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CD2AP and p130Cas localize to different F-actin
structures in podocytes
T. WELSCH,* N. ENDLICH,* W. KRIZ, AND K. ENDLICH
Institute of Anatomy and Cell Biology I, University of Heidelberg, D-69120 Heidelberg, Germany
Welsch, T., N. Endlich, W. Kriz, and K. Endlich.
CD2AP and p130Cas localize to different F-actin structures
in podocytes. Am J Physiol Renal Physiol 281: F769–F777,
2001.—Mice lacking the 80-kDa CD2-associated protein
(CD2AP) develop progressive renal failure that starts soon
after birth with proteinuria and foot process effacement by
unknown mechanisms. CD2AP has been identified and
cloned independently by virtue of its interaction with the T
cell protein CD2 and with the docking protein p130Cas. In
the present study we examined the localization of CD2AP
and p130Cas in the mouse glomerulus and in cultured podocytes. In glomeruli, CD2AP and p130Cas immunofluorescence were observed in podocytes, where they colocalized
with F-actin in foot processes. In addition, p130Cas was
strongly expressed in mesangial cells. Immunoelectron microscopy demonstrated that CD2AP was present in podocyte
foot processes without a prevailing localization. In cultured
podocytes, p130Cas was enriched at sites of focal adhesions,
where it colocalized like vinculin with F-actin at stress fiber
ends. In contrast, CD2AP colocalized with F-actin at the
leading edge of lamellipodia and in small spots, which were
unevenly distributed in the cytoplasm. The spot-shaped Factin structures were also stained by antibodies against the
actin nucleation Arp2/3 complex and cortactin, both contributing to dynamic actin assembly. Moreover, CD2AP spots in
cultured podocytes were in close spatial association with
actinin-4, but not actinin-1. Our results suggest that CD2AP
and p130Cas, which both colocalize with F-actin in podocytes
in situ, possess different functions. Whereas p130Cas is
found in focal adhesions, CD2AP seems to be involved in the
regulation of highly dynamic F-actin structures in podocyte
foot processes.
CD2AP/CMS; actin cytoskeleton; focal adhesion
THE 80-KDA CD2-ASSOCIATED PROTEIN
(CD2AP) was initially
detected because of its association with the transmembranous protein CD2 in T cells (3), being necessary for
cytoskeletal polarization in these cells. It raised even
* T. Welsch and N. Endlich contributed equally to this work.
Address for reprint requests and other correspondence: K. Endlich,
Institut für Anatomie und Zellbiologie I, Universität Heidelberg,
INF 307, D-69120 Heidelberg, Germany (E-mail karlhans.endlich
@urz.uni-heidelberg.de).
http://www.ajprenal.org
more interest when it was shown that CD2AP-deficient
mice develop progressive glomerulosclerosis and die
at the age of 6–7 wk (26). In the same study, CD2AP
was located to podocytes by colocalization with the
podocyte-specific protein synaptopodin. CD2AP contains an NH2 terminally located actin-binding site,
a proline-rich region (PXXP), and three SH3 domains,
one of which interacts with CD2 (3). Nephrin, like
CD2 a protein of the immunoglobulin superfamily and
the putative main component of the glomerular
slit diaphragm (21, 27), was coimmunoprecipitated
with CD2AP in HeLa cells. Besides its interaction with
nephrin, CD2AP was observed in colocalization with
actin in lamellipodia and membrane ruffles (11, 12).
These sites of dynamic actin assembly are further
characterized by the presence of the actin nucleation
complex Arp2/3 and other specific proteins (1, 9, 12, 25,
29, 32, 33) as well as by the appearance of transitory
motile F-actin spots (25).
Independently, Cas ligand with multiple SH3 domains (CMS), the human homolog of CD2AP, was
identified by a search for interacting molecules with
p130Cas (11). p130Cas is a 130-kDa docking protein
composed of multiple tyrosine residues forming SH2binding motifs and one SH3 domain, which mediates
interaction with CD2AP. It was first recognized as a
highly phosphorylated protein in v-src- and v-crktransformed cells (22, 23, 30) and is also phosphorylated subsequently to integrin-mediated cell adhesion
to the extracellular matrix (20, 31). Interaction with
focal adhesion kinase (FAK) and c-Crk as well as its
localization to focal adhesions (5, 13, 16, 23) indicate a
role for p130Cas in signaling pathways from cell adhesion sites to the cytoskeleton. Although detected in
glomeruli in context with proteinuric diseases (2),
p130Cas has not been studied in podocytes as yet.
In the present study, we identify the subcellular
localization of CD2AP and p130Cas in podocytes of the
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.
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society
F769
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Received 9 January 2001; accepted in final form 31 May 2001
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CD2AP AND P130CAS IN PODOCYTES
mouse glomerulus by immunofluorescence and immunoelectron microscopy. Utilizing cultured podocytes,
we show that CD2AP and p130Cas are targeted to
different cytoskeletal structures. Whereas p130Cas is
located at focal adhesions, CD2AP overlaps with actinin-4 and F-actin spots that colocalize with the actin
assembly proteins ARP2/3 and cortactin. This study
permits a further understanding of the important roles
of CD2AP and p130Cas in podocytes because alterations in both proteins are correlated with renal malfunction (2, 26).
MATERIALS AND METHODS
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Podocyte cell cultures. Cultivation of conditionally immortalized mouse podocytes was performed as previously reported (4). In brief, podocytes were maintained in RPMI-1640
(Life Technologies, Karlsruhe, Germany) supplemented with
10% FBS (Boehringer Mannheim), 100 U/ml penicillin, and
0.1 mg/ml streptomycin (Life Technologies). To propagate
podocytes, cells were cultivated at 33°C, and the culture
medium was supplemented with 10 U/ml mouse recombinant
␥-interferon (Life Technologies) to enhance expression of the
temperature-sensitive large T antigen. To induce differentiation, podocytes were maintained at 38°C without ␥-interferon for at least 1 wk before further preparation.
In addition, immunofluorescence studies were performed
on primary podocyte cell culture. To this end, glomeruli were
isolated from mouse kidneys by three-step sieving of renal
cortices (250, 100, and 70 ␮m). Isolated mouse glomeruli
were then maintained at 38°C, and outgrowth of podocytes
started at day 3 or 4. After 8 days, glomeruli were removed
and podocyte phenotype was confirmed by WT-1 immunofluorescence (15). For further immunofluorescence studies, conditionally immortalized as well as primary cultured podocytes were seeded on collagen I-coated glass coverslips
(Biochrom, Berlin, Germany).
Immunofluorescence and confocal laser scanning microscopy. Mice were perfused with 2% paraformaldehyde (PFA)
in PBS before the kidneys were removed and snap-frozen.
Kidneys were sectioned at 9-␮m thickness and blocked for 1 h
at room temperature in blocking solution (2% FBS, 2% BSA,
0.2% fish gelatin in PBS). Further washing and antibody
incubation procedures were identical to those for cultured
podocytes as recently described (4). Briefly, cells were fixed
(2% PFA, 4% sucrose), permeabilized (0.3% Triton X-100),
and blocked in blocking solution followed by antibody incubation. For colocalization with F-actin, fluorochrome (Alexa)conjugated phalloidin (Molecular Probes, Eugene, OR) was
incubated with a further specific primary antibody. The following antibodies were used: rabbit anti-CD2AP (3) (kindly
provided by Dr. A. S. Shaw, Washington University School of
Medicine, St. Louis, MO) and another rabbit anti-CD2AP
(H-290, Santa Cruz Biotechnology, Santa Cruz, CA) that
yielded identical results; rabbit anti-cortactin (34) (kindly
provided by Dr. X. Zhan, American Red Cross); mouse antip130Cas (Transduction Laboratories, Lexington, KY); mouse
anti-vinculin (Sigma, Deisenhofen, Germany); rabbit antiWT-1 (C-19, Santa Cruz Biotechnology); a rabbit antibody
against the p41 subunit of the Arp2/3 complex (kindly provided by Dr. H. Higgs, Salk Institute for Biological Studies);
mouse anti-actinin-1 clone BM-75.2 (Sigma); and mouse antiactinin-4 clone NCC-Lu-632 (1, 8) (kindly provided by Dr. S.
Hirohashi, National Cancer Center Research Institute, Tokyo, Japan). Antigen-antibody complexes were visualized
with Cy2- and Cy3-conjugated secondary antibodies (Di-
anova, Hamburg, Germany). Specimens were viewed with a
confocal laser scanning microscope (TCS-SP, Leica Microsystems, Heidelberg, Germany).
Inhibitors. Actin polymerization was inhibited by incubation of cells with 1 ␮g/ml cytochalasin D (Sigma). Microtubules were depolymerized with 1 ␮g/ml colcemid (Alexis,
Grünberg, Germany), and stress fibers were disrupted by
treating cells with 10 ␮M of the specific Rho kinase inhibitor
Y-27632 (28) (Yoshitomo Pharmaceutical Industries, Iruma-Shi
Saitama, Japan). All incubations were performed at 38°C.
Transmission electron microscopy. Mice were perfused
with 4% PFA in PBS through the left ventricle for 2 min and
afterward with 18% sucrose for 3 min. Kidney tissue was
then inhibited in 25% polyvinylpyrrolidine (molecular wt
10,000; Sigma) in 2.3 M sucrose for 2 h before being frozen in
liquid nitrogen. Kidney sections were cut at 90-nm thickness,
blocked in blocking solution (see Immunofluorescence and
confocal laser scanning microscopy) for 1 h, and finally incubated with primary antibody overnight. After being rinsed
with washing buffer (PBS containing 0.1% BSA), goat antirabbit IgG coupled to 10 nm colloidal gold (Sigma) was
applied for 1 h at room temperature. After being rinsed with
washing buffer and PBS, sections were postfixed with 2%
glutaraldehyde and 0.5% tannic acid and counterstained
with 2% OsO4 in PBS. After being stained with 2% uranyl
acetate for 2–5 min, the sections were absorption-stained
with 0.003% lead citrate in 2% polyvinyl alcohol (Sigma).
After being air dried at room temperature, the sections were
observed under a Phillips EM 301 electron microscope.
Western blotting. Podocytes were lysed in PBS containing 6
M urea and 1% Triton X-100. Protein samples were heated to
100°C for 3 min in SDS gel-loading buffer (50 mM Tris 䡠 Cl,
pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol
blue, 10% glycerol), and 30 ␮g protein/lane were separated in
a 8% SDS-polyacrylamide gel. After blotting on a polyvinylidene difluoride membrane (Millipore, Eschborn, Germany),
membranes were blocked overnight at 4°C in blocking buffer
(5% nonfat dry milk, 0.9% NaCl, 20 mM Tris, pH 7.5, 0.05%
Tween). The blot was then incubated with primary antibodies diluted in blocking buffer for 1 h at room temperature,
rinsed for 30 min with washing buffer (0.9% NaCl, 20 mM
Tris, pH 7.5, and 0.05% Tween), and incubated with horseradish peroxidase-labeled secondary anti-mouse or anti-rabbit (Sigma) antibodies for 1 h at room temperature. After a
further washing, the blot was incubated for 1 min in chemiluminescence solution (ECL, Amersham Pharmacia Biotech
UK, Buckinghamshire, UK) and finally exposed to X-ray film
(Hyperfilm ECL, Amersham Pharmacia Biotech).
RNA isolation and RT-PCR. RNA isolation of cultured
podocytes and total mouse kidneys was performed with a
mixture of guanidine thiocyanate and phenol (TRI Reagent,
Sigma) according to the manufacturer’s protocol. For detection of p130Cas and CD2AP, sequence-specific primers were
designed [p130Cas (GenBank accession no. U28151): sense
5⬘- CCA CCG TAG CCC ACC TTC TG-3⬘; antisense 5⬘-ACC
CCC TTC ACT GTT CTC-3⬘, yielding a 563-bp PCR product;
CD2AP (GenBank accession no. NM009847): sense 5⬘-CGA
GTT GGG GAA ATC ATC AG-3⬘; antisense 5⬘-TGA GGT
AGG GCC AGT CAA AG-3⬘, yielding a 504-bp PCR product].
PCR products were polymerized for 40 cycles at 58°C annealing temperature. RT-lacking reaction mixes served as negative control whereas sequence specificity of the amplicons
was confirmed by endonuclease restriction analysis (EcoRI,
KpnI: Sigma; NheI, PvuII: MBI Fermentas, St. Leon-Rot,
Germany).
CD2AP AND P130CAS IN PODOCYTES
RESULTS
Double-staining of CD2AP with F-actin predominantly showed colocalization in podocyte foot processes
around capillary loops in mouse glomeruli (Fig. 1, a–c).
Podocyte cell bodies also stained clearly for CD2AP.
CD2AP was practically absent in mesangial areas,
which possessed a high content of F-actin. Immunoelectron microscopy of mouse kidney sections demonstrated that CD2AP is randomly distributed in podocyte foot processes. CD2AP can be found close to the
slit diaphragm, at the sole plate, and elsewhere (Fig. 1,
d and e). The dominant immunofluorescence signal of
p130Cas, a CD2AP-interacting protein (11), was observed in mesangial cells in colocalization with F-actin
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(Fig. 1, f–h). Furthermore, p130Cas was present in
podocyte cell bodies and also colocalized with F-actin in
podocytes circumferentially around capillaries, but
less intensely than CD2AP.
To further study the subcellular localization of
CD2AP and p130Cas, we used a permanent podocyte
cell line. The expression of both mRNAs in cultured
podocytes was detected with RT-PCR (Fig. 2, a and b).
Specific reaction of two antibodies with the 80-kDa
CD2AP and the 130-kDa protein p130Cas used for
immunofluorescence was demonstrated by Western
blot analysis (Fig. 2, c and d). The predominant feature
of the CD2AP staining is the spotlike pattern, in addition to an occasional enrichment at the cell edge,
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Fig. 1. Localization of CD2AP and p130Cas in the mouse glomerulus. CD2AP colocalizes with F-actin around
capillary loops (arrowheads in a–c). CD2AP staining is also observed in podocyte cell bodies (arrow in c). CD2AP
is randomly distributed in podocyte foot processes, as revealed by immunoelectron microscopy (d–e). p130Cas
colocalizes with F-actin predominantly in mesangial areas but also around capillary loops (arrowheads in f–h).
p130Cas is further present in podocyte cell bodies (arrow in h). Panel width represents 110 ␮m in a–c and f–h.
Magnification in d–e: ⫻34,000.
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CD2AP AND P130CAS IN PODOCYTES
whereas p130Cas shows a diffuse cytoplasmic distribution with accumulation at distinct sites visible as short
stripes (Fig. 2, e and f). Identical distribution patterns
were observed in podocytes of primary culture (not
shown).
p130Cas colocalized with F-actin stress fiber ends,
reminiscent of anchoring proteins (Fig. 3, a–c). To
identify these sites as of focal adhesion origin, we
colocalized F-actin with the focal adhesion protein vinculin that links the ends of stress fibers to integrins
(Fig. 3, d–f). Next, we double-stained podocytes for
CD2AP and vinculin and found that the CD2AP-labeled spots do not colocalize with vinculin and that
both proteins are clearly separated from each other
(Fig. 3, g–i). Moreover, CD2AP is not found at the ends
of actin stress fibers, but it shows overlapping expression with stress fiber-independent F-actin spots in the
cytoplasm and with F-actin-enriched cell borders (Fig.
4, a–c). These F-actin spots are localized to neither
vinculin-mediated adhesion sites nor with the diffuse
cytoplasmic staining of p130Cas. After treatment for
30 min with the fungal drug cytochalasin D, which
prevents actin from polymerizing by binding to the
plus-end of actin, F-actin was retracted to a few actinrich centers accompanied by largely overlapping distribution and accumulation of CD2AP (Fig. 4, d–f), indicative of direct or indirect interaction with F-actin. In
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DISCUSSION
In HeLa cells, CD2AP has been shown to bind to the
cytoplasmic domain of nephrin (26), which is thought
to be the main component of the slit diaphragm (27).
Various mutations in NPHS1, the gene encoding nephrin, lead to the congenital nephrotic syndrome of the
Finnish type (21) whereas the absence of CD2AP in
mice resulted in severe podocyte injury associated with
proteinuria, leading to progressive glomerulosclerosis
and death (26). It was therefore speculated that
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Fig. 2. Expression of CD2AP and p130Cas in cultured podocytes.
RT-PCR yielded a 504-bp product for CD2AP (a) and a 563-bp
product for p130Cas (b). Lane 1, total kidney; lanes 2 and 3, cultured
podocytes; lane 4, cultured podocytes without RT. Specific reaction of
antibodies was confirmed by Western blotting with anti-CD2AP (c)
and anti-p130Cas (d). Antibodies gave characteristic staining patterns in podocytes, which were spotted for CD2AP (e), and diffuse/
stripelike in the case of p130Cas (f). Panel width represents 140 ␮m.
contrast, cytochalasin D treatment did not alter the
distribution of microtubuli and vimentin, and the
CD2AP spotlike pattern was unchanged after treatment for 30 min with the microtubuli-disrupting toxin
colcemid (not shown). Inhibition of Rho-kinase, involved in regulating the actin stress fibers by activation of the small GTPase Rho for 30 min with Y-27632
(28), did not affect F-actin spots and their colocalizaton
with CD2AP whereas stress fibers disintegrated (not
shown). Because of the association of CD2AP with
F-actin spots, we further examined the distribution of
the actin-nucleating complex Arp2/3 and of cortactin,
which have been shown to play a major role in dynamic
actin assembly especially in lamellipodia and transitory motile F-actin spots (9, 25, 29, 32, 33). All three
proteins, CD2AP, the p41 subunit of the Arp2/3 complex (p41ARC), and cortactin, showed similar staining
patterns of cytoplasmic spots and cell edges in colocalization with F-actin (Fig. 4, g–i). Vertical sectioning
with the confocal microscope indicated a cytoplasmic
localization of the F-actin spots that had an average
diameter of ⬃1 ␮m.
Recently, mutations in the actinin-4 gene were discovered to cause familial focal segmental glomerulosclerosis (10). We therefore examined the localization
of actinin-1 and actinin-4 with respect to the localization of CD2AP in cultured podocytes (Fig. 5). Podocytes
reacted with monoclonal antibodies BM-75.2 and NCCLu-632, which bind specifically to actinin-1 and actinin-4, respectively (1, 8). Actinin-1 was associated predominantly with stress fibers in a punctate pattern
(Fig. 5b). The highest intensity of actinin-1 fluorescence was observed at focal adhesions at the end of
stress fibers (Fig. 5b). Actinin-1 did not colocalize with
CD2AP spots (Fig. 5, c, g, and i). Actinin-4 localized
also to stress fibers; however, stress fibers were stained
in a continuous pattern (Fig. 5e). Different from actinin-1, actinin-4 staining was most intense in sharply
extending cell processes (Fig. 5e). Moreover, actinin-4
showed a rather strong perinuclear and granular cytoplasmic staining. Although CD2AP spots did not
strictly overlap with the actinin-4 distribution, almost
all CD2AP spots were in close spatial association with
actinin-4 (Fig. 5, f, h, and i). CD2AP and actinin-4 often
appeared to complement each other in forming subcellular structures, some of which were ringlike (Fig. 5h).
Actinin-1 and actinin-4 colocalization with F-actin
stress fibers and focal adhesions was confirmed by
double-labeling with phalloidin (not shown).
CD2AP AND P130CAS IN PODOCYTES
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CD2AP, interacting with the actin cytoskeleton (3, 11),
stabilizes nephrin at the slit diaphragm and thus is
necessary for the proper integrity of the filtration slit.
Our results obtained by immunoelectron microscopy
are quite compatible with a partial association of
CD2AP and the slit diaphragm. However, developing
CD2AP ⫺/⫺ mice first exhibit normal foot processes
and slit membranes and show no alteration in nephrin
expression (14), suggesting that CD2AP is neither necessary for the correct localization of nephrin at the slit
diaphragm nor for the development of proper foot processes. Moreover, our immunoelectron microscopic
analysis of the glomerular localization of CD2AP reveals that CD2AP is also present elsewhere in the
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cytoplasm of foot processes, proposing additional interaction sites.
Because CD2AP was also detected as CMS in a yeast
two-hybrid screen with the docking protein p130Cas,
we examined the glomerular expression and the subcellular localization in cultured podocytes of p130Cas
in comparison with CD2AP. p130Cas belongs to a family of docking proteins (19) and seems to be involved in
integrin-mediated signaling, becoming tyrosine phosphorylated on cell adhesion to extracellular matrix (17)
and on flow-induced shear stress (18). It is localized
both cytoplasmically and partly to focal adhesions in
untransformed NIH3T3 fibroblasts and COS-7 cells
(16). Studies in primary fibroblasts from p130Cas-de-
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Fig. 3. Localization of p130Cas to focal adhesions in cultured podocytes. p130Cas shows a diffuse cytoplasmic
staining with enrichment and F-actin colocalization at stress fiber ends (a–c). The latter localization at the ends
of stress fibers is also typical of the focal adhesion protein vinculin (d–f). In contrast, CD2AP staining exhibits
distinct spots that are clearly separated from vinculin (g–i). Panel width represents 175 ␮m.
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CD2AP AND P130CAS IN PODOCYTES
ficient mice, which show impaired cardiovascular development, revealed that p130Cas is important in actin
stress fiber formation and cell motility (6, 7). In the
present study, we assigned p130Cas to a location consistent with podocyte foot processes (Fig. 1) and demonstrated its presence in cultured podocytes as well
(Fig. 2), where it localized diffusely to the cytoplasm
with accumulation at ends of F-actin stress fibers, as is
common for focal adhesion proteins like vinculin (Fig.
3, d–f). p130Cas, lacking an actin-binding domain, is
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recruited to focal adhesions via its interaction with
FAK (19), a protein that is also expressed in podocytes
in situ (2). In accordance with the observation of increased phosphotyrosine and increased expression of
p130Cas in podocytes of human proteinuric patients
(2), these findings indicate a decisive role for p130Cas
in podocyte physiology.
In contrast, CD2AP was totally absent at focal adhesions (Fig. 3, g–i) and was not found at the ends of
stress fibers but colocalized with F-actin in cytoplasmic
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Fig. 4. Localization of CD2AP to F-actin spots in cultured podocytes. CD2AP colocalizes with F-actin in spots and
at cell edges (a–c). After treatment with cytochalasin D for 30 min, several cytoplasmic areas are devoid of CD2AP
and F-actin (d and e); F-actin becomes condensed and retracted at a few actin-rich centers but remains bound to
CD2AP, as demonstrated by colocalization (f). F-actin colocalizes at cell edges and in spots with CD2AP (g), with
the p41 subunit of the Arp2/3 complex (e), and with cortactin (f) comparably. Panel width represents 125 ␮m in a–c,
190 ␮m in d–f, and 40 ␮m in g–i.
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Fig. 5. Colocalization of CD2AP with actinin-1 and actinin-4 in cultured podocytes. Actinin-1 localizes to focal
adhesions and to stress fibers in a punctate pattern (arrows in b). CD2AP spots (a) do not colocalize with actinin-1
(c). Actinin-4 localizes to sharply extended cell processes and to stress fibers (arrows in e). CD2AP spots (d)
partially overlap with the granular cytoplasmic distribution of actinin-4 (f). At higher magnification, it is clearly
visible that CD2AP (red) and actinin-1 (green) assume separate localizations (g), whereas CD2AP (red) and
actinin-4 (green) are in close spatial association (arrows in h). This is further documented by the intensity profiles
(i) along the lines as indicated in g and h. Panel width represents 140 ␮m in a–f and 40 ␮m in g and h.
spots and at the leading edge of lamellipodia (Fig. 4,
a–c), sheetlike membrane extensions with a dense
F-actin scaffold in motile cells. Immunofluorescence
analysis succeeding treatment with cytochalasin D
proved that CD2AP is connected to F-actin. Whether
CD2AP, possessing an actin-binding site (3, 11), interacts directly or indirectly with F-actin in spots, cannot
be answered by these experiments. CD2AP spots did
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not colocalize with vinculin and even seemed to be
more numerous at cellular sites with less vinculin
distribution, proposing a different role than for cell
adhesion. Double-labeling the F-actin spots with the
p41 subunit of the actin nucleation complex Arp2/3 and
with cortactin served to further classify the actin spots
because both proteins colocalized with the F-actin
spots and exhibited an enrichment at the cell mem-
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CD2AP AND P130CAS IN PODOCYTES
We thank Claudia Kocksch for skilled and committed technical
assistance, Hiltraud Hosser for expert preparation of specimen for
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electron microscopy, and Rolf Nonnenmacher for excellent artwork.
The podocyte cell line was kindly provided by Dr. Peter Mundel. We
further thank Dr. Henry N. Higgs, Dr. Setsuo Hirohashi, Dr. Andrew
S. Shaw, and Dr. Xi Zhan for providing antibodies.
Y-27632 was a gift of Yoshitomi Pharmaceutical Industries, Ltd.
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brane similar to that of CD2AP. The Arp2/3 complex is
essential for dynamic actin assembly at the leading
edge and was shown to colocalize with and to be activated directly by cortactin (29, 32). Time-lapse imaging
of fibroblasts recently revealed that cortactin-labeled
cytoplasmic spots are motile, and some of them are
associated with endosomes (9). Because of the same
morphology and size and because of the colocalization
with Arp2/3 and cortactin, we hypothesize that the
F-actin structures bound to CD2AP in podocytes are
identical to actin spots described and characterized as
transitory motile spots related to dynamic actin assembly (25). Furthermore, expression of an activated form
of the small GTPase ADP-ribosylation factor-6 induced
the formation of motile F-actin tail structures, containing CD2AP at one end (24). Another approach to encircle the function of CD2AP is perhaps given by the
recently detected colocalization of CD2AP and the protooncogenic protein c-Cbl in transfected podocytes
overexpressing both proteins (12). c-Cbl, being bound
to CD2AP and being a substrate for tyrosine kinases,
might be involved in a signaling cascade to regulate the
actin cytoskeleton (12). All of these findings provide
evidence that small F-actin spots in podocytes are sites
of motility-related actin assembly mediated by a cluster of proteins including the adaptor protein CD2AP
that, by nature of its binding site composition, may be
involved in regulating mechanisms.
Recently, Kaplan et al. (10) presented evidence that
mutations in the gene encoding actinin-4 cause an
autosomal dominant form of focal segmental glomerulosclerosis in humans (10). Using the specific antibodies BM-75.2 and NCC-Lu-632 directed against mouse
actinin-1 and actinin-4, respectively (1, 8), we demonstrate a close spatial association of CD2AP with actinin-4 but not with actinin-1 in podocytes. Actinin-4
was discovered by means of the monoclonal antibody
NCC-Lu-632, which bound a protein that was upregulated on enhanced cell movement (8). Moreover, actinin-4 localizes to circular ruffles and is necessary for
macropinocytosis in mouse macrophages, suggesting a
function of actinin-4 in motile F-actin-based structures
(1). Because actinin-4 mutations in focal segmental
glomerulosclerosis result in enhanced F-actin crosslinking (10), it is tempting to speculate on the basis of
our results that CD2AP and actinin-4 share a critical
function in podocytes involving actin-driven motility.
To summarize, we characterized the glomerular localization of CD2AP and p130Cas, which were shown
to interact with each other in transfected human 293T
kidney epithelial cells (11). In podocytes, the subcellular distribution of p130Cas implies an integrin-dependent signaling function, whereas CD2AP was not localized to focal adhesions. According to its localization in
foot processes, its overlapping expression with p41ARC
and cortactin in F-actin spots, and its spatial association with actinin-4, we suggest a role for CD2AP in
dynamic actin assembly in podocytes.
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