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KIBRA Modulates Directional Migration of Podocytes

Kerstin Duning,* Eva-Maria Schurek,*† Marc Schlu¨ter,* Michael Bayer,* Hans-Christian Reinhardt,* Albrecht Schwab,‡ Liliana Schaefer,§ Thomas Benzing,† ʈ Bernhard Schermer,† Moin A. Saleem, Tobias B. Huber,¶ Sebastian Bachmann,** Joachim Kremerskothen,* Thomas Weide,* and Hermann Pavensta¨dt*

*Medizinische Klinik und Poliklinik D and ‡Institut fu¨r Physiologie II, Universita¨tsklinikum Mu¨nster, Mu¨nster, §Universita¨tsklinikum Frankfurt, Pharmazentrum, Frankfurt/Main, †Universita¨tsklinikum Ko¨ln, Innere Medizin IV, Nephrologie und Allgemeine Innere Medizin, Ko¨ln, ¶Medizinische Universita¨tsklinik, Abteilung Innere Medizin IV, Freiburg, and **Charite´–Universita¨tsmedizin Berlin, Institut fu¨r Vegetative Anatomie, Berlin, Germany; and ʈ Academic and Children’s Renal Unit, University of Bristol, Bristol, United Kingdom

ABSTRACT Asymmetric delivery and distribution of macromolecules are essential for cell polarity and for cellular functions such as differentiation, division, and signaling. Injury of podocytes, which are polarized epithelial cells, changes the dynamics of the actin meshwork, resulting in foot process retraction and proteinuria. Although the spatiotemporal control of specific protein–protein interactions is crucial for the establishment of cell polarity, the mechanisms controlling polarity-dependent differentiation and division are incompletely understood. In this study, yeast two-hybrid screens were performed using a podocyte cDNA library and the polarity protein PATJ as bait. The protein KIBRA was identified as an interaction partner of PATJ and was localized to podocytes, tubular structures, and collecting ducts. The last four amino acids of KIBRA mediated binding to the eighth PDZ domain of PATJ. In addition, KIBRA directly bound to synaptopodin, an essential organizer of the podocyte cytoskeleton. Stable knockdown of KIBRA in immortalized podocytes impaired directed cell migration, suggesting that KIBRA modulates the motility of podocytes by linking polarity proteins and cytoskeleton-associated protein complexes.

J Am Soc Nephrol 19: 1891–1903, 2008. doi: 10.1681/ASN.2007080916

Cell polarity regulates important processes, such as the actin cytoskeleton, resulting in foot process re- asymmetric cell division, cellular morphology, in- traction (foot process effacement) and proteinuria. tracellular signaling, and cell migration. So far, it is Obviously, a regulated cell polarity is essential for known that specific protein–protein interactions podocyte function. Nevertheless, the expression, are important for these processes, but the detailed molecular mechanisms that control cell polarity are Received August 20, 2007. Accepted April 11, 2008. poorly understood. Podocytes are highly polarized Published online ahead of print. Publication date available at epithelial cells that play a key role in the mainte- www.jasn.org. nance of the size-selective filtration barrier of the 1 K.D. and E.-M.S. contributed equally to this work, and T.W. and kidney. They consist of a cell body with primary H.P. contributed equally to this work. and highly branched secondary foot processes, Correspondence: Dr. Thomas Weide, UKM, Medizinische Klinik leading to a complex “neuron-like” cell architec- und Poliklinik D, Abteilung: Molekulare Nephrologie, Domagk- ture. The interdigitating secondary foot processes strasse 3a, D-48149 Mu¨nster, Germany. Phone: ϩ49-251-83- mediate the adhesion to the glomerular basement 57939; Fax: ϩ49-251-83-57943; E-mail: [email protected]; or Prof. Hermann Pavensta¨dt, UKM, Medizinische Klinik und membrane and form the slit diaphragm, unique Poliklinik D, Albert-Schweizer Strasse 33, D-48149 Mu¨nster, cell–cell contacts that serve as a final filtration bar- Germany. Phone: ϩ49-251-83-47516; Fax: ϩ49-251-83-46979; rier.1 E-mail: [email protected] Injury of podocytes leads to dynamic changes of Copyright ᮊ 2008 by the American Society of Nephrology

J Am Soc Nephrol 19: 1891–1903, 2008 ISSN : 1046-6673/1910-1891 1891 BASIC RESEARCH www.jasn.org function, and cross-talk of polarity regulators and the molec- RESULTS ular links between polarity proteins and downstream effects such as signaling, differentiation, and directional migration of KIBRA Directly Interacts with Cell Polarity Protein podocytes are unknown. PATJ During the past decade, two polarity complexes have been During the past decade, it was shown that the Pals1-PATJ- described, the aPKC-PAR3-PAR6 (aPKC for atypical protein Crb3 and aPKC-PAR3-PAR6 cell polarity complexes are espe- kinase C; PAR for partitioning defective) and the Pals1-PATJ- cially important in diverse epithelia of various tissues; there- Crb complex (Pals1 for protein-associated with Lin7–1; PATJ fore, we first tested whether these core proteins of the apical for Pals1-associated tight junction protein; and Crb3 for polarity complexes are expressed in immortalized cultured Crumbs3).2–4 It has been shown that these complexes are part podocytes.22 We found mRNA expression of PATJ; Pals1; both of an evolutionarily conserved system that regulates apicobasal Crumbs3 isoforms (Crb3a/b); and aPKC␨, PAR3, and three polarity, tight junction formation, signaling, and directional PAR6 isoforms in podocyte cDNA library PCR reactions (Fig- migration of eukaryotic cells. All core components of the com- ure 1A). This expression pattern might be a first hint that the plexes carry multiple protein–protein interaction modules, cell polarity of podocytes could be regulated in a similar man- suggesting that they are part of multiprotein complexes.2–4 ner as already shown for many other cell types. Interestingly, PATJ (also called INADL or CIPP) was first iden- For further examinations, we focused on the multi-PDZ tified as a highly abundant protein in brain and kidney.5–7 In ad- protein PATJ and performed a Y2H screen, using a podocyte dition to the N-terminal L27 domain, PATJ contains ten PDZ (for cDNA library and PATJ as bait. One of the isolated yeast clones PSD95/discs large/zonula occludens 1 [ZO-1]) domains, suggest- encoded an N-terminal deletion of KIBRA.23 Mapping studies ing that PATJ acts as a scaffolding protein that is able to bind to using a set of PATJ deletion mutants in the Y2H system (Figure many cellular partners through these domains.3,6 Previously, it 1, B and C) and GST pull-down assays revealed that PDZ8 was shown that PATJ associates with neuronal proteins and chan- mediates the PATJ–KIBRA interaction (Figure 1D). nels (e.g., neurexin, neuroligin, members of the Kir-family, The identified KIBRA clone (clone 29) of the Y2H screen ASIC3, 5HT2A), indicating that it recruits receptors and struc- lacks the two N-terminal WW domains (amino acids [aa] 1 tural proteins at synaptic sites.8,9 A recent study reported that through 39 and 54 through 86) but contains a putative cal- PATJ interacts with the tuberous sclerosis complex protein 1 and cium-sensitive C2-like domain (aa 726 through 787), a gluta- 2 (TSC1/2) and thereby links the mammalian target of rapamycin mate-rich region (aa 845 through 868), and the aPKC␨-bind- (mTOR) signaling pathway to the Pals1-PATJ-Crb3 cell polarity ing domain (aa 953 through 996; Figure 1B, bottom). The complex, supporting the idea that cell polarity proteins are also C-terminal four aa of KIBRA (ADDV, single-letter code) con- involved in signaling pathways.10 tain a putative class III PDZ-binding site.24 To find out In addition, PATJ directly binds to the tight junction pro- whether the KIBRA-PATJ interaction is mediated by this mo- teins ZO-3 and claudin 1 via the sixth and eighth PDZ do- tif, we performed co-immunoprecipitation (Co-IP) assays mains, emphasizing that PATJ plays an important role in the with lysates from HEK293T cells expressing FLAG-tagged full- maintenance of cell polarity and tight junction establishment length KIBRA with and without the ADDV motif and EYFP- of epithelial cells.11–14 Recently, it was shown that PATJ is part tagged full-length PATJ, respectively. Only KIBRA containing of a huge Rich/Amot complex by interacting with members of this motif was able to precipitate EYFP-tagged PATJ in these the Amot protein family.12,15 This family plays a role in the assays (Figure 1E). In addition, lysates from HEK293T express- regulation of cell–cell junctions and cell motility.16 In this con- ing the GFP-tagged C-terminus of KIBRA with and without text, it is interesting that the knockdown of PATJ in epithelial the ADDV motif (aa 1045 through 1113/1109) were incubated cells results in an impaired migration of MDCK II cells, sug- with recombinant GST PDZ7–10 fusion proteins. Again, only gesting that PATJ regulates not only cell polarity and tight KIBRA deletion mutants that contained the ADDV motif in- junction establishment but also the directional migration of teracted with PATJ (Figure 1F). epithelial cells. In this study, we performed a yeast two-hybrid (Y2H) KIBRA Is Expressed in Various Renal Tissues screen, using a cDNA library from immortalized podocytes KIBRA and PATJ both are expressed in immortalized podo- and PATJ as bait. We found KIBRA (for kidney brain), a pro- cytes (Figure 2A). Furthermore, immunofluorescence analysis tein with high expression in the brain and kidney that probably revealed that KIBRA and PATJ co-localize throughout the cy- plays a central role in human memory, as interaction partner of toplasm in a predominant perinuclear pattern. Minor frac- PATJ.17–21 tions of both proteins were also found at the lamellipodia lead- Our investigations revealed that in the kidney, KIBRA is ing edge, similar to the recently described PATJ distribution in expressed in glomerular podocytes, in some tubules, and in the migrating MDCK II cells (Figure 2B).25 In addition, we found collecting duct. In addition to the PATJ interaction, we found that KIBRA partially co-localizes with the actin and tubulin that KIBRA binds to synaptopodin, an essential protein of podo- cytoskeleton at these leading edges (Figure 2C, a through f). cytes. Furthermore, a knockdown of the KIBRA expression re- By contrast, vimentin, a marker for intermediate filaments, sulted in an impairment of podocyte directional migration. does not co-localize with KIBRA (Figure 2C, g through i). After

1892 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1891–1903, 2008 www.jasn.org BASIC RESEARCH

Figure 1. KIBRA directly interacts with PDZ8 domain of cell polarity protein PATJ. (A) Expression of apical cell polarity proteins in a human podocyte cell line. A PCR analysis of a cDNA library derived from the human differentiated podocytes (AB 8 cells) demonstrates the expression of apical cell polarity proteins in podocytes. cDNA for PATJ (lanes 1 through 3), PAls1 (lanes 4 through 5), Crumbs isoforms Crb3a ad Crb3b (lanes 6 through 7), PAR3 (lanes 8 through 9), aPKC␨ (lane 10), and three PAR6 isoforms (␣␤␥, lanes 11 through 13) were detected (details in Supplementary Table 2). (B) Domain architecture of PATJ, KIBRA, and used deletion mutants. Full-length PATJ (1801 aa) consists of one N-terminal L27 (blue) and 10 PDZ domains (orange). KIBRA (1113 aa) comprises two N-terminal WW domains (pink), a C2-like domain (magenta), a glutamate-rich region (green), an aPKC-binding domain (light blue), and a C-terminal PDZ-binding motif (yellow). An N-terminal deletion mutant of KIBRA (clone 29 [aa 359 through 1113]) was isolated in a Y2H screen, using the last four PDZ domains of PATJ as bait. (C) Y2H assays using different PATJ mutants and prey plasmids (clone 29/full-length KIBRA) to map the binding site. (D) KIBRA binds to PATJ PDZ 8 in a GST pull-down assay. GFP-tagged KIBRA C-terminus (aa 1045 through 1113) were incubated with equal amounts of various GST-PATJ fusion proteins. GST was used as a control. (E and F) Co-IP experiments (E) and GST pull-down assays (F) revealed that the last four aa of KIBRA (ADDV) mediate the interaction with PATJ. treatment of the cells with tubulin or actin de-polymerizing tubules seemed to express only low levels of KIBRA. These data agents (nocodazole and cytochalasin D, respectively), a frac- are supported by a Western blot analysis of IHKE-1 cells, an tion of KIBRA accumulates with retracted tubulin or actin immortalized cell line of the human proximal tubule. Here, (Supplemental Figure S1). KIBRA shows only low expression levels when compared with An in situ hybridization analysis of total human kidney re- lysates from total kidney or the podocyte cell line (Supplemen- vealed an mRNA expression of KIBRA in podocytes and tubu- tal Figure S2C).26 lar structures (Figure 3). These observations were confirmed An additional analysis using rodent tissue confirmed this by immunohistochemistry analyses on human kidney sections KIBRA expression pattern. In the adult rat, KIBRA staining of showing a predominant KIBRA expression in glomerular and the glomerular tuft was restricted to the podocytes. These cells tubular cells (Supplemental Figure S2B). Cells of the proximal were identified by double staining of the filtration slit using

J Am Soc Nephrol 19: 1891–1903, 2008 Role of KIBRA in Podocytes 1893 BASIC RESEARCH www.jasn.org

Figure 2. Expression of KIBRA and PATJ in a human podocyte cell line. (A, left) Affinity-purified KIBRA antiserum (Supplemental Figure S2A) specifically recognize KIBRA in extracts from proliferating (lane 3) and differentiated human podocytes (lane 4). Preimmune serum shows no reactivity to KIBRA (lanes 1 and 2). (A, right) PATJ is also expressed in both proliferating (lane 5) and differentiated (lane 6) human podocytes. (B) Co-localization of myc-KIBRA and EYFP-PATJ. Both proteins display a cytosolic distribution with perinuclear enrichment in proliferating and differentiated human podocytes. Small amounts of both proteins are localized at the cell periphery (arrow). (C) Myc-KIBRA co-localizes at lamellipodia of the leading edge (arrows) with actin (a through c) and tubulin (d through f) but not with vimentin (g through i). anti–ZO-1 antibodies (Figure 4A, a through c) and of their differences between cortical collecting duct and medullar col- nuclei with anti–Wilms Tumor Antigen-1 (WT-1) (Figure 4A, lecting duct principal cell staining patterns (Figure 4B, d d through f). In the glomeruli, all podocytes seemed to be through i). KIBRA positive, albeit at a relatively low signal intensity com- pared with collecting duct staining. KIBRA Binds Directly to the Slit Diaphragm Protein Parietal cells of the “bottleneck” region of Bowman’s cap- Synaptopodin sule and parietal epithelium at the transition to the proximal The PATJ–KIBRA interaction raises the question of whether tubule were regularly positive with two to four cells stained on KIBRA might act as a linker protein that mediates the binding each side in transverse sections through the glomerulus (Figure of PATJ-containing complexes to proteins that specifically in- 4B). Equally strong staining was seen in principal cells of the teract with the N-terminal WW domains of KIBRA. WW do- entire connecting tubule (Figure 4B, b and c) and collecting mains are protein–protein interaction modules that bind to duct (Figure 4B, d through i). Apparently, the intercalated cells PPxY aa motifs.27 This is interesting because KIBRA directly were negative throughout. The KIBRA signal was strong at the binds to the actin cytoskeleton-associated protein dendrin that luminal cell membrane, equally significant at the lateral cell was recently described as a slit diaphragm protein.28–30 Den- membrane, and weaker and diffuse intracellularly and at the drin directly interacts with CD2AP and nephrin and shares basal cell aspects of the principal cells. Double staining with several properties with synaptopodin that is involved in migra- anti–aquaporin-2 antibodies revealed co-localization with tion of podocytes and the development of kidney diseases.31,32 KIBRA chiefly at the luminal cell aspect. There were no major In addition, dendrin and synaptopodin are found in the den-

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Figure 3. In situ hybridization analysis of KIBRA and PATJ mRNA expression. Representative pictures after incubation with specific antisense probes against KIBRA mRNA revealed the expression of KIBRA in the glomeruli, distal tubule, and collecting duct of the kidney (A, overview; B, tubule). Higher magnification of human glomeruli showed a KIBRA (D and E) and PATJ (G and H) expression in podocytes (arrows). (C, F, and I) Sense negative control. drites of neurons and are associated with the cytoskeleton via mutant (P37A/P84A) did not. A KIBRA mutant lacking the binding to actin and ␣-actinin isoforms.31–34 WW domains (aa 85 through 1113) and an empty bait or prey An alignment of synaptopodin and dendrin showed no ob- vectors were used as controls (Figure 5C). These results were vious homology between both proteins; however, the PPxY confirmed by GST pull-down experiments (Figure 5D). For sites of human synaptopodin and dendrin elucidated a striking these, recombinant HA-tagged synaptopodin (903 aa) was in- similarity between aa 140 through 179 of dendrin and aa 318 cubated with GST or GST KIBRA-WW fusion proteins. Again, through 340 of synaptopodin (Figure 5A, green boxes). Both the wild-type KIBRA WW domains bind to HA-synaptopodin, proteins contain two flanking PPxY sites with a serine or thre- whereas the WW P37A/P84A double mutant failed. Fusion onine at the third position and have additional identical aa proteins that composed only a single mutated WW domain (TAPxxxW) inside these flanking PPxY motifs (Figure 5A, ma- were still able to interact with synaptopodin, suggesting that genta boxes). To test whether KIBRA binds not only to dendrin one KIBRA WW domain is sufficient to recognize the PPxY but also to synaptopodin, we performed Y2H assays with var- motifs of synaptopodin. The interaction of KIBRA and synap- ious synaptopodin deletion mutants and full-length KIBRA. topodin was further supported by our findings that both pro- Only deletion mutants (aa 9 through 527 and aa 245 through teins display a high degree of co-localization in podocytes (Fig- 527) that include the two PPxY sites (aa 318 through 340) ure 5E). interact with KIBRA (Figure 5, B and C, bottom). This inter- action was further investigated by truncated KIBRA mutants A Knockdown of KIBRA Disturbs Directional Cell that encode only the N-terminal two WW domains (aa 2 Migration through 85). We applied the wild-type WW domains as well as Previous studies reported that synaptopodin regulates the point mutants for single (P37A or P84A) or both WW domains bundling activity of the actin cross-linking protein ␣-actinin in (P37A/P84A) that should abrogate the binding to PPxY mo- an isoform-specific manner.32 Furthermore, synaptopodin tifs.23 The binding assays revealed that the wild-type WW do- regulates the integrity of the cytoskeleton and the cell motility mains of KIBRA bind to synaptopodin, whereas the double of podocytes, because gene silencing of synaptopodin leads to

J Am Soc Nephrol 19: 1891–1903, 2008 Role of KIBRA in Podocytes 1895 BASIC RESEARCH www.jasn.org

Figure 4. KIBRA expression in rodent renal tissue. (A) KIBRA immunostaining of the glo- merular tuft. (a through c) Double staining of glomeruli for KIBRA (a) and ZO-1 (b) is dis- played. Only the podocytes are immuno- stained, which is indicated by signals in epimembranous and membranous location; ZO-1 shows the slit membrane. The position of one representative podocyte is indicated by arrowheads. (c) Merged image. (d through f) Double staining of glomeruli for KIBRA (d) and podocyte marker protein WT-1 (e). All WT-1– positive podocytes are also KIBRA positive; two representative, double-stained podocytes are marked by arrowheads. (f) Merged image. (B) KIBRA immunostaining of the renal cortex and medulla. KIBRA immunoperoxidase stain- ing shows significantly labeled cells at the “bot- tleneck” region near the urinary pole of the glomerular parietal epithelium (a, arrowheads). Note that immunoreactive profiles of cortical collecting ducts (CCD) show strong epithelial staining with luminally enhanced signal. (b and c) Double staining for KIBRA (b) and aquaporin (AQP-2; c) of a connecting tubule profile. Sig- nals overlap at the luminal cell pole, whereas only KIBRA staining extends to the lateral cell borders and the intracellular/basolateral as- pect. Intercalated cells (arrowheads) are nega- tive for either staining. (d through i) Double staining for KIBRA (d and g) and AQP-2 (e and h) of a CCD profile (d through f) and medullary collecting duct profiles (MCD; g through i). As in CNT, principal cells show overlap at the lu- minal cell pole, whereas only KIBRA staining extends to the lateral cell borders and the in- tracellular/basolateral aspect in CCD and MCD principal cells. Intercalated cells (arrowheads in d through f) are negative. (f and i) Merged images. defects in directional podocyte migration.31 In addition, a re- normalize for transfection efficiency, expression level, and cell cently published study showed that a PATJ knockdown (k/d) number. ShRNA 1 and 2 resulted in at least 60% k/d of the resulted in an impaired migration of epithelial cells, suggesting reporter luciferase (Figure 6A). The expression levels of that PATJ regulates not only tight junction formation but also KIBRA-interacting proteins PATJ, synaptopodin, and aPKC␨ the mobility of migrating cells. This leads to the intriguing and the cytoskeleton-regulating protein rhoA were not hypothesis that KIBRA may take part in podocyte migration, changed in KIBRA k/d cells (Figure 6B). because KIBRA directly binds to both of these motility regu- shRNA 1 and 2 were subcloned into a lentiviral vector to k/d lating proteins. KIBRA expression selectively in human podocytes (Supple- We generated KIBRA k/d podocytes to investigate a possi- mental Figure S3). Relying on a highly efficient retroviral gene ble involvement of KIBRA in cell migration. For testing the transfer method, we obtained a pool of polyclonal cells harbor- efficiency of various short hairpin RNA (shRNA), human ing integration of the transgene at various positions in the ge- KIBRA cDNA was cloned into the bicistronic luciferase vector nome. Expression of shRNA was monitored by simultaneous (psiCHECK2; Promega, Madison, WI) encoding a fusion pro- coexpression of GFP from the same construct (Supplemental tein with Renilla reniformis luciferase. Within this system, R. Figure S3). reniformis luciferase activity is a quantitative parameter of Next, we performed wound-healing assays clearly showing RNA degradation mediated by co-transfected shRNA. Coex- the incapability of KIBRA k/d podocytes to close a wound via pressed firefly (Photinus pyralis) luciferase served as control to directed migration within 10 h. By contrast, control cells

1896 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1891–1903, 2008 www.jasn.org BASIC RESEARCH

Figure 5. KIBRA binds directly to synaptopodin via the WW domains. (A) Synaptopodin and dendrin carry two independent PPxY motifs with an S/T at the third position. The regions between the flanking PPxY motifs contain additional identical aa (magenta). Dendrin comprises one further PPxY motif (green italic letters). (B) Schematic structure of KIBRA and synaptopodin deletion constructs used for the binding assays. The N-terminal region of KIBRA contains two WW domains (pink/white boxes; aa 7 through 39 and aa 54 through 84). The substitution of a conserved proline into alanine (P37A and/or P84A) abrogates binding of the KIBRA WW domains. The deletion mutant lacking the WW domains (aa 85 through 1113) was used as negative control. The long variant of synaptopodin (903 aa) that is expressed in podocytes contains two internal PPxY sites (yellow), two PEST sequences (light blue), an actin-binding domain (red), and four ␣-actinin–binding regions (actinin BR, purple). (C) Y2H assay with bait constructs expressing different synaptopodin deletion mutants and prey plasmids encoding KIBRA, illustrating a direct interaction between KIBRA and synaptopodin. (control: empty bait/prey plasmid). (D) KIBRA WW domains bind to synaptopodin. HA-tagged synaptopodin was incubated with equal amounts of GST-fusion proteins that comprise KIBRA wild-type or mutated WW domains as shown in B. (E) Co-localization of KIBRA and synaptopodin in podocytes. Differentiated AB 8 cells coexpressing HA-synaptopodin and myc-KIBRA display a partial co-localization of both proteins.

Figure 6. KIBRA k/d in podocytes. (A) For determina- tion of the KIBRA k/d efficiencies of different shRNA, luciferase assays using the psiCHECK2 (Promega) sys- tem were performed. shRNA 1 and 2 resulted in ap- proximately 60 to 65% k/d of the reporter luciferase (Supplemental Figure S3). (B) The expression level for PATJ, synaptopodin, aPKC␨, and rhoA is not changed in KIBRA k/d podocytes (lane 2) compared with control cell lines (empty vector–transduced cells, lane 1), em- phasizing that the migration impairment is caused by the decreased endogenous KIBRA expression.

J Am Soc Nephrol 19: 1891–1903, 2008 Role of KIBRA in Podocytes 1897 BASIC RESEARCH www.jasn.org achieved wound healing within the same time (Figure 7, A and control cells versus 0.5 Ϯ 0.09 ␮m/min in KIBRA k/d cells; P ϭ B[n ϭ 3; 41.6 Ϯ 8.4 in control cells versus 9.6 Ϯ 1.0 in KIBRA 0.01, t test; means of 21 (control) or 17 (KIBRA k/d) cells are k/d cells; P ϭ 0.04, t test], and Supplemental Movies). To in- given]). Nevertheless, podocytes from both cell lines cover ap- vestigate further whether KIBRA k/d podocytes are impaired proximately the same distance within the same period (10 h; in the migration process itself or alternatively in polarized mi- Figure 7D, displacement). This apparent discrepancy can be gration (toward a wound), we analyzed trajectories of single accounted for by the frequent occurrence of changes in the cells, migration velocity, and the distance covered within the direction of movement of KIBRA k/d cells. These data point to experimental period. Control cells migrate in a persistent way KIBRA’s being required for efficient directed migration. into the wounded area and rarely make major changes in the direction of movement. Figure 7C, left, displays original paths of migrating control cells (magnification ϫ20). In contrast, DISCUSSION KIBRA k/d cells migrate ineffectively with frequent turns (Fig- ure 7C, right; magnification ϫ20). Surprising, our data show It is still an open question of which mechanism links cell po- KIBRA k/d podocytes to migrate significantly faster than con- larity to the diverse cellular programs such as cell differentia- trol cells (Figure 7D, migration velocity [0.3 Ϯ 0.01 ␮m/min in tion and motility in podocytes. Here we used the cell polarity

Figure 7. KIBRA regulated cell migration of podocytes. (A) A wound was scraped into confluent cell culture monolayers of control and KIBRA k/d podocytes (top, 0 h). In contrast to control cells, KIBRA k/d podocytes are incapable of closing a wound within 10 h via directed migration (bottom, 10 h). Square fields of identical size were superimposed to count cells that had migrated into the wound area. Bar ϭ 50 ␮m. (B) The number of cells that had migrated into equal areas within 10 h was plotted (square fields in A). KIBRA k/d podocytes are severely impaired in migrating into the denuded area. Experiments were performed in triplicate. (C) Typical original trajectories of control and KIBRA k/d cells are superimposed on the starting images of wound-healing assays. Migration of control podocytes is characterized by a high degree of persistence, and cells rarely change the direction of migration (left). In contrast, KIBRA k/d podocyte migration is ineffective and direction of movement changes frequently (right). (D) Statistical evaluation of migration velocity and displacement. KIBRA k/d podocytes move significantly faster (P Ͻ 0.05; P ϭ 0.01) than control cells. Despite the higher velocity of KIBRA k/d podocytes, their displacement (Ø 152.8 Ϯ 22.9 ␮m) is not significantly different (P ϭ 0.09) from that of control cells (Ø 112.8 Ϯ 7.0 ␮m). This can be accounted for by the frequent turns in the direction of movement of KIBRA k/d cells. Means of 21 (control) or 17 (KIBRA k/d) cells are given.

1898 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1891–1903, 2008 www.jasn.org BASIC RESEARCH and scaffold protein PATJ to screen a podocyte library for in- cytoskeleton, it has been demonstrated that KIBRA directly teracting partners. We identified KIBRA as a new interacting binds to dynein light chain 1 (DLC1) and sorting nexin 4 partner of PATJ and described the expression and localization (SNX4).38,39 The DLC1-SNX4-KIBRA complex associates with of KIBRA in the kidney. KIBRA directly interacts with the the minus-end directed microtubule motor dynein complex.38 eighth PDZ domain of PATJ. The C-terminal ADDV motif of These studies support our observation that KIBRA at least in- KIBRA is similar to that of the previously characterized PATJ directly associates with the microtubule network; therefore, interacting protein claudin 1 (KDYV), emphasizing that the KIBRA might act as a bridge between actin- and/or microtu- eighth PDZ domain favors class III PDZ-binding motifs.11 In bule-associated networks and cell polarity complexes. addition, we elucidated that the N-terminal WW domains of In this context, it is worth mentioning that in podocytes, KIBRA directly interact with synaptopodin, a podocyte protein MAGI-1 also interacts with dendrin and synaptopodin.28,40,41 that plays a role as cytoskeletal organizer and that is also asso- MAGI-1 belongs to the MAGUK (for membrane associated ciated with synaptic plasticity in neurons.34,35 In the kidney, we guanylate kinase) family and has a guanylate kinase domain found KIBRA expression in glomeruli, tubules, and collecting and two WW domains that are flanked by one N- and five duct. Furthermore, we described for the first time that KIBRA C-terminal PDZ domains. Similar to KIBRA, MAGI-1 associ- is important for directional migration. ates with synaptopodin and dendrin via the WW domains. In Initially, KIBRA was identified as an interacting partner of addition, the PDZ2 and PDZ3 domains of MAGI-1 bind to the dendrin, a protein that controls synaptic plasticity and that has C-terminus of the slit diaphragm protein nephrin, suggesting been recently identified as a slit diaphragm–associated pro- that MAGI-1 provides a molecular link between the slit dia- tein.23,29 Interestingly, dendrin binds to nephrin and CD2AP, phragm and cytoskeletal-associated proteins40; however, be- two other essential components of the slit diaphragm.30 Nota- cause of the different domain architecture of KIBRA and bly, protein kinase aPKC that is part of the aPKC-PAR3-PAR6 MAGI-1, it is likely that they have only partial analogous func- complex directly binds to and phosphorylates KIBRA.36 The tions in podocytes. C-terminus of KIBRA, which carries the aPKC␨ binding as well Interestingly, in brain, KIBRA represents a component of as the PATJ binding site, might be involved in the assembly or the postsynaptic density (data not shown). Thus, our data sup- the maintenance of both apical polarity complexes (see model port the hypothesis that the motility of podocyte foot processes Figure 8).37 and the flexibility of synaptic contacts of neurons could be The N-terminal part of KIBRA contains two WW domains regulated by an analogous set of molecules, including proteins that mediate the binding of KIBRA to dendrin and synaptopo- such as KIBRA, synaptopodin, dendrin, actin, and ␣-actinin. din. It remains to be shown whether the PPxY motifs of both Both foot processes and dendrites are long F-actin–rich, proteins compete or cooperate in binding to KIBRA; however, cellular extensions, suggesting a role for KIBRA in the contin- both KIBRA interactors, synaptopodin as well as dendrin, bind uous regeneration and plasticity of both cell types. Remark- to actin and ␣-actinin isoforms, thereby acting as regulators of ably, several recently published studies reported that the mem- the actin-based cytoskeleton.30–32 ory performance that depends on synaptic plasticity correlates In addition to the putative indirect association to the actin with different KIBRA alleles.17–20,38 Hence, the functional data provided may also provide a first hint of how synaptic plasticity could be regulated in neurons and how KIBRA might be in- volved in learning and memory processes on a molecular level. Foot processes of podocytes are highly flexible and dynamic structures that play a key role in withstanding the continuous filtration pressure. In addition, it is assumed that foot process retraction is a migration event caused by podocyte injury.42 Key aspects of migratory processes are rearrangements of the actin and microtubule cytoskeleton and polarization of the cell along the front-rear axis. We propose that a possible physio- logic function of KIBRA is to channel actin/microtubule cy- toskeleton dynamics into a polarized way. Interestingly, KIBRA k/d does not influence expression levels of interaction partners PATJ, synaptopodin, aPKC␨, and associated protein rhoA, indicating that the observed phenotype of KIBRA k/d cells is directly based on KIBRA gene silencing and not on Figure 8. Model of the cellular function of KIBRA. KIBRA directly interacts with dendrin, synaptopodin (SYNPO), and PATJ and indirect effects. Here we observed that KIBRA k/d leads to an aPKC (red arrows). Thus, KIBRA could serve as a linker molecule increased velocity without affecting the displacement of cells. between polarity proteins and components of the cytoskeleton That is, directed migration of KIBRA k/d cells is less efficient (actin, ␣-actinin, CD2AP, synaptopodin, and dendrin), thereby than that of control cells. Our data suggest that the higher regulating cell motility of podocytes. migration velocity displayed by KIBRA k/d cells is due to in-

J Am Soc Nephrol 19: 1891–1903, 2008 Role of KIBRA in Podocytes 1899 BASIC RESEARCH www.jasn.org creased dynamics of lamellipodia. Any rearrangement of the Lingueglia, Institute of Pharmacology, Valbonne, France) were trans- cytoskeleton resulting in protrusions or retraction of lamelli- formed with a cDNA library derived from a human podocyte cell line podia leads to a shift of the cell center (i.e., cells may reach high (differentiated AB 8) in pEXP-AD502 (Invitrogen) according to the migration velocities without truly covering a distance [“run- manufacturer’s instructions. For co-transformation assays, yeast cells ning on the spot”]). Thus, the inefficient migration of KIBRA were simultaneously transformed with bait and prey plasmids encod- k/d podocytes results in the impairment of closing a wound via ing various deletion mutants of human KIBRA, human and murine directed movement. PATJ, and human synaptopodin (Supplemental Table 1). Interaction Our data provide evidence that KIBRA is part of multipro- of bait and prey molecules were tested by growth of yeast cells on tein complexes that probably link cell polarity components selective medium containing 75 mM 3-amino 1,2,4,-triazole but lack- with cytoskeleton networks. Future studies are required to elu- ing leucine, tryptophan, and histidine and by LacZ-assays with filter- cidate the detailed composition of these complexes and their immobilized cells. spatially and temporally controlled interactions in the different cell types and how they regulate efficient directional migration. Generation of Polyclonal Antibodies against Human KIBRA Polyclonal KIBRA antibodies were raised in rabbits against a purified CONCISE METHODS recombinant KIBRA fragment (aa 661 to 796) fused to GST (Euro- gentec, Cologne, Germany). The obtained antisera were affinity-pu-

All procedures performed were in accordance with the ethics com- rified using a GST-KIBRA661 to 796 affinity column and were tested by mission guidelines of the University of Mu¨nster Ethic Commission. Western blot analysis (Supplemental Figure S2A).

Plasmid Constructs Extract Preparation and Western Blotting To generate human PATJ cDNA (accession no. NM_176877.2) mu- Cellular lysates were prepared by scraping cells into IP buffer (1% tants by PCR, we used the pEYFP-PATJ construct as a template (pro- Triton-X 100, 20 mM Tris-HCl [pH 7.5], 25 mM NaCl, 50 mM NaF, vided by Dr. Ben Margolis, Division of Nephrology, Department of 15 mM Na4P2O7, and 1.5 mM EDTA) containing protease inhibitor Internal Medicine, The University of Michigan Health System, Ann (Complete; Roche, Mannheim, Germany). Lysates were centrifuged Arbor, MI). KIBRA cDNA truncation mutants have been described at 10,000 ϫ g for 30 min at 4°C. Supernatants were removed and previously or were amplified from pSV42-myc-KIBRA (Supplemen- stored at Ϫ80°C until further use. For Western blot analysis, samples tal Table 1). Full-length human KIBRA (accession no. NM_15238.1) were mixed with Laemmli buffer and boiled, and proteins were sepa- cDNA or fragments encoding deletion mutants were cloned into rated by 6 to 12% SDS-PAGE (Bio-Rad, Munich, Germany). After modified pcDNA6 (details available from T.B.) or pEGFP-C2 (Clon- transfer of proteins onto a polyvinylidene fluoride membrane (Milli- tech, Heidelberg, Germany) expression plasmids, respectively. The pore, Schwalbach, Germany), reactive sites were blocked for 30 min at corresponding PCR products were subcloned into yeast GAL4 DNA- 37°C with 5% skim milk powder dissolved in TBS containing 0.05% binding or GAL4-activation domain vectors (pDB-Leu, pEX-AD502, Tween-20 (TBS-T). Primary antibodies were diluted in blocking and pDEST32/22 from Invitrogen (Karlsruhe, Germany) or pAS2–1 buffer and incubated with membranes for1hat37°C or overnight at and pACT2 from Clontech, respectively) or into a modified 4°C. KIBRA and rabbit anti-PATJ antibodies were used at 1:500 dilu- pGEX-KG expression construct. Vectors for the expression of HA- tions. The other primary antibodies used in this work were as follows: tagged synaptopodin or synaptopodin bait fragments for yeast co- Rabbit anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA), mouse transformation assays were described previously.33 Details of con- anti–glyceraldehyde-3-phosphate dehydrogenase (Covance, Muen- structs (Supplemental Table 1) and primers (Supplemental Tables 2 ster, Germany), mouse anti-FLAG (Sigma-Aldrich, Munich, Ger- and 3) are also available from T.W. or H.P. many), mouse anti-HA-tag (Roche), rabbit anti-aPKC␨ (Sigma), rab- bit anti-RhoA (Santa Cruz Biotechnology), mouse anti-synaptopodin Cell Culture and Transient Transfection (Progen, Heidelberg, Germany), mouse anti–␤-tubulin (Sigma), and Human immortalized podocytes (AB 8 cells) were cultivated as de- rabbit anti-V5 (Abcam, Heidelberg, Germany). After washing in scribed previously.22 In brief, cells were grown in standard RPMI 1640 TBS-T, the membranes were incubated with a secondary horseradish medium containing 10% FCS and supplements either at the permis- peroxidase–coupled antibody directed against the primary antibody sive temperature of 33°C (in 5% CO2) to promote cell propagation or (Dianova, Hamburg, Germany). Finally, the membranes were washed at the nonpermissive temperature of 37°C (in 5% CO2) to allow the and developed using a chemiluminescence detection reagent (Roche). terminal differentiation. HEK293T cells were cultivated and trans- fected as described previously.23 Transient transfection of human Bacterial Protein Synthesis and In Vitro Binding Assays podocytes was performed using the nucleofector technology (Amaxa, Constructs for bacterial expression of PATJ and KIBRA fragment mu- Cologne, Germany) according to the manufacturer’s instructions. tants are described in Supplemental Table 1. Synthesis of recombinant proteins in Escherichia coli strain BL21 was induced with 1 mM IPTG Yeast Two-Hybrid Screen and Yeast Co-transformation for3hat30°C. Affinity purification of GST fusion proteins was per- Assays formed according to the manufacturer’s instructions (GE Healthcare, S. cerevisiae MaV203 yeast cells containing the bait plasmid pDB-Leu Munich, Germany). Purified proteins were stored at Ϫ80°C until fur- encoding the last four PDZ of murine PATJ (provided by Dr. Eric ther use. For GST-binding assays, equal amounts of GST fusion pro-

1900 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1891–1903, 2008 www.jasn.org BASIC RESEARCH teins (approximately 5 ␮g) were immobilized on GST-Sepharose many) fluorescence microscope equipped with a SPOT camera and beads (GE Healthcare) for2hat4°C. The beads were washed five Visitron (Puchheim, Germany) software. Human kidney slices were times with PBS and were subsequently incubated at 4°C with cell fixed in 4% PFA, embedded in paraffin, and cut into 4-␮m-thick lysates containing recombinant FLAG-KIBRA, GFP-KIBRA frag- slices. Slices were deparaffinized in Xylol for 1 h, gradually hydrated ments, or HA-tagged synaptopodin, respectively. Loaded beads were through graded alcohols (100 to 50%), and washed in deionized wa- washed five times in PBS, and bound proteins were eluted in SDS ter. After incubation in 3% H2O2 for 20 min, slices were rehydrated sample buffer by boiling samples for 5 min at 95°C. Finally, probes with PBS. Antigen unmasking was performed by incubation of the were analyzed by SDS-PAGE and Western blot analysis as described slices in 30% FCS and 3% BSA in PBS for 20 min at room tempera- already. ture. Furthermore, slices were incubated for 3 min at 120°C in 0.01 M citrate/PBS. Thereafter, sections were incubated overnight in a hu- Co-immunoprecipitation midified chamber at 4°C, with rabbit anti-KIBRA (1:400) or with For Co-IP assays, single or co-transfected HEK293T cells were first KIBRA serum preincubated for1hatroom temperature with 10 ␮gof lysed in IP buffer. Aliquots of lysates were incubated with anti-FLAG blocking agent (Supplemental Figure S1A), respectively. Rabbit anti– affinity gel (Sigma) overnight at 4°C on a rocking platform. In a next WT-1 (1:400; Santa Cruz Biotechnology) was applied as positive con- step, beads were washed five times in IP buffer. For elution of bound trol. Slices were washed extensively with PBS and incubated for 40 proteins, beads were resolved in sample buffer and boiled for 5 min at min with an anti-rabbit antibody included in the commercially avail- 95°C. Samples were subsequently subjected to SDS-PAGE and West- able Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Slices ern blot analysis as described already. were washed with PBS, incubated with avidin-biotin for 40 min, and stained with diaminobenzidine. Sections were examined with a con- Staining of Cells and Tissues ventional light microscope (Zeiss, Cologne, Germany). For indirect immunofluorescence analysis on cultured podocytes, cells were grown on collagen-coated coverslips and fixed in 4% para- In Situ Hybridization formaldehyde (PFA) supplemented with 4% sucrose in PBS at room DNA probes comprising nucleotides (nt) 1402 through 1680 of hu- temperature for 20 min. Samples were washed with PBS and then man KIBRA cDNA and nt 5026 through 5407 of human PATJ cDNA incubated for 10 min in 50 mM NH4Cl in PBS to quench reactive were cloned in pGEM-T (Promega, Madison, WI). Recombinant amino groups. After washing with PBS, cells were permeabilized in RNA probes were transcribed in vitro from linearized pGEM-T using PBS containing 0.2% Triton-X 100 for 5 min and washed three times digoxygenin-labeled UTP and the DIG RNA Labeling Kit (Roche). In in PBS containing 0.2% Triton-X 100 and 0.2% gelatin (PBS-TG). situ hybridization experiments on kidney sections were performed as Next, samples were blocked with 10% goat serum diluted in PBS-TG described previously44 except that the probes were hybridized at 50°C for 20 min at room temperature. Immunofluorescence staining was for 16 h. performed by incubating the coverslips for1hatroom temperature with primary antibodies diluted in PBS-TG containing 2% goat se- RNA Interference Experiments rum. Primary antibodies were the polyclonal, affinity-purified anti- shRNA were designed with publicly available prediction programs KIBRA serum, mouse anti–␤-tubulin (Sigma), mouse anti-vimentin and are summarized in Supplemental Table 3. shRNA were cloned (Sigma), and phalloidin–Alexa 594 (Molecular Probes, Eugene, OR). into the RNA expression vector pSuper (Oligoengine, Seattle, WA). For detection of overexpressed KIBRA and synaptopodin, the mAb To determine the efficiency of shRNA-mediated KIBRA k/d, we used against HA-tag (Roche) and myc-tag (Abcam, Santa Cruz Biotech- psiCHECK2 (Promega) in which the coding sequence and the 3Ј un- nology) were used. Coverslips were washed after antibody incubation translated region of KIBRA were fused to the coding sequence of R. in PBS-TG and incubated 20 min at room temperature with fluoro- reniformis luciferase as an artificial 3Ј untranslated region. In addition chrome-conjugated secondary antibodies (Molecular Probes) diluted to R. reniformis luciferase, psiCHECK2 encodes for firefly luciferase as in PBS-TG containing 2% goat serum. After washing in PBS, cover- internal control. A total of 50 ng of the reporter plasmid and 50 ng of slips were rinsed in water and cells were mounted in Crystal Clear the respective pSuper shRNA constructs were co-transfected into Mount Medium (Sigma). Images were obtained using a Leica pho- HEK 293T cells in a 96-well format using Lipofectamine 2000 (In- tomicroscope attached to a Spot 2 slider digital camera. Immunoflu- vitrogen). Luciferase activities were measured by a dual-luciferase re- orescence stainings of rat kidneys were performed as described previ- porter assay system (Promega) in a luminometer (Mithras LB940; ously.43 Adult male Sprague-Dawley rats (250 g body wt) were Berthold Technologies, Pforzheim, Germany) 24 h after transfection. perfusion-fixed using 3% PFA/cacodylate. Tissues were prepared for Transfections and measurements were performed in triplicate. Se- histochemical analysis using paraffin or cryotechniques. Sections lected hairpins (shRNA 1 and 2) were then subcloned into pLVTHM were incubated with specific antisera and fluorescence- or peroxi- for stable lentiviral expression in human podocyte cell lines. Empty dase-labeled secondary antibodies. For signal enhancement, an am- vector–transduced podocytes served as control cells as described pre- plification kit (CSA; Dako, Glastrup, Denmark) was used. Specific viously.45 antibodies comprised anti-KIBRA (see “Generation of Polyclonal An- tibodies against Human KIBRA”), goat anti–aquaporin-2 (Santa Wound-Healing Assays Cruz Biotechnology), mouse anti–WT-1 (Dako), and mouse anti– Migration of empty vector–transduced control cells and KIBRA k/d ZO-1 (Zymed). Signals were evaluated using a Leica (Bensheim, Ger- podocytes was assessed with wound-healing assays. Cells were grown

J Am Soc Nephrol 19: 1891–1903, 2008 Role of KIBRA in Podocytes 1901 BASIC RESEARCH www.jasn.org in collagen-coated (50 ␮g/ml) tissue culture flasks to confluence. A 2. Suzuki A, Ohno S: The PAR-aPKC system: Lessons in polarity. J Cell Sci wound was scraped into the cell layers, using a sterile 10-␮l pipette tip. 119: 979–987, 2006 Then cells were washed with PBS and incubated with fresh medium 3. Margolis B, Borg JP: Apicobasal polarity complexes. J Cell Sci 118: 5157–5159, 2005 for1hintheincubator to allow recovering of the cells. Thereafter, the 4. Shin K, Fogg VC, Margolis B: Tight junctions and cell polarity. Annu cells were placed in a heating chamber (37°C) on the stage of an Rev Cell Dev Biol 22: 207–235, 2006 inverted microscope (Axiovert, Zeiss, Cologne, Germany). Constant 5. Roh MH, Fan S, Liu CJ, Margolis B: The Crumbs3-Pals1 complex participates in the establishment of polarity in mammalian epithelial CO2 contents for the length of the experiments were ensured by gas- tight culture flasks. Migration was monitored with a video camera for cells. J Cell Sci 116: 2895–2906, 2003 6. Roh MH, Makarova O, Liu CJ, Shin K, Lee S, Laurinec S, Goyal M, 10 h (Hamamatsu, Hersching, Germany) and controlled by HiPic Wiggins R, Margolis B: The Maguk protein, Pals1, functions as an software (Hamamatsu). Images were taken at 10-min intervals. adapter, linking mammalian homologues of Crumbs and Discs Lost. The number of cells that had migrated into same-sized square J Cell Biol 157: 161–172, 2002 fields after 10 h was counted. Migration itself was quantified on a 7. Kurschner C, Mermelstein PG, Holden WT, Surmeier DJ: CIPP, a novel single-cell level. To this end, the outlines of individual cells were multivalent PDZ domain protein, selectively interacts with Kir4.0 family members, NMDA receptor subunits, neurexins, and neuroligins. Mol marked semiautomatically at each time step throughout the entire Cell Neurosci 11: 161–172, 1998 image stacks with Amira software (Mercury Computer Systems, 8. Anzai N, Deval E, Schaefer L, Friend V, Lazdunski M, Lingueglia E: The Duesseldorf, Germany) as described previously.46,47 These segmenta- multivalent PDZ domain-containing protein CIPP is a partner of acid- tion data were used for further processing. Migration was quantified sensing ion channel 3 in sensory neurons. J Biol Chem 277: 16655– as the movement of the cell center. The x and y coordinates of the cell 16661, 2002 9. Becamel C, Gavarini S, Chanrion B, Alonso G, Galeotti N, Dumuis A, center were calculated as geometric means of equally weighted pixel Bockaert J, Marin P: The serotonin 5-HT2A and 5-HT2C receptors positions within the cell outlines as function of time. We used two interact with specific sets of PDZ proteins. J Biol Chem 279: 20257– parameters to reveal the effect of KIBRA k/d on podocyte migration: 20266, 2004 Velocity and displacement. The velocity of migrating cells (␮m/min) 10. Massey-Harroche D, Delgrossi MH, Lane-Guermonprez L, Arsanto JP, was calculated for each time interval by applying a three-point differ- Borg JP, Billaud M, Le BA: Evidence for a molecular link between the ␮ tuberous sclerosis complex and the crumbs complex. Hum Mol Genet ence quotient. The displacement ( m) is the distance between the 16: 529–536, 2007 position of cells at the beginning and at the end of the experiment 11. Roh MH, Liu CJ, Laurinec S, Margolis B: The carboxyl terminus of zona (after 10 h). Because the formation of lamellipodia leads to a shift of occludens-3 binds and recruits a mammalian homologue of discs lost the cell center, it has to be mentioned that cells may reach high mi- to tight junctions. J Biol Chem 277: 27501–27509, 2002 gration velocities without really covering a distance (“running on the 12. Wells CD, Fawcett JP, Traweger A, Yamanaka Y, Goudreault M, Elder K, Kulkarni S, Gish G, Virag C, Lim C, Colwill K, Starostine A, Metalni- spot”). Experiments were performed in triplicate. Representatively, kov P, Pawson T: A Rich1/Amot complex regulates the Cdc42 GTPase original trajectories of control cells and KIBRA k/d podocytes are and apical-polarity proteins in epithelial cells. Cell 125: 535–548, 2006 displayed in Figure 7C. 13. Shin K, Straight S, Margolis B: PATJ regulates tight junction formation and polarity in mammalian epithelial cells. J Cell Biol 168: 705–711, 2005 ACKNOWLEDGMENTS 14. Lemmers C, Medina E, Delgrossi MH, Michel D, Arsanto JP, Le BA: hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells. J Biol Chem 277: This study was supported by the Deutsche Forschungsgemeinschaft 25408–25415, 2002 Pa 483/14-1 and IZKF Schw2/030/08 (A.S.). 15. Sugihara-Mizuno Y, Adachi M, Kobayashi Y, Hamazaki Y, Nishimura M, We thank Nina Meyer, Katja Brinkmann, and Sabine Mally for Imai T, Furuse M, Tsukita S: Molecular characterization of angiomotin/ excellent technical assistance and Kerstin Riskowsky for expert help in JEAP family proteins: Interaction with MUPP1/Patj and their endoge- immunohistochemistry and imaging. We thank Drs. Hsiang-Hao nous properties. Genes Cells 12: 473–486, 2007 16. Bratt A, Birot O, Sinha I, Veitonmaki N, Aase K, Ernkvist M, Holmgren Hsu and Roman Preston for critical reading of the manuscript and all L: Angiomotin regulates endothelial cell-cell junctions and cell motil- members of our laboratory for helpful comments and discussions. We ity. J Biol Chem 280: 34859–34869, 2005 are grateful to Hermann Kra¨hling for technical support during the 17. Almeida OP, Schwab SG, Lautenschlager NT, Morar B, Greenop KR, migration assays. In addition, we thank Dr. Eric Lingueglia (Val- Flicker L, Wildenauer D: KIBRA genetic polymorphism influences ep- bonne, France) for the mCIPP construct and Dr. Ben Margolis (Ann isodic memory in later life, but does not increase the risk of mild cognitive impairment. J Cell Mol Med January 11, 2008 [epub ahead Arbor, MI) and Dr. Elior Peles (Rehovot, Israel) for human PATJ of print] cDNA and PATJ antibody gifts. 18. 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J Am Soc Nephrol 19: 1891–1903, 2008 Role of KIBRA in Podocytes 1903