pathophysiology of ischemic alterations. Am J Physiol Renal Physiol 272:F430-F433,1997 ./ D: }7 S2 r: x6 y
' r7 l) F3 }; L: Y( M5 T0 P

4 J; z# a4 i+ R- {  K; v  j  }+ x  N( a
22. Molitoris, BA,Chan LK,Shapiro JI,Conger JD,andFalk SA. Loss of epithelial polarity: a novel hypothesis for reduced proximal tubule Na   transport following ischemic injury. J Membr Biol 107:119-127,1989  .2 @9 h; F3 s* Q
: m2 \$ r3 G9 [) P) _; j6 b
4 r% C4 ?) A- ?' ^
( o& D4 ^0 {, f' H8 \# P! x
23. Molitoris, BA,Falk SA,andDahl RH. Ischemia-induced loss of epithelial polarity: role of tight junction. J Clin Invest 84:1334-1339,1989  .
4 G- S, T2 j- ^' ]1 [! ^2 }$ D* I- k* w- Z& ?0 _- s  j- B! g" O- S

* ~- z) o+ x) K" _: ?# ?
6 O3 I) m" C5 w8 X* p  f% c24. Molitoris, BA,Geerdes A,andMcIntosh JR. Dissociation and redistribution of Na  ,K   -ATPase from its surface membrane actin cytoskeletal complex during cellular ATP depletion. J Clin Invest 88:462-469,1991  .
4 t4 N4 [2 l: c/ D! y+ L8 Y4 }: n" t# u; f( @+ W$ m# H1 O

6 n! A% s  C' T! F  p0 r) o3 q0 v0 I
. J0 B5 g2 B7 D( x8 b25. Morgan, TE,Lockerbie RO,Minamide LS,Browning MD,andBamburg JR. Isolation and characterization of a regulated form of actin depolymerizing factor. J Cell Biol 122:623-633,1993 .: m4 d, n' G/ U+ p$ [, M

+ N/ ^$ }/ t6 }+ e
; S7 b  s( j! @. I0 v& z3 G) X; g; [% X4 T" ?4 B0 M7 f
26. Niwa, R,Nagata-Ohashi K,Takeichi M,Mizuno K,andUemura T. Control of actin reorganization by slingshot, a family of phosphatases that dephosphorylate ADF/cofilin. Cell 108:233-246,2002  .6 ^& u& E, T: T" v

, t; g& v+ o( D% c+ h1 D# v+ _; S+ e7 j' F3 P* `% c) P
4 v( A2 n  e) W' j$ U6 f
27. Pfannstiel, J,Cyrklaff M,Habermann A,Stoeva S,Griffiths G,Shoeman R,andFaulstich H. Human cofilin forms oligomers exhibiting actin bundling activity. J Biol Chem 276:49476-49484,2001 .3 F8 \, x* G0 P
7 F/ [; u: h" }* c( i0 T! [

/ T0 H& X. c# L$ Q
( o- F% w' R+ w. m5 `2 C4 {28. Phelps, PC,Smith MW,andTrump BF. Cytosolic ionized calcium and bleb formation after acute cell injury of cultured rabbit renal tubule cells. Lab Invest 60:630-642,1989  .3 t; S' Z% U  J' |. p7 h

/ I; p: E7 g+ o' ^% i! y  }+ ^* |2 w& C7 u5 i+ d0 W* `0 j

6 S: N  O+ S, v9 g29. Schwartz, N,Hosford M,Sandoval RM,Wagner MC,Atkinson SJ,Bamburg JR,andMolitoris BA. Ischemia activates actin depolymerizing factor: role in proximal tubule microvillar actin alterations. Am J Physiol Renal Physiol 276:F544-F551,1999 .0 Y; I' d1 |. k& C5 a& H: H: p
2 V: T) G: K+ W* `+ W# A& J8 d
( s6 K. M7 J( F) }" ^  i
$ @+ m+ g6 D& d1 Y$ ?8 P+ W
30. Sutton, TA,andMolitoris BA. Mechanisms of cellular injury in ischemic acute renal failure. Semin Nephrol 18:490-497,1998  .
, E3 F* B6 n! [/ b/ Q6 v; o- g9 t% b6 z4 Z
2 L  a/ `" Y9 d8 J4 D4 C0 E
/ h! }1 ^$ \# q8 z1 i7 _: Q. s3 o# l; ]
31. Toshima, J,Toshima JY,Amano T,Yang N,Narumiya S,andMizuno K. Cofilin phosphorylation by protein kinase testicular protein kinase 1 and its role in integrin-mediated actin reorganization and focal adhesion formation. Mol Biol Cell 12:1131-1145,2001 .
' `9 R/ ^. V3 ^" @" v2 j* U+ A
2 x) a% P; U5 ]5 N; z2 g: c5 S( @8 L+ e3 n8 U$ h7 _) l
. e. B% B* V. {) a
32. Vartiainen, MK,Mustonen T,Mattila PK,Ojala PJ,Thesleff I,Partanen J,andLappalainen P. The three mouse actin-depolymerizing factor/cofilins evolved to fulfill cell-type-specific requirements for actin dynamics. Mol Biol Cell 13:183-194,2002 .
# P& Q% F0 j; g+ Y' \/ O9 s( L2 k3 t) O. f8 G

9 V7 q% [' S5 H. {6 f
4 M% K7 ~" x: j# j0 k0 a33. Venkatachalam, MA,Jones DB,Rennke HG,Sandstrom D,andPatel Y. Mechanism of proximal tubule brush border loss and regeneration following mild renal ischemia. Lab Invest 45:355-365,1981  .
0 c' {( T3 a* h( c- w- u! Z
/ @! R/ c" O+ i9 P
" O' b, O" g4 o5 y- X6 ]+ s# K) B9 d
34. Yang, N,Higuchi O,Ohashi K,Nagata K,Wada A,Kangawa K,Nishida E,andMizuno K. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 393:809-812,1998 .

tuanzi

¸Éϸ°ûÖ®¼ÒÊDz»´íµÄÍøÕ¾

ÅÝÅÝÓã

¸Éϸ°ûÐÐÒµÃÅ»§ ¸Éϸ°ûÖ®¼Ò

ÍÃÍÃ

HOHO~~~~~~  

ê¿ê¿

Äã¼ÓÓÍ°É  

³ÈζÂ̲è

ÕæÊÇÅå·þµÃÁùÌåͶµØ°¡  

foxok

ºÙºÙ  

beautylive

ºÙºÙ  

xuguofeng

¼Ò²ÆÍò¹á»¹µÃ»ØºÜ¶àÌùŶ  

ÃüÔ˵ijè¶ù

ÕâÄêÍ·£¬·Ö²»ºÃ׬°¡