Institute of Anatomy and Cell Biology I, University of Heidelberg, D-69120 Heidelberg, Germany
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ABSTRACT |
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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 F-actin 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
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INTRODUCTION |
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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 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 SH2-binding motifs and one SH3 domain, which mediates interaction with CD2AP. It was first recognized as a highly phosphorylated protein in v-src- and v-crk-transformed 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 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).
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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.
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-µm 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 anti-p130Cas (Transduction Laboratories, Lexington, KY); mouse anti-vinculin (Sigma, Deisenhofen, Germany); rabbit anti-WT-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 anti-actinin-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 (Dianova, 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 µg/ml cytochalasin D (Sigma). Microtubules were depolymerized with 1 µg/ml colcemid (Alexis, Grünberg, Germany), and stress fibers were disrupted by treating cells with 10 µM 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 anti-rabbit 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 · Cl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol), and 30 µg 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).
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RESULTS |
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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 (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.
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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, 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).
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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 actin-rich centers accompanied by largely
overlapping distribution and accumulation of CD2AP (Fig. 4,
d-f), indicative of direct or indirect
interaction with F-actin. In 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
µm.
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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 NCC-Lu-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).
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DISCUSSION |
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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 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 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-deficient 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 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 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 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 membrane 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 cross-linking (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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ACKNOWLEDGEMENTS |
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We thank Claudia Kocksch for skilled and committed technical assistance, Hiltraud Hosser for expert preparation of specimen for 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.
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FOOTNOTES |
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* T. Welsch and N. Endlich contributed equally to this work.
Y-27632 was a gift of Yoshitomi Pharmaceutical Industries, Ltd.
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{at}urz.uni-heidelberg.de).
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.
Received 9 January 2001; accepted in final form 31 May 2001.
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