1 Division of Nephrology, Department of Medicine, Indiana University, and Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 46202-5116; 2 Department of Biology, University of North Dakota, Grand Forks, North Dakota 58201; and 3 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870
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ABSTRACT |
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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.
ischemia; microvilli; actin-depolymerizing factor; XAC-GFP
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INTRODUCTION |
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ISCHEMIA-INDUCED CELL INJURY of polarized proximal tubule cells results in severe biochemical, physiological, and morphological alterations (13, 20, 33). The extent of cellular injury is affected by the length and severity of the ischemic insult (14). Cellular changes in surface membrane polarity, junctional complexes, and the actin cytoskeleton are among the earliest observed alterations (14, 22, 23, 30). Within 5 min of ischemic injury induction, renal proximal tubule actin cytoskeletal alterations begin with 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 the proximal tubule cytoplasm or lost into the proximal tubule lumen. The cellular mechanisms responsible for the microfilament alterations are not known. In addition to microvillar F-actin rearrangement in proximal tubule cells in response to ischemia, cytosolic F-actin redistributes with formation of F-actin aggregates (12, 15, 24).
Our previous in vivo data are consistent with a role for the actin-depolymerizing factor (ADF)/cofilin family of proteins in proximal tubule apical microvillar breakdown (3, 29). The ADF/cofilin family of proteins is necessary for eukaryotic cell survival, although the number and type of isoforms may vary between cell types (32). These proteins are among the most important cellular regulators of actin filament dynamics. They bind F-actin in a pH-dependent manner and have been shown to mediate F-actin severing and depolymerization (5). Under physiological conditions, ADF has a diffuse cytoplasmic distribution with little or no localization in the apical region of proximal tubule cells, but with induction of ischemia, this distribution pattern changes dramatically. Within 15 min of ischemia, the phosphorylated or inactive form of the ADF protein (25) is rapidly dephosphorylated (29) and translocated from the cytoplasm into the terminal web and apical microvilli (3). Both actin and ADF have been localized to luminal membrane vesicles that have been lost from the apical surface during ischemic injury. Although these data are consistent with participation of ADF/cofilin in destruction of the F-actin core of microvilli in response to ischemic injury of proximal tubule cells, direct proof for this role is lacking.
Therefore, the present studies were undertaken to directly evaluate the role ADF plays in F-actin destruction and reorganization during ischemic cell injury. To accomplish this goal, we utilized the proximal 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 in substrate-depleted medium (4, 10, 15, 24). Recently, adenoviral constructs containing cDNAs of the wild-type (wt) ADF/cofilin isoform, Xenopus ADF/cofilin, XAC(wt)-green fluorescent protein (GFP), the constitutively active mutant, XAC(S3A)-GFP, and the inactive mutant, XAC(S3E)-GFP, have become available (1, 18) and allowed for expression of these proteins in LLC-PK cells. These unique tools have been successfully used for expression of the wild-type ADF/cofilin isoform to directly demonstrate XAC(wt)-GFP-mediated alterations in actin dynamics in cells (6, 18). With the use of these probes, we manipulated expression of wild-type and mutant XAC-GFP isoforms and studied the effects of their expression on the actin cytoskeleton in proximal tubule cultured cells under physiological and ATP-depleted conditions. Our data indicate a direct role for ADF/cofilin proteins in mediating the severe actin cytoskeletal alterations observed in response to cellular ATP depletion in addition to dramatically impacting the rate and extent of these cellular alterations.
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METHODS |
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Cell culture. Cell culture experiments were performed on three proximal tubule cell lines, two of which were porcine cell lines clonally derived from LLC-PK(wt) (LLC-PK10 and LLC-PKA4.8), and the S1 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-PKA4.8 cell line was maintained in a low-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, at 37°C in 5% CO2 incubators. The LLC-PK10 cell line was maintained and expanded on plastic tissue culture dishes in DMEM (JRH Biosciences, no. 56-498) containing 10% FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin, pH 7.4. The S1 cells were cultured in a 50:50 mixture of Ham's F-12:DMEM supplemented with 2 mM L-glutamine, 10 mM sodium-HEPES, 2 mM sodium pyruvate, insulin, sodium selenite, and sodium bicarbonate and 7% fetal 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 ATP depletion were incubated in 0.1 µM antimycin A diluted in substrate-free DMEM (no glucose, pyruvate, serum, or amino acids), pH 7.4, or in depletion buffer, 1× PBS containing 0.5 mM CaCl2, and 1.0 mM MgCl2, pH 7.4, for designated time intervals.
Adenoviral construction. XAC-GFP (wt, S3A mutant and S3E mutant) clones were constructed in the Clontech phGFP-S65T vector by H. Abe, Chiba University, and generously shared with us. The 1,300-bp XAC-phGFP (wt, S3A and S3E) inserts were removed from the phGFP plasmid with SacI and XbaI (SacI site was blunt ended by degrading the 3' overhang with mung bean nuclease). The XAC-phGFP inserts (wt, S3A or S3E) were cloned into the XbaI and blunt ended KpnI site of the shuttle vector plasmid for adenovirus production by homologous recombination in HEK-293 cells as previously described (18). The fusion proteins were expressed under control of the immediate early promoter of the cytomegalovirus.
Adenoviral infection. The cells were infected at 40-60% confluency with a viral multiplicity of infection of 25 for 18 h with adenovirus expressing GFP, XAC(wt)-GFP, the constitutively active mutant XAC(S3A)-GFP, or the inactive mutant form XAC(S3E)-GFP. Cell cultures were harvested at 18, 28, and 51 h postinfection with cell extracts prepared and examined by SDS-PAGE, followed by Western blot analysis. By 24 h postinfection, 70-80% of the treated cells were expressing XAC-GFP isoforms as observed by epifluorescence microscopy. All studies were done at 24 h postinfection unless otherwise stated.
SDS-PAGE and Western analysis. LLC-PK or S1 cellular proteins were extracted in a 2% SDS buffer (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. For immunodetection, the rabbit primary antibodies to XAC (1:10,000), to the 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), were utilized and followed by horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (1:30,000). Protein bands were detected by enhanced chemiluminescence (Pierce, Rockford, IL) or stained with 4-chloro-1-napthol and quantified by densitometry.
Microscopy. LLC-PKA4.8 cells were fixed in 4% paraformaldehyde or 3.7% formaldehyde for 1 h and permeabilized with 0.1% Triton X-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 confocal microscope (Bio-Rad, Hercules, CA) using a Nikon Diaphot 200 inverted microscope with a ×100, 1.4-numerical aperture (NA) oil-immersion objective or a ×60, 1.2-NA water-immersion objective. Live cell images were captured with a Nikon Diaphot inverted microscope with a ×40, 0.85-NA objective and a PXL cooled charge-coupled device camera with a Kodak 1400 chip (Photometrics, Tucson, AZ). Metamorph software (Universal Imaging, West Chester, PA) was used to process the images and to reconstruct basal-to-apical three-dimensional reconstructions.
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RESULTS |
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ATP depletion of LLC-PKA4.8 cells reduces pcofilin
signal.
Initial studies were undertaken to determine the effect of ATP
depletion on the phosphorylation status of cofilin in
LLC-PKA4.8 cells. ADF and cofilin are two highly conserved
and related proteins, but differentially expressed proteins with
similar, but distinct, actin-binding properties belonging to the same
family of actin-associated proteins (5, 32). With the use
of isoform-specific antibodies and an anti-phosphoepitope antibody that
recognizes the phosphorylated form of each isoform, the cellular
expression of these proteins can be determined. With the use of these
probes, we found that the endogenous expression of ADF and cofilin
isoforms in porcine proximal tubule cell lines was not
equivalent. The LLC-PK4.8 cells had ample expression of
cofilin and little or no expression of ADF (Fig.
1A). The LLC-PK10
cell line expressed ADF with no expression of cofilin, whereas the
S1 mouse cell line expressed both isoforms.
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Expression of XAC-GFP through adenoviral infection of
LLC-PKA4.8 cells.
To obtain direct evidence regarding the role of cofilin-mediated
cellular actin destruction and reorganization and microvillar F-actin
core degeneration during ATP depletion, we utilized adenoviral vectors
containing GFP, XAC(wt)-GFP, XAC(S3A)-GFP, or XAC(S3E)-GFP cDNA to
express GFP or ADF/cofilin protein isoforms in LLC-PKA4.8 cells. In characterization studies, LLC-PKA4.8 cells
infected with adenovirus containing the cDNA for XAC(wt)-GFP
demonstrated expression of the XAC(wt)-GFP fusion protein as early as
18 h postinfection, as detected by GFP fluorescence (Fig.
2A) and
Western blotting techniques (Fig. 2B). The fraction of
GFP-expressing cells increased from ~70 to 90% over the 51-h period
postinfection (Fig. 2A). The level of XAC(wt)-GFP expression
increased linearly over the 51-h period postinfection (Fig.
2B). The level of phosphorylated XAC(wt)-GFP, as detected by
Western blot analysis, also increased linearly over the 51-h period
postinfection (Fig. 2B). Therefore, as the wild-type XAC-GFP
protein was expressed, it was regulated through phosphorylation by a
cellular kinase. The level of endogenous cofilin, as detected by
Western blot analysis, remained constant during the first 18 h
postinfection of XAC(wt)-GFP, but decreased endogenous cofilin levels
were observed at 28 and 51 h postinfection.
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XAC-GFP expression did not alter F-actin under physiological
conditions.
To determine the effect of XAC(wt)-GFP expression on the F-actin
cytoskeleton of LLC-PKA4.8 cells, cells were stained with rhodamine or Texas red-phalloidin 24 h postinfection with
adenovirus containing XAC(wt)-GFP. In Fig.
3, A-F, reconstructed
basal-to-apical images and single-plane basal images of
uninfected control cells (A and B) and
XAC(wt)-GFP-expressing cells (C-F) are presented. In
Fig. 3, C-F, comparison of actin cytoskeletal stress
fibers, microvillar microfilaments, and cortical actin
network can be drawn between high (a), medium
(b), and low (c) XAC(wt)-GFP-expressing cells and
nonexpressing cells (d). These data demonstrate that adenoviral infection and XAC(wt)-GFP expression did not affect the
distribution or composition of the dense F-actin bundles that compose
basal stress fibers, apical microvillar microfilament cores, or the
cortical actin orientation of LLC-PKA4.8 cells, suggesting
XAC(wt)-GFP expression does not alter cellular actin architecture under
physiological conditions, implying physiological regulation and
function of the XAC(wt)-GFP proteins.
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XAC(wt)-GFP translocates to the surface membrane domain in response
to ATP depletion.
Basal-to-apical reconstructions (x-z axes images)
demonstrate that, under physiological conditions (Fig.
4A), F-actin primarily located
to basal and lateral aspects of the cell and in the microvilli at the
apical surface. The expression of XAC(wt)-GFP under physiological conditions (Fig. 4B) was primarily detected in the cytoplasm
of the LLC-PKA4.8 cells with little or no colocalization of
fluorescence with Texas red-phalloidin F-actin staining in the apical,
basal, or lateral cellular regions. However, XAC(wt)-GFP-expressing
cells that were ATP depleted for 25 min (Fig. 4C) had
intense XAC(wt)-GFP fluorescence, and colocalization of XAC(wt)-GFP
with 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 in
agreement with and extend our previous observations showing rapid
relocalization of ADF to the apical domain of rat proximal tubule cells
during ischemia (3).
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XAC mediates F-actin aggregation and rod formation during ATP
depletion.
We next sought to determine the effect of XAC expression and ATP
depletion on the F-actin cytoskeleton. Cell monolayers infected with
the XAC(wt)-GFP adenovirus were ATP depleted with antimycin A in
depletion media for 25 min and stained for F-actin using Texas
red-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, ATP
depleted for 25 min (Fig. 5, A-C, arrows), were
characterized by minimal disturbance in the fine-mesh cortical and
stress fiber F-actin staining (Fig. 5A, arrow). These data are similar to what we previously described under physiological or
short-term, ATP-depleted conditions (24). However, in
XAC(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 greater
extent than cells with lower expression levels. Colocalization of
XAC(wt)-GFP and F-actin, as demonstrated by intense yellow fluorescence
(Fig. 5C, open square), was apparent in
XAC(wt)-GFP-expressing cells. The F-actin- and XAC-GFP-stained
aggregates had a much brighter GFP signal than the Texas red-phalloidin
F-actin signal. We believe this difference in staining properties
results from the known competition between XAC-GFP and phalloidin for
F-actin binding (5, 17).
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ATP depletion induces rapid formation of aggregates and rods in
cells expressing XAC(wt)-GFP.
Next, we sought to determine the time course of F-actin alterations in
control and XAC(wt)-GFP-expressing cells in response to ATP depletion.
Rapid and extensive appearance of XAC-GFP/F-actin aggregates and rods
would directly indicate an important and early role for ADF/cofilin
proteins in mediating F-actin disruption. To test this hypothesis, we
undertook ATP depletion studies of cells infected with either
XAC(wt)-GFP, GFP, or XAC(S3E)-GFP. In GFP- and XAC(S3E)-GFP-expressing
cells, as well as in uninfected cells, we did not observe alterations
to the actin cytoskeleton comparable with the severe alterations
observed in XAC(wt)-GFP-expressing cells in response to the same time
of ATP depletion (Figs. 5 and 6). XAC(wt)-GFP-, GFP-, and
XAC(S3E)-GFP-expressing cells all demonstrated a high percentage of GFP
signal, indicating a similar level of infection and GFP protein
expression. In addition, the wild-type XAC(wt)-GFP-expressing cells
appeared similar in morphology to uninfected cells or cells infected
with GFP or the inactive S3E mutant. During ATP depletion, the GFP
intensity and distribution at 2 min were comparable in the
XAC(wt)-GFP-, GFP-, XAC(S3E)-GFP-expressing cells (Fig. 7,
A, D, and
G). A homogenous cytosolic
distribution of GFP was observed, and nuclear localization was also
noted. 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- or
XAC(S3E)-GFP-expressing cells (data not shown). By 20 min, cells
expressing XAC(wt)-GFP had a reduction in the homogenous cytosolic
XAC(wt)-GFP signal and an accumulation of cytoplasmic XAC(wt)-GFP-stained aggregates (Fig. 7B, arrows). By 40 min,
this effect was further enhanced in the XAC(wt)-GFP-infected cells (Fig. 7C), but the GFP- and XAC(S3E)-GFP-expressing cells
still demonstrated no change in the diffuse GFP fluorescence (Fig. 7, F and I).
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DISCUSSION |
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This is the first study to directly demonstrate that the ADF/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 by severing 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-actin polymerization affected to a greater extent than ATP-actin polymerization (11). The actin-binding properties of this family of proteins are primarily regulated by phosphorylation and dephosphorylation. Also, ADF/cofilin proteins compete for F-actin binding with other actin-binding proteins and phalloidin. Two kinase families have been identified to specifically phosphorylate ADF/cofilin on serine-3, each with different upstream regulators. The Lim kinase family, the first identified ADF/cofilin-specific kinase, is phosphorylated, and its kinase activity is significantly increased through downstream effects of the Rho family of small GTPases, Rac, Rho, and Cdc42. In turn, the activated Lim kinase phosphorylates and inactivates the ADF/cofilin protein family (2, 34). The second family of ADF/cofilin-specific kinases, the testicular protein kinase family (TESK1 or TESK2), includes serine/threonine kinases stimulated through the integrin-mediated signaling pathway (31). Phosphorylated ADF/cofilin proteins can no longer bind F- or G-actin to regulate actin dynamics (9, 25). Recently, the ADF/cofilin-specific phosphatase slingshot has been shown to dephosphorylate and activate ADF/cofilin at serine-3 (26).
Our previous studies suggested the ADF/cofilin family of proteins played a significant role in ischemia-induced renal cell injury of proximal tubule cells (3, 29). Acute renal failure mediates functional changes in the biochemical, physiological, and morphological 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 extremely sensitive because they contain the majority of F-actin in these cells (14, 15, 24). Clinical consequences resulting from ischemic injury include tubular obstruction from apical membrane blebbing, back-leak between cells that have loss their junctional complex integrity, reduced Na+ reabsorption from redistribution of ion pumps in the membrane, and abnormal tubuloglomerular feedback (30).
Changes in the actin cytoarchitecture occur early and precede the other observed 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, we observed 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-induced injury, the apical membrane begins to coalesce and form luminal or cytoplasmic blebs or vesicles containing high concentrations of ADF and G-actin. In addition, microvillar microfilament destruction is concurrent with increased G-actin concentration in the apical membrane region. These events occur in a time frame to suggest that ADF locates to this region to participate through F-actin severing and depolymerization in the breakdown of the microvillar microfilament core. In addition to microvillar microfilament changes, aggregates of F-actin have been observed in the cytoplasm of injured proximal tubule cells (12, 15, 24).
Although our previous studies suggested dephosphorylation/activation
and relocalization of ADF were coincident with microvillar microfilament core disintegration in response to ischemic
injury, we could not directly test the involvement of ADF in this
process. Therefore, to directly evaluate the role of the ADF/cofilin
family of proteins in proximal tubule cell actin alterations, we
expressed the ADF/cofilin isoform XAC(wt)-GFP by adenoviral infection
in the proximal tubule cultured cell line LLC-PKA4.8. In
these cells, endogenous cofilin expression is <0.1% of the total
protein concentration (data not shown). With expression of XAC(wt)-GFP,
we observed a decrease in endogenous cofilin levels, suggesting that
endogenous cofilin played a minimal role in actin alterations in
response to ATP depletion in XAC(wt)-GFP
LLC-PKA4.8-expressing cells. Although expression of GFP,
XAC(S3E)-GFP, or XAC(wt)-GFP in these cells did not alter the integrity
of their actin cytoskeleton, inducing ATP depletion in the
XAC(wt)-GFP-expressing cells resulted in extremely rapid and extensive
changes in the actin cytoarchitecture (Figs. 3, 5, and 6) comparable to
the phenotype observed in uninfected cells that underwent a much longer
ischemic 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 were
not observed in uninfected cells until after >30 min of ATP depletion.
These aggregates were primarily located in the cytoplasm, although rods
were also observed in the nucleus. As the number of XAC(wt)-GFP/actin
aggregates increased, stress fibers and the fine meshwork of the
cortical F-actin disappeared, suggesting XAC(wt)-GFP bound F-actin to
depolymerize, sever, and redistribute the characteristic F-actin
meshwork into dense aggregates of F-actin bound by XAC(wt)-GFP. Because
XAC(wt)-GFP competes with phalloidin for F-actin binding, increased
concentrations of Texas red-phalloidin were utilized to insure
phalloidin binding and, therefore, visualization of F-actin. Also, with
ATP depletion, the XAC(wt)-GFP relocalized into basal and apical
regions of the cells. Therefore, with ATP depletion, XAC(wt)-GFP signal
significantly increased and rapidly moved from a diffuse cytoplasmic
distribution into aggregates along with F-actin. To achieve this
remodeling, XAC(wt)-GFP must be activated from its predepletion state
and relocalized to bind F-actin with subsequent F-actin
depolymerization and severing activity, followed by localization of
XAC(wt)-GFP along with F-actin to new abnormal actin aggregate and rod
structures (Fig. 8). These data extend
our kidney in vivo studies by providing direct evidence that
XAC(wt)-GFP relocalizes and participates in F-actin destruction and
remodeling. Finally, in cells infected with the constitutively active
form of XAC(S3A)-GFP, spontaneously occurring aggregates and rods were
seen postinfection, and 24 h later, the entire actin cytoskeleton
was disrupted. This resulted in cell detachment and death (Fig. 6).
These data, and the lack of F-actin disruption in response to ATP
depletion in GFP- and XAC(S3E)-GFP-expressing cells (Fig. 6), further
demonstrate that activation of ADF/cofilin is required to bring about
these cytoskeletal alterations.
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The mechanism for formation of ADF/cofilin rods and aggregates is unknown, although recent studies by Pfannstiel and coworkers (27) suggest cofilin oligomers may induce actin bundling activity, leading to aggregate formation. At present, there are no data to support this in LLC-PKA4.8 cells that have been ATP depleted. Although it is possible that XAC(wt)-GFP proteins may form oligomers in response to long-term ATP depletion in oxidizing conditions, short-term ATP depletion results in a drop in intracellular pH that is not consistent with reported conditions for cofilin oligomer formation (27).
In summary, these studies strongly suggest ATP depletion induced ADF dephosphorylation/activation and relocalization to mediate F-actin alterations. By expressing the ADF/cofilin protein, and through its GFP fluorescent tag, we were able to follow its activity in response to ATP depletion. With the use of this powerful tool, we demonstrated that ATP depletion rapidly stimulated movement of the XAC(wt)-GFP signal from a diffuse cytoplasmic distribution to localize at sites of F-actin and to newly formed actin aggregates and rod structures. These data strongly suggest XAC(wt)-GFP bound, depolymerized, and severed F-actin to remodel actin into XAC(wt)-GFP-containing aggregates and rods. These data further substantiate a mechanistic role for ADF/cofilin proteins in mediating the rapid actin cytoskeletal remodeling that leads to the functional changes observed in the biochemical, physiological, and morphological aspects of the proximal tubule cells in response to ischemia-induced injury.
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ACKNOWLEDGEMENTS |
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We thank Laurie Minamide and Melanie Hosford for technical expertise and helpful discussions.
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FOOTNOTES |
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This research was supported by National Institutes of Health (NIH) Grants 1P01-DK-53465, 1R01-DK-41126, and Veterans Affairs Merit Review grants (to B. A. Molitoris), American Paralysis Association Grant BB2-9601 (to P. J. Meberg), and NIH Grants GM-35126 and NS-40371 (to J. R. Bamburg).
Address for reprint requests and other correspondence: B. Molitoris, Division of Nephrology, Indiana Univ. School of Medicine, 1120 South Dr., FH 115, Indianapolis, IN 46202-5116 (E-mail: bmolitor{at}iupui.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 3, 2002;10.1152/ajprenal.00210.2002
Received 5 June 2002; accepted in final form 22 November 2002.
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