* Department of Clinical Biology and Medicine, University of Tokushima Graduate School of Medical Sciences, Tokushima; Department of Structural Pathology, Institute of Nephrology, Graduate School of Medical and Dental Sciences; Cooperative Laboratory for Electron Microscopy, Niigata University, Niigata; and Department of Anatomy, Juntendo University School of Medicine, Tokyo, Japan
Correspondence: Dr. Hiroyasu Tsukaguchi, Department of Clinical Biology and Medicine, The University of Tokushima Graduate School of Medical Sciences, 3-18-15 Kuramoto, Tokushima, 770-0042, Japan. Phone: +81-88-633-7184; Fax: +81-88-633-9245; hiroyasu{at}clin.med.tokushima-u.ac.jp
Received for publication October 3, 2006.
Accepted for publication May 22, 2007.
The predominant type of cellular junction between normal podocytefoot processes is the slit diaphragm. Under nephrotic conditions,however,foot process effacement leads to the loss of slit diaphragmsand the new formationof tight junctions composed of the proteinscoxsackievirus and adenovirus receptor (CAR) and zonula occludens1 (ZO-1). Podocin, a protein that plays a key role in maintainingthe integrity of the slit diaphragm, has also been localizedto these tight junctions, but its function at this site is unknown.In this study, we confirmed that podocin colocalizes with CARand ZO-1 at the tight junction between foot processes in nephroticrats. Using primary cultures of rat podocytes, as well as celllines that co-expressed podocin and CAR, we observed that podocinwas recruited to sites of cell–cell contact and that itco-localized with CAR and ZO-1. Immunoprecipitation suggestedthat these three junctional proteins from a multi-protein complex.Consistent with this, we found that podociin facilitated thecoalescence of preassembled lipid rafts containing CAR and restrictedtheir lateral mobility, the latter likely a result of dynamicactin reorganization and subsequent tethering of CAR-podocincomplexes to the cytoskeleton. In conclusion, in addition toserving as a structural protein of the slit diaphragm of normalpodocytes, our data suggest that podocin may also serve as ascaffold that links tight junction proteins to the actin cytoskeletonin nephrotic foot processes.
The slit diaphragm (SD) is maintained by multiple molecularinteractions that ensure stable anchorage of the membrane complexesto the actin cytoskeleton.1, 2 Several podocyte proteins playa key role in the maintenance of SD integrity. Nephrin is atransmembrane protein with eight Ig-like domains and constitutesa major structural backbone of the SD.3 Podocin is a 42-kD integralmembrane protein that interacts with nephrin at the SD.4, 5It has a hairpin-like structure, the membrane topology of whichis reminiscent of caveolin.6 Similar to caveolin, podocin ispresent in high-order oligomeric complexes and localizes inso-called "rafts," the specialized membrane microdomains wheresphingolipids and cholesterol are highly enriched.5, 7 The multiproteinSD complexes are assembled through the rafts.8
There are two types of cellular junctions in the foot process:SD and tight junction (TJ).9, 10 TJ is defined by the morphologicfeatures of fusion points on the opposing membranes. The characteristicsare distinguishable from SD that forms the junctions with theusual 20- to 50-nm intercellular gap. In normal foot processes,the SD constitutes a major cellular junction, whereas the TJis rarely encountered. Under nephrotic conditions of human11and rat,9 the TJ complex newly appears at the interface of footprocesses as it replaces the vanishing SD and/or emerges belowthe apically dislocated SD.9, 12 The TJ complex represents immatureassembly that evolves into the SD during glomerular development,and these two junctions share the common structural elementzonula occludens-1 (ZO-1), a cytosolic scaffold protein.10 Studiesin rats with puromycin aminonucleoside (PAN) nephrosis revealedthat coxsackievirus and adenovirus receptor (CAR), a transmembraneprotein with two extracellular Ig-like domains, serves as aTJ component in nephrotic foot processes.13 Besides the originallyidentified function as a virus receptor,14 CAR serves as a TJprotein that links the adhesion complexes to F-actin throughinteraction with ZO-1.15
Localization of podocin at the TJ complex has reportedly beenobserved,16 but the biologic relevance and underlying molecularbasis remain to be elucidated. These observations led us toexplore whether podocin is implicated in TJ complex formation.In this study, we investigated the role of podocin in TJ complexby focusing on the interaction with a new binding partner CARand ZO-1.
Podocin Co-localizes with CAR at the TJ-Like Structures in PAN Glomeruli
Ultrastructural analysis of normal rat glomeruli demonstratedthat the vast majority of podocin is primarily localized atthe cytoplasmic face of the plasma membrane adjacent to theSD. In contrast, CAR was not found anywhere in the foot process,with the exception of the TJ-like structures that were onlyrarely encountered under normal conditions. ZO-1 was concentratedalong the cytoplasmic surfaces of the SD in association withpodocin (data not shown). In PAN glomeruli at day 4, CAR accumulatedexclusively at TJ-like structures between adjacent foot processesbut was rarely located at the SD (Figure 1, A through D). Incontrast, podocin became more frequently co-localized with CARat sites of cell–cell contacts in opposing foot processes,whereas some podocin remained in its usual position at the SD.Quantification revealed that podocin distributes to the SD andTJ-like structures in almost equal frequency (Table 1). In PANglomeruli at day 11 (Figure 1, E through I), podocin and CARaccumulated along the regions of close contact between adjacentfoot processes, where ZO-1 co-distributed. These data suggestthat CAR is preferentially involved in the formation of TJ complexesbut not SD, whereas podocin is implicated in the assembly ofboth SD and TJ complexes.
Figure 1. Podocin localizes to various junctions in nephrotic foot processes. (A through D) Double-immunogold staining of ultrathin sections from the periodate-lysine-paraformaldehyde–fixed rat glomeruli at 4 d after puromycin aminonucleoside (PAN) injection. There are a series of intercellular junctions with varying gap distance: Normal slit (A), tight junctions between adjoining foot processes (C and D), and both (B). Podocin (arrow, 5 nm gold) and coxsackievirus and adenovirus receptor (CAR; arrowhead, 15 nm gold) are depicted. Bar = 0.2 µm. (E and F) Cryosections of rat glomeruli at 11 d after PAN injection are double labeled with zonula occludens-1 (ZO-1; arrows, 5 nm gold) and CAR (arrowheads, 10 nm gold). Note that CAR preferentially localizes along the regions of close contact between adjacent foot processes. Bar = 0.1 µm. (G through I) Cryosections of rat glomeruli on PAN 11 are double labeled with ZO-1 (arrows, 5 nm gold) and podocin (arrowheads, 10 nm gold). *Apically dislocated slit diaphragm (SD). Note that podocin distributes to both shifted SD and tight junction (TJ)-like structures. Bar = 0.1 µm. Expression of CAR upregulated by approximately four-fold at 4 d after PAN injection, whereas that of podocin did not significantly alter during 10 d (Supplementary Figure 1).
Table 1. Frequency of immunogold-positive junctions of nephrotic foot processesa
Podocin Is Recruited to the Cell–Cell Junctions in Cultured Cells
In COS-7 cells co-expressing CAR and podocin, both proteinsprimarily exhibit a diffuse punctate pattern throughout thecytoplasm, although some expression also is distributed alongthe edge of the cell as well (Figure 2A). The quantificationof intracellular vesicles revealed that 30 to 40% of podocin-positivepuncta co-localize with CAR-labeled spots and vice versa (Figure 2B),suggesting that a significant proportion of the two proteinsmay be sorted into the same vesicles. It is notable that podocinis remarkably concentrated along cell–cell borders whenneighboring cells came into contact (Figure 2A). A similar distributionof podocin was observed for primary cultured podocytes (Figure 2C),showing that the endogenous podocin was expressed at sites ofcell–cell contacts, at which ZO-1 concomitantly accumulated.However, podocin was virtually absent at the cellular free edges.These observations suggested that cell–cell attachmentis required for translocation of podocin to the cell–cellinterface. When MDCK cells co-expressing CAR and podocin weresparsely plated at a lower density, both proteins showed a diffusecytoplasmic punctate vesicle pattern. Once these cells cameinto cell contact and acquired cell polarity, both CAR and podocinconcentrated focally at the points of cell–cell contactswith ZO-1 (Figure 2D). The data indicate that a proportion ofpodocin is recruited to cell–cell contacts in associationwith CAR and ZO-1.
Figure 2. Podocin is recruited to the cell–cell junctions and co-localizes with CAR and ZO-1. (A) Double labeling of CAR (red) with podocin (green) in transiently transfected COS-7 cells. Both CAR and podocin co-localize in the cytoplasmic vesicles (arrowheads) and preferentially concentrate along cell–cell contacts (arrows). (B) Proportion of a CAR–podocin double-labeled cytoplasmic vesicle within the total number of intracellular vesicles expressing either CAR or podocin is presented. Bar = 10 µm. Results are representative of three independent experiments. (C) Primary cultured podocytes migrating from the decapsulated rat glomeruli. Podocin co-localizes with ZO-1 along cell–cell contacts (arrows) and not at the cellular free edges (arrowheads). Bars = 10 µm. CAR co-merges with ZO-1 along cell–cell contacts (data not shown). (D) Polarized epithelial MDCK cells stably expressing podocin. A series of sections are shown at the apical portion (marker gp135, red), at the TJ (marker ZO-1, red), and at the basolateral portion underneath the TJ (co-staining with ZO-1, red). (Top) Vertical section view. Podocin (green) localizes broadly alongside the basolateral membrane and accumulates focally at the cell–cell contacts with ZO-1. Note that no podocin staining is seen on the apical membrane. Bars = 10 µm.
Podocin Physically Interacts with the TJ Markers
Immunoprecipitation assays revealed that podocin co-precipitatedwith CAR in lysates from CAR and podocin co-expressing COS-7cells and vice versa (Figure 3A). Pull-down assays with lysatesfrom COS-7 cells and rat glomeruli revealed that at least thelast C-terminal 65 amino acids of CAR are essential to mediatethe interaction with podocin (Figure 3, B and C) and that theC-terminal 122 amino acids of podocin are crucial for the bindingto CAR (Figure 3, D and E). Endogenous ZO-1 co-precipitatedpodocin as a complex from COS-7 cells expressing podocin (Figure 3F)and also co-precipitated CAR from those expressing CAR (Figure 3G).In reciprocal experiments, podocin and CAR co-precipitated endogenousZO-1. These data indicate that podocin physically interactswith CAR and ZO-1 and forms a multiprotein complex, througheither a direct or an indirect association in these cells.
Figure 3. Podocin interacts with CAR and ZO-1. (A) Podocin and V5-tagged CAR co-immunoprecipitate each other. Lysates from COS-7 cells coexpressing V5-tagged CAR and podocin were precipitated with a control IgG or indicated antibodies. Arrowheads mark the immunoprecipitation products. IgG, IgG heavy chains. Membrane topology of CAR (B) and podocin (D). TM, transmembrane domain. (C) The C-terminus of CAR interacts with podocin. GST pull-downs using lysates from podocin-expressing COS-7 cells and rat glomeruli reveal that CAR binds podocin through its C-terminus. (E) The C-terminus of podocin is associated with CAR. GST pull-down assay using lysates from L cells stably expressing CAR (L-CAR) shows that the C-terminus of podocin interacts with CAR. (F and G) CAR and podocin interact with endogenous ZO-1 and vice versa. Lysates from COS-7 cells expressing either FLAG-tagged podocin (F) or V5-tagged CAR (G) are immunoprecipitated with a control IgG or polyclonal antibodies against podocin, CAR (H300), and ZO-1.
CAR Interacts with Podocin through Lipid Rafts
Antibody cross-linking is a reliable technique that allows detectionof even weak association to the rafts.17, 18 Upon antibody-inducedcross-linking, CAR preferentially co-patched with the raft markerganglioside GM1 (labeled by Cholera toxin B subunit, 39.8 ±8.3%; n = 5) rather than the nonraft marker transferrin receptorTfR del 4 to 51 (21.2 ± 7.3%; n = 6; Figure 4, A throughD and G). The cholesterol-depleting agent methyl cyclodextrininhibited antibody-induced clustering of CAR (data not shown),indicating that CAR forms a large cluster in a cholesterol-dependentmanner. When CAR-podocin co-expressing COS-7 cells were cross-linked,podocin co-localized with clustered CAR at the cell surface(Figure 4, E and F), but coexpression of podocin per se didnot further augment the extent of co-localization of CAR withthe raft marker GM1 (39.8 ± 8.3% without podocin versus40.3 ± 7.2% with podocin; n = 4 to 6; Figure 4G). Theresults indicate that CAR clusters via a raft-mediated mechanismand that podocin supports the coalescence of preexisting smallrafts containing CAR.
Figure 4. Clustered CAR preferentially partitions in lipid rafts in living cells. Surface of COS-7 cells expressing CAR alone (A) and of those coexpressing CAR and V5-tagged transferrin receptor (TfR; C) is cross-linked by anti-RmcB mAb, Cholera toxin B subunit (CT-B) biotin, and anti-V5 polyclonal antibodies. (Insets) Non–cross-linked control. (B and D) Higher magnification of boxed regions in A and C, respectively. Cross-linked CAR (green) co-patches with the raft marker GM1 (B) but co-patches to a lesser degree with the nonraft marker TfR (D). (E) Clustered CAR co-patches with GM1 and podocin. (F) Higher magnification of boxed portion in E. Arrowheads indicate discrete spots where CAR, podocin, and GM1 co-localize. (G) Proportion of CAR that co-merges with the raft or nonraft markers is quantified. CAR co-clusters more frequently with GM1 than with TfR. Bars = 10 µm.
Podocin Enhances Raft Association and Oligomerization of CAR
We examined the biochemical property of detergent-resistantmembrane containing CAR.19 The floating analysis with lysatefrom COS-7 cells and isolated rat glomeruli revealed a remarkablepartitioning of CAR into cholesterol-rich fractions (Figure 5and Supplementary Figure 2). Clustering and oligomerizationof raft proteins reportedly promote the coalescence of morerafts and thereby stabilize the microdomains.17 We thereforeinvestigated the effects of podocin coexpression and antibodycross-linking on raft association of CAR. Antibody cross-linkingof CAR or podocin coexpression alone led to a slight increasein raft association of CAR of 1.2-fold (32.7 ± 6.2%;n = 2) and 1.8-fold (48.4 ± 2.4%; n = 3), respectively,compared with control (26.3 ± 3.2%; n = 5; Figure 6,A and B). However, the combination of cross-linking and podocincoexpression resulted in increased CAR floating to the rafts(69.3 ± 1.2% versus control; P < 0.01; n = 5; Figure 6,A and B). Continuous sucrose gradient ultracentrifugation analysisdemonstrated that both podocin and CAR form oligomers in high-ordercomplex (Figure 6C; data not shown for podocin). Podocin coexpressionled to a slight shift of CAR into the higher molecular weightfraction (n = 5; P < 0.05; Figure 6, C and D). Our data indicatethat CAR ectodomain engagement and podocin coexpression cooperativelyenhance raft partitioning of CAR by promoting its oligomerization.
Figure 5. Podocin and CAR partition into the raft fractions in COS-7 cells. (A) Floating assay using 1% Brij 98 extracted lysates from COS-7 cells coexpressing CAR and podocin reveals that CAR and podocin co-fractionate to the low-density, detergent-resistant membrane (DRM) fractions with the raft marker of caveolin-1 and GM1. (B) Cholesterol content in each fraction is shown as a proportion in the total amount from all fractions. The preliminary data indicated that CAR has a considerably weaker affinity to rafts than nephrin or podocin and that the Brij 98 is a suitable detergent for the floating assay (Supplementary Figure 3.
Figure 6. Raft partitioning of CAR is enhanced by antibody-induced cross-linking of CAR and podocin coexpression. (A) The effects of podocin coexpression and CAR ectodomain cross-linking on the raft partitioning of CAR were examined by the floating assay using 0.2% Triton-X100 resistant membrane preparations. Either podocin coexpression or cross-linking alone did not largely influence the raft affinity of CAR. Both treatments synergistically augment raft association of CAR. Raft fractions (R) are separated from nonraft (NR) fractions by the sucrose density gradient centrifugation of 0.2% TX-insoluble material. CAR was detected by a polyclonal antibody directed against the ectodomain of CAR (H-300). The detailed methods of raft isolation are provided in Supplementary Figure 2. (B) Proportion of raft-associated CAR (R) in the total amount of proteins (R+NR) in all fractions is quantified (*P < 0.01; n = 5). (C) Effects of podocin on oligomerization property of CAR are examined by flotation on velocity gradient centrifugation. Molecular mass standards are indicated by arrows. (D) Proportion of higher order multimers comprising fractions 11, 13, and 15 (High MW) in the total amount from all fractions is shown (*P < 0.05; n = 5).
Podocin Restricts the Diffusional Mobility of CAR in Living Cells
We next examined the diffusion property of green fluorescenceprotein (GFP)-tagged CAR (Figure 7A) in living cells by fluorescencerecovery after photobleaching. Mouse L cells stably expressingpodocin (L-pod) were used to ensure equal abundance of podocinfor each experiment. Wild-type L cells (L-wt) or the L-pod weretransiently transfected with GFP-CAR, and fluorescence recoverywas compared between L cells expressing GFP-CAR alone (L-wtGFP-CAR) and those coexpressing GFP-CAR and podocin (L-pod GFP-CAR).As shown in Figure 7, B and C, in L-wt expressing GFP-CAR (L-wtGFP-CAR), fluorescence intensity reached a plateau of 83.8 ±12.2% of the prebleached value with a recovery time of 300 s(n = 6). In contrast, L-pod GFP-CAR displayed a significantlyhigher percentage of the immobile fraction (47.0 ± 9.5%)than L-wt GFP-CAR (16.2 ± 12.2%; P < 0.001; n = 6;Figure 7D). These data indicate that podocin confines the lateralmobility of CAR in a living plasma membrane.
Figure 7. Podocin restricts the lateral diffusion of green fluorescence protein (GFP)-CAR in living mouse L cells. (A) Membrane model of GFP-CAR. GFP is inserted just underneath the TM domain of CAR, leaving an essential cytoplasmic motif intact: Putative palmitoylation sites (Cys 259, 260), tyrosine phosphorylation site (Tyr 269), and PDZ binding motif (Ser-Ile-Val 363 to 365). (B) Merged images from bright-field and fluorescence observations of L cells expressing GFP-CAR alone (L-wt GFP-CAR) and those with stable expression of podocin (L-pod GFP-CAR). Fluorescence recovery after photobleach is measured in boxed regions of 15-µm width. Bar = 10 µm. (C) Kinetics of recovery of L-wt GFP-CAR () and L-pod GFP-CAR () cells. Each bar shows the mean ± SEM from six cells from three individual experiments. (D) The calculated percentages of the immobile fraction.
Clustering of CAR Leads to the Redistribution of F-actin and Podocin
The restricted lateral diffusion of surface proteins may bedue in part to their tethering to the actin cytoskeleton.20To test this hypothesis, we examined the effects of CAR ectodomainengagement on the distribution of F-actin and podocin. In CAR-podocincoexpressing COS-7 cells, antibody-mediated clustering of CARresulted in rearrangement of the actin cytoskeleton from itsoriginal filament-like pattern to a larger discrete punctatespot (Figure 8, A and B)—a morphologic pattern similarto the "actin comets" that represent the sites of dynamic actinreorganization.20 Notably, phalloidin staining indicated thatthe focal accumulation of F-actin coincided with the patcheswhere CAR and podocin co-localized (Figure 8A). Quantitativeanalysis revealed that podocin coexpression with CAR furtherincreases the proportion of cells exhibiting actin filamentreorganization (Figure 8C). However, an actin co-sedimentationassay with COS-7 cells showed that podocin does not alter thebinding affinity of CAR to F-actin (Supplementary Figure 4).Taken together, these data suggest that clustering of CAR inducesfocal cytoskeletal responses in association with podocin (Figure 9).
Figure 8. Clustered CAR induces podocin recruitment and F-actin reorganization. (A) Clustered CAR recruited podocin to the actin spots. Upon cross-linking CAR in COS-7 cells coexpressing CAR (green) and podocin (red), actin filaments are reorganized from a filament-like (top, arrows) to discrete punctate pattern (bottom), and podocin is recruited to the spots. Actin filament is visualized by phalloidin (blue). Bars = 10 µm. (B) Clustered CAR induced actin reorganization in association with podocin. White dashed lines denote the contour of untransfected cells. Note that cells devoid of CAR have normal bundle-like stress-fiber distribution even upon cross-linking of CAR. Insets show higher magnification of boxed portion. Arrowheads denote the punctate structures where actin is reorganized in association with CAR and podocin. (C) Quantitative analysis of actin reorganization. The images were binned into three main morphologic subtypes by inspection in terms of actin filament distribution: (I) Predominant stress fibers with few or no actin spots, (II) stress fibers mixed with focal actin spots, and (III) remarkable focal actin spots with loss of stress fibers. The diagrams show the means ± SEM of the distribution of the three phenotypes. The percentage of cells falling into the respective categories was based on more than 80 cell profiles from five independent experiments, thereby ensuring objectivity and reproducibility of the scoring. *P < 0.01.
Figure 9. Model illustrating a TJ complex assembly mediated by podocin. CAR and podocin normally reside within individual raft microdomains, with a certain proportion (30 to 40%) being sorted as preassembled CAR/podocin hetero-oligomer complexes. These membrane domains are small and highly dispersed at the cell surface, thereby representing "nonfunctional" or "inactive" microdomains. Antibody-mediated clustering induces the formation of "active" microdomains through the coalescence of neighboring lipid rafts. Such processes selectively recruit and sequester specific sets of interacting partners needed for TJ formation. The properties of podocin such as oligomerization and its high lipid affinity enhance raft association of CAR. Thus, podocin facilitates sequestration of CAR and its downstream signaling molecules central to actin reorganization (e.g., Fyn). In this way, podocin facilitates the efficient conversion of a cell adhesion signal into remodeling of cellular architecture.
Our data provide evidence that CAR is assembled and organizedas a multiprotein complex via membrane rafts in associationwith podocin and ZO-1. The transmembrane components for thepodocyte TJ complex have remained an open question. Neitheroccludin nor claudin has been detected in the podocytes, whereasother Ig-like proteins, including nephrin and JAM4, have neverbeen evident at the TJ.21 The N-terminus Ig-like domain of CARforms homodimers,22 suggesting that the initial cell–cellrecognition is mediated by its homodimerization in vivo. However,such "head-to-head" homodimerization does not normally occurat the SD because the ectodomain of CAR, consisting of justtwo Ig domains, is not long enough to bridge the slit gap (20to 50 nm). Therefore, engagement of the CAR ectodomains in transoccurs only when the opposing membranes come into close proximityafter injury. The cytoplasmic tail of CAR has a putative palmitoylationsite (Cys at 259 and 260) that facilitates sequestration ofrafts lipid23 and a putative tyrosine phosphorylation site (Tyr269). These features of CAR favor its ability to form a signalingplatform that attracts specific sets of raft-associated signalingmolecules (e.g., Src-family kinases7, 24). It is therefore conceivablethat CAR integrates the initial cell recognition signal intomorphologic reorganization and serves as the initial primingof nascent cell junction formation. CAR–podocin complexeswould be implicated in the formation of cell–cell junctionsof the nephrotic foot process. However, the physiologic relevanceneeds to be further evaluated by other approaches such as tissue-and stage-specific knockouts.25
Podocin enhances raft association of CAR by promoting its oligomerizationin an analogous mechanism to that reported for the other raftproteins.17 Podocin forms a large oligomeric complex,5 and itsraft association is stabilized in part via its C-terminus containinga prohibitin homology domain, reportedly important for lipid–proteininteractions.26 These properties facilitate the ability of podocinas a raft organizer that sequestrates CAR into the rafts, eventuallyfacilitating intimate contacts between signaling molecules byminimizing diffusion of binding partners. Because podocyteshave a huge surface area reflecting numerous interdigitatingprocesses, podocin-mediated sequestration of TJ components seemsto be physiologically relevant.
We demonstrated that clustering of CAR induces the formationof focal actin spots and concomitant recruitment of podocin.Together with the previous observation that CD2AP localizesthe actin spots,27 our data suggest that podocin may serve asa scaffold that links the membrane complexes to the cytoskeleton.In the rat PAN model, drastic actin reorganization was reportedin the foot processes after TJ assembly.28 Actin concentratesalong junctional complexes and regulates permeability of TJthrough its contraction. That foot processes are invariablyexposed to the hydrodynamic filtration pressure substantiatesa need for reinforcement of TJ complexes by anchoring them tothe cytoskeleton. These observations suggest that interactionsbetween the SD complex and the actin cytoskeleton play a crucialrole in the regulation of SD integrity and foot process architecture.
Podocin remains associated with the cell–cell junctionalcomplex even in nephrotic podocytes. It might be a secondaryor passive process reflecting the close structural and developmentalrelationship between the TJ and SD. During glomerular development,the SD is thought to have originated from apically located TJin prepodocytes and to have replaced the TJ in mature podocytes.2,10 The premature, differentiating podocytes bear a number ofmorphologic characteristics of the nephrotic podocytes.10, 12Effacement or retraction may represent a "simplification" and"dedifferentiation" to a more primitive organization and representthe reversal of normal developmental processes.
Another explanation for podocin localization in TJ complexesis that it represents the adaptive or reparative responses tothe nephrotic condition and injury.8, 29 In podocin-null mice,30foot processes develop normally and TJ-like structures are visible,suggesting that podocin is not absolutely necessary to formthe immature cell–cell junctions. Notably, the mice lackany SD at birth and develop severe cell damage during the next7 d and die from renal failure at 6 to 7 wk. We therefore speculatethat podocin-null podocytes are structurally labile and highlysusceptible to injury because of the inability to scaffold thejunctional complexes. Given the unusual feature of the footprocess, being continuously exposed to the threat of a highhydrostatic pressure, even minor impairment of junctional assemblymight thus be sufficient to produce foot process damage. Inpatients harboring podocin mutations, dysfunctional orchestrationof the integrity of multiple junctions may represent the molecularbasis of FSGS.
Constructs, Cells, and Antibodies
The details are provided as supplementary information.
Immunofluorescence Labeling
Surface proteins on living cells were first reacted with theprimary antibodies or Cholera toxin B subunit–biotin (bindsto ganglioside GM1) conjugate at 4°C for 20 min and thenfurther incubated at 12°C for 30 min.17 The secondary antibodieswere applied at 12°C for 1 h, and the cells were permeabilizedafter fixation.
Immunoelectron Microscopy
The tissues from periodate-lysine-paraformaldehyde–perfusedkidneys were placed in the periodate-lysine-paraformaldehydefixative for 4 h and embedded in hydrophilic methacrylate resin.13,31 Immunogold labeling of ultrathin cryosections was performedas described previously.9, 21
Immunoprecipitation and GST Pull-Down
For co-immunoprecipitation studies, transfected COS-7 cellswere lysed in RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl,0.5% sodium deoxycholate, 0.1% SDS, and 1% NP-40). Lysates (500µg) were rocked at 4°C for 2 h with the appropriateprimary antibody. Then 40 µl of protein G–Sepharosebeads was added and rocked at 4°C for 1 h.5
Flotation Gradient Centrifugation
A detergent-insoluble membrane fraction of COS-7 cells and ratisolated glomeruli was isolated as shown in Supplementary Figure 2.
Velocity Gradient Centrifugation
Transfected COS-7 cells were homogenized in Mes-buffered saline(25 mM Mes [pH 6.5] and 150 mM NaCl). The homogenate was centrifugedat 1000 x g for 10 min, and the supernatant was centrifugedat 200,000 x g for 30 min. The pellet was dissociated in Mes-bufferedsaline containing 60 mM octyl glucoside. Solubilized materialwas loaded atop a 5 to 40% linear sucrose gradient and centrifugedat 50,000 rpm for 16 h.
Fluorescence Recovery in Photobleaching Analysis
Bleaching of the outlined regions of interest was done at 37°Cwith the 488-nm laser line at full power and full transmissionfor 15 s. The recovery values were normalized by dividing themwith the mean prebleach values for each region of interest (LSMPascal software, Carl Zeiss Inc., Jena, Germany). The mobilefractions were estimated at the time points, where the recoveryof fluorescence reached a plateau.
Statistical Analysis
The unpaired t test with a P < 0.05 was taken as significant.
This work was supported by a Grant in Aid for Scientific ResearchB 16390245 and C 14571026 from Japan Society for the Promotionof Science.
Part of this article was presented in abstract form at the annualmeeting of the American Society of Nephrology; November 12 through17, 2003; San Diego, CA.
We are grateful to Drs. K. Taguchi, N. Tanimura, K. Kawai, andY. Taketani for assistance of rafts experiments.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Supplemental information for this article is available onlineat http://www.jasn.org/.
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Abstract
Introduction
cyclodextrin inhibited antibody-induced clustering of CAR (data not shown), indicating that CAR forms a large cluster in a cholesterol-dependent manner. When CAR-podocin co-expressing COS-7 cells were cross-linked, podocin co-localized with clustered CAR at the cell surface (Figure 4, E and F), but coexpression of podocin per se did not further augment the extent of co-localization of CAR with the raft marker GM1 (39.8 ± 8.3% without podocin versus 40.3 ± 7.2% with podocin; n = 4 to 6; Figure 4G). The results indicate that CAR clusters via a raft-mediated mechanism and that podocin supports the coalescence of preexisting small rafts containing CAR. 
) and L-pod GFP-CAR (
) cells. Each bar shows the mean ± SEM from six cells from three individual experiments. (D) The calculated percentages of the immobile fraction.
