original article http://www.kidney-international.org & 2009 International Society of Nephrology

Palladin is a dynamic -associated in podocytes Nicole Endlich1, Eric Schordan1, Clemens D. Cohen2, Matthias Kretzler3, Barbara Lewko4, Thilo Welsch5, Wilhelm Kriz6, Carol A. Otey7 and Karlhans Endlich1; European Renal cDNA Bank (ERCB) Consortium

1Department of Anatomy and Cell Biology, University of Greifswald, Greifswald, Germany; 2Nephrology Clinic and Institute of Physiology, University of Zurich, Zu¨rich, Switzerland; 3Division of Nephrology, University of Michigan School of Medicine, Ann Arbor, Michigan, USA; 4Department of Immunopathology, Medical University of Gdansk, Gdansk, Poland; 5Department of Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany; 6Department of Anatomy and Cell Biology, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany and 7Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Palladin, a cytoskeletal protein with essential functions for During the last decade it became more and more obvious stress fiber formation, is found in developing and mature that podocytes have a key function in renal disease because of tissues, including the kidney. To define its role in the kidney, glomerulopathies. The exceptional three-dimensional archi- we measured its expression in mouse kidney and found it tecture of podocytes covering the outer aspect of glomerular co-localized with F-actin in smooth muscle cells of renal capillaries is the result of the complex interdigitation of their arterial vessels, mesangial cells, and podocytes but not in foot processes and the base for proper blood filtration. The tubular epithelium. Using immunoelectron microscopy, we actin and actin-associated are crucial confirmed that palladin was present in podocytes. In cultured for the function of the filtration unit. Therefore, mutations of mouse podocytes, palladin co-localized with F-actin in dense a-actinin-4 or CD2-associated protein (CD2AP), which are regions of stress fibers, focal adhesions, cell–cell contacts and both actin-binding proteins, result in podocyte foot process motile cell margins. Transfection with the N-terminal half of effacement accompanied by proteinuria and the development palladin targeted it to F-actin-containing structures in of focal segmental glomerulosclerosis.1,2 Elucidation of the podocytes while the C-terminal half accumulated in the mechanisms of the actin organization in podocytes will be nucleus, a result also found for endogenous palladin in crucial for the understanding and prevention of glomerulo- cultured cells after leptomycin B was used to block nuclear sclerosis.3 export. Green fluorescent protein (GFP)-tagged palladin was The actin-associated protein palladin, first described by found in dynamic ring-like F-actin structures and ruffles in Parast and Otey,4 is expressed in different isoforms in a cultured podocytes after stimulation with epidermal growth variety of embryonic and adult tissues as well as in different factor. Inhibition of palladin expression by transfection of an cell lines.4,5 It was shown that palladin is a key regulator antisense construct reduced the formation of ring-like of stress fiber organization in rat choriocarcinoma cells structures. Photo-bleaching analysis showed that GFP- (Rcho-1) and in cultured mouse embryonic fibroblasts.4 The palladin turned over with a half-time of 10 s in focal reduction of palladin expression in fibroblasts via antisense adhesions and dense regions of stress fibers, suggesting that virus infection resulted in a complete loss of actin stress fibers palladin is a dynamic scaffolding protein. Our study shows and focal adhesions, leading to rounding of cells.4 Over- that palladin is expressed in podocytes and plays an expression of palladin in astrocytes and COS-7 cells induced important role in actin dynamics. formation of robust actin bundles.6,7 Furthermore, it was Kidney International (2009) 75, 214–226; doi:10.1038/ki.2008.486; demonstrated that palladin is highly expressed in the central published online 1 October 2008 nervous system,8 where it has important functions. On the KEYWORDS: actin; cytoskeleton; foot processes one hand palladin is essential for growth cone formation during neurite outgrowth.9 On the other hand palladin is significantly upregulated in migrating neural crest cells and in astrocytes in response to injury.6,10 The knockout of palladin in mice is associated with a defect in neuronal tube Correspondence: Nicole Endlich, Institut fu¨r Anatomie und Zellbiologie, closure during development which is lethal around E15.5 Universita¨t Greifswald, Friedrich Lo¨fflerstr. 23c, D-17487 Greifswald, and indicates the important role of palladin during 11 Germany. E-mail: [email protected] development. 7 Received 14 November 2007; revised 20 June 2008; accepted 24 July Ronty et al. have demonstrated that palladin interacts 2008; published online 1 October 2008 with a-actinin through a novel a-actinin binding motif in the

214 Kidney International (2009) 75, 214–226 N Endlich et al.: Role of palladin in actin dynamics original article

N-terminal half of palladin. Further it was shown by podocyte cell line by time-lapse microscopy and fluorescence Boukhelifa12 that vasodilator-stimulated phosphoprotein recovery after photobleaching (FRAP) analysis in detail. (VASP), which is involved in actin assembly independent of Arp2/3 complex, binds to two proline-rich regions in the N- RESULTS terminal half of palladin. VASP and a-actinin-1 colocalize Palladin is expressed in podocytes, mesangial cells, and with palladin in dense regions along stress fibers and in focal vessels in kidney adhesions.4 Recently it was found that palladin binds profilin, Palladin expression was detected in glomeruli and vessels in a protein that controls actin polymerization. Furthermore kidney sections from mice. In glomeruli, palladin immunor- Goicoechea et al.13 have recently presented data that palladin eactivity was highest around capillary loops where the actin- enhances the formation of dorsal ruffles and podosomes. based foot processes of podocytes are located (Figure 1a–c). These results suggest that palladin is directly involved in The glomerular mesangium stained only weakly for palladin. dynamic processes and plays a role as an actin-bundling In addition, palladin colocalized with F-actin in smooth protein. To clarify the role of palladin in podocytes we muscle cells of arterial vessels of different size; fibroblasts in studied the localization and dynamics of palladin in our the adventitia showed weak immunoreactivity (Figure 1d–f).

Palladin F-Actin Merge

Glom

a b c

Vessel

d e f

RT–PCR

g h Kidney ++ –Ladder PCL Figure 1 | Analysis of renal palladin expression. Panels a–f show double staining of mouse kidney sections with an antibody against palladin (a, d) and with Alexa-488-conjugated phalloidin to label F-actin (b, e). Palladin is strongly expressed in podocytes in colocalization with F-actin in podocyte foot processes around glomerular capillaries (a–c). Glomerular mesangial cells, which are strongly positive for F-actin, exhibit only a weak staining for palladin (a–c). Arterial vessels (panels d–f show a cortical radial artery giving rise to an afferent arteriole) express palladin in smooth muscle cells (d) in colocalization with F-actin (e, f). In addition, palladin is weakly expressed in fibroblasts in the adventitia of arterial vessels (d). RT–PCR of total RNA isolated from mouse kidney and podocyte cell line (PCL) confirm expression of palladin (g). Lane 4 shows the control reaction in the absence of reverse transcriptase (). Localization of palladin by immunoelectron microscopy is shown in panel h. Gold particles (25 nm) were found in foot processes and to some extent in the nucleus. The red circle indicates a gold particle near the slit membrane. Panel width represents 250 mm(a–f) and 1.2 mm(h).

Kidney International (2009) 75, 214–226 215 original article N Endlich et al.: Role of palladin in actin dynamics

Reverese transcription (RT)–PCR of total RNA isolated from 3d–f). It is important to point out that VASP, like cortactin mouse kidney confirmed renal expression of palladin (Figure (Figure 3g–i), is involved in actin dynamics and polymeriza- 1g). Furthermore, we detected palladin expression in our tion processes. established podocyte cell line14 by RT–PCR as well as in primary culture. By electron microscopy we were able to Colocalization studies between palladin and demonstrate that palladin is localized in foot processes podocyte-specific proteins (Figure 1h). To some extent palladin was also found in the We studied the spatial relationship between palladin and vicinity of the slit membrane (red circle in Figure 1h) and in podocyte-specific proteins. Figure 4 shows a complete the nuclei. overlap between the staining pattern of palladin and synaptopodin along stress fibers, an actin-associated protein Palladin is downregulated in focal segmental expressed in podocytes and neurons.15 However along the glomerulosclerosis cell margin only palladin was found (Figure 4a and c). In To assess whether glomerular palladin expression is tran- contrast, CD2AP, a podocyte-specific protein located in scriptionally regulated in human disease, we measured highly dynamic F-actin spots in cultured podocytes,16 glomerular mRNA levels of palladin vs 18S rRNA levels by showed a staining pattern being distinct from that of palladin quantitative PCR in glomerular cDNA, isolated from biopsies (Figure 4d–f). Our podocyte cell line possesses typical of patients with diabetic nephropathy (DN; n ¼ 16) or focal features of the unique cell–cell contacts between podocytes segmental glomerulosclerosis (FSGS; n ¼ 14). Material from in vivo, that is a simplified pattern of interdigitating foot living kidney donors served as controls (n ¼ 8). Palladin processes14 and stable expression of nephrin, a transmem- mRNA levels were significantly downregulated in FSGS by brane protein of the slit diaphragm.17 In our podocyte cell 68% (P ¼ 0.001), but not in DN (P ¼ 0.32; Supplementary line palladin is partially colocalized with nephrin at the Figure S1). cell–cell junction (Figure 4g–i). Strong accumulation of palladin at areas of cell–cell contact was also observed in Palladin localizes to stress fibers and focal adhesions in confluent primary podocyte cultures (Supplementary Figure cultured podocytes S3). Palladin was described to be localized along actin stress fibers in a punctate pattern, at focal adhesions and cell–cell contacts Domains in the C- and N-terminal halves of palladin mediate in fibroblast cell lines and embryonic chick pigmented differential targeting epithelial cells.4 To gain insight into the subcellular distribu- To find out which part of palladin is responsible for tion of palladin in podocytes, we studied the localization of association of palladin with the various F-actin structures palladin in our conditionally immortalized podocyte cell in podocytes, we transfected podocytes with the N-terminal lines by immunofluorescence. In colocalization studies with and C-terminal halves of palladin, respectively. The domains F-actin, we observed that palladin assumes various patterns in the N-terminal half of palladin were sufficient to achieve in cultured podocytes. Palladin was located along and at the localization at focal adhesions and cell margins (Figure 5d–f), end of stress fibers but did not colocalize with tubulin which was similar to the distribution of full-length palladin (Figure 2). Higher magnification of podocytes stained with (Figure 5a–c). In contrast, the C-terminal half of palladin, an antibody directed against palladin demonstrated that containing three IgC2 domains, showed only a diffuse palladin was distributed in a periodic, punctate manner along cytosolic staining, but a very intense staining of the nucleus stress fibers (Figure 2j–l). This pattern is typical for (Figure 5g–i). The accumulation of the C-terminal half of components of dense regions of stress fibers. The distance palladin in the nucleus suggested that the N-terminal half between single palladin spots was approximately 2 mm. In could be necessary for nuclear export. Therefore, we blocked green fluorescent protein (GFP)-palladin-transfected podo- nuclear export by treatment with leptomycin B (20 nM). cytes, GFP-palladin puncta on stress fibers colocalized with a- Treatment with leptomycin B resulted in clear accumulation actinin-1 and actinin-4, which are well-known components of endogenous palladin in the nucleus (Figure 5m–o) as of dense regions. Further palladin was found to be located in compared to untreated cells (Figure 5j–l). Factors that might cloud-forming small spots in those podocytes that exhibited induce nuclear shuttling of palladin are still unknown. a motile phenotype with a clear lamellipodium (asterisk in Mechanical strain, inhibition of the phosphatidylinositol Figure 2f). 3-kinase or treatment with cytochalasin D, respectively, Furthermore palladin was present at focal adhesions. which were described to introduce nuclear accumulation of Therefore, we performed colocalization experiments of other proteins, did not increase nuclear localization of palladin and the focal adhesion proteins vinculin and VASP; palladin in cultured podocytes (data not shown). the latter one has recently been described to bind to the proline-rich regions of palladin.12 GFP-palladin colocalized Palladin is highly expressed in dynamic structures such as with vinculin at focal adhesions albeit in a varying ratio RiLiS and ruffles (Figure 3a–c). In contrast to vinculin, GFP-palladin and VASP Time series of GFP-palladin in living podocytes are shown in colocalized at focal adhesions in a constant ratio (Figure Figure 6. Similar to the localization of palladin in fixed cells,

216 Kidney International (2009) 75, 214–226 N Endlich et al.: Role of palladin in actin dynamics original article

F-Actin Palladin Merge abc

Merge Merge Merge

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Tubulin Palladin Merge ghi

F-Actin Palladin Merge

jkl

Figure 2 | Localization of palladin in cultured podocytes. Palladin exhibits different staining patterns in podocytes. Colocalization is shown for F-actin (a, j) and palladin (b, k). Merged pictures are presented in panels c–f and l. Palladin is localized in a punctate pattern along F-actin filaments (b, c, k, l), at the end of stress fibers (d), at focal adhesions (e), and in clouds consisting of small spots (asterisk in f). No overlap between palladin and tubulin is seen (g–i). Palladin spots are localized along F-actin filaments in a punctate pattern with a distance between single spots of approximately 2 mm(j–l). Panel width represents 160 mm(a–i) and 20 mm(j–l).

GFP-palladin localized to dense regions along stress fibers. growth factors such as epidermal growth factor (EGF; Figure Due to the localization of GFP-palladin in dense regions, 6e–l; Supplementary Movie 3). RiLiS are circular clouds of contractions and relaxations of single stress fibers over time small actin-containing spots moving highly dynamic through were clearly visible (Supplementary Movie 1). GFP-palladin the cells and disappearing after a few minutes (Figure 6i–l). was also present in focal adhesions. Some focal adhesion-like At higher levels of stimulation with EGF, RiLiS convert into structures exhibited rapid movement toward the cell margin ruffles (Figure 6e–h). Besides vinculin, VASP and other actin- (arrow in Figure 6a–d; Supplementary Movie 2). associated proteins, we found that palladin is localized in Cultured podocytes develop highly dynamic ring-like RiLiS. Furthermore, we observed that palladin colocalizes actin strutures (RiLiS) and ruffles after stimulation with with the Arp2/3 complex, cortactin, and CD2AP to a high

Kidney International (2009) 75, 214–226 217 original article N Endlich et al.: Role of palladin in actin dynamics

Palladin Merge abc Vinculin

def VASP

ghi Cortactin

Figure 3 | Colocalization of palladin with vinculin and VASP, but not with cortactin. GFP-palladin colocalizes with vinculin (a–c) and VASP (d–f) in focal adhesions. Note the varying ratio between the staining intensities of vinculin and of GFP-palladin in different focal adhesions. No colocalization was observed for palladin and cortactin (g–i), except for the leading edge of the lamellipodium (arrow in panel h marks a palladin cloud). Panel width represents 160 mm. extent in RiLiS only (data not shown). In contrast to FRAP occurred with similar time characteristics in focal podosomes, RiLiS are on the dorsal surface of the cell (Figure adhesions (Figure 8f) and dense bodies (Figure 8g). 7a and b). By confocal laser scanning microscopy we were Fluorescence intensity in reference areas (Figure 8e) initially able to show that the highest intensity of actin spots is apical. dropped due to fluorescence loss in photobleaching, and This was confirmed by scanning electron microscopy (SEM). then slightly decreased over time (about 0.5% per image) The function of RiLiS is still unknown, but these structures caused by laser illumination for imaging. Rectangular might represent some kind of lamellipodia in the apical areas of different sizes (10 mm 10 mm ¼ 100 mm2, direction. 7.2 mm 7.2 mm ¼ 52 mm2, and 5 mm 5 mm ¼ 25 mm2) were Transfection of podocytes with a palladin antisense photobleached to distinguish whether diffusion or turnover construct resulted in a decrease of stress fibers. More was the rate-limiting step of FRAP. FRAP followed a mono- importantly, formation of RiLiS in response to 30 nM EGF exponential function (Figure 8) with half-times of 11±2s for 7 min was significantly blunted in podocytes double (100 mm2, n ¼ 12), 9±2 s (52 mm2, n ¼ 7), and 6±2s transfected with GFP-antisense palladin and red fluorescence (25 mm2, n ¼ 6). If FRAP were limited by diffusion, half-time protein-actin. RiLiS formation was observed in 46±5% of would be proportional to area.18 However, there was no GFP-palladin-transfected cells in contrast to 14±4% of GFP- significant correlation between half-time and area (r2 ¼ 0.11, antisense palladin-transfected podocytes (Figure 7c–i). P ¼ 0.1, n ¼ 25). Thus, FRAP of palladin is mainly due to binding and unbinding of palladin in actin structures. The Photobleaching experiments reveal a rapid turnover of parameters of FRAP for the pooled data (n ¼ 25) were a half- palladin time of 10±1 s and an immobile fraction of 18±4% with a Palladin turnover in focal adhesions and in stress fibers was mean squared regression coefficient of 0.91±0.02 for the analyzed by FRAP analysis in GFP-palladin-transfected exponential fit. Free diffusion of proteins was verified in GFP podocytes (Figure 8). Recovery of palladin fluorescence in expressing podocytes. FRAP of GFP in the cytoplasm is the bleached areas was rapid. More than half of the governed only by diffusion with a half-time of about 80 ms fluorescence was recovered within 17 s (Figure 8d, f and g). for a spot area of 20 mm2.19 As we did not detect any time

218 Kidney International (2009) 75, 214–226 N Endlich et al.: Role of palladin in actin dynamics original article

GFP-Palladin Synaptopodin Merge abc

Palladin CD2AP Merge def

Palladin Nephrin Merge ghi

Figure 4 | Colocalization studies between palladin and podocyte-specific proteins. GFP-palladin colocalizes with the podocyte-specific actin-associated protein synaptopodin in dense regions along stress fibers (a–c). CD2AP, an essential actin-associated podocyte protein, did not colocalize with palladin (d–f). In confluent podocyte monolayers, palladin is concentrated at cell–cell contacts in our podocyte cell line (g). Nephrin, a transmembrane protein essential for the formation of the slit diaphragm, shows a high degree of overlap with palladin in areas of cell–cell contact (h, i). Panel width represents 160 mm(a–f) and 250 mm(g–i). dependency of FRAP (100 mm2 area) in GFP expressing essentially to the glomerular filtration barrier by their podocytes (Figure 8h) with the maximal time resolution of interdigitating foot processes. The complex architecture of our equipment (41 s), diffusion of proteins appears to be interdigitating foot processes depends on the actin cytoske- unhindered in podocytes. Taken together, these data leton and on specific actin-associated proteins. Mutation or demonstrate a rapid turnover of palladin in focal adhesions knockout of actin-binding proteins may result in podocyte and stress fibers. Comparison of FRAP between fixed and foot process effacement and severe kidney disease, especially living cells (Supplementary Figure S4) further supports the FSGS. In this study, we demonstrate that palladin is rapid turnover of palladin in actin structures. significantly downregulated in patients with FSGS. Whether palladin plays a major role in the development of FSGS will DISCUSSION be the subject of further investigations. Palladin expression has been detected in various tissues, In localization studies of palladin, performed in con- including the kidney, by northern and western blot ditionally immortalized and primary podocytes, we observed analyses.4,5 However, the cellular localization of palladin in on one hand the staining pattern described in previous adult tissues has been investigated only for the stomach and reports, that is the punctate pattern along stress fibers, the central nervous system so far.5,6,8 In this paper we show localization in focal adhesions, and at areas of cell–cell for the first time that, besides vascular smooth muscle cells of contact.4 On the other hand, palladin was found to localize to renal vessels and the mesangium, podocytes in vivo and in the cell margin, to dorsal membrane ruffles, to RiLiS and to cell culture exhibit a strong staining for palladin. Podocytes clouds in lamellipodia. These structures were identified to be are highly specialized epithelial cells,20 which contribute highly dynamic by time-lapse microscopy in GFP-palladin-

Kidney International (2009) 75, 214–226 219 original article N Endlich et al.: Role of palladin in actin dynamics

F-Actin Merge abc Palladin

def N-terminal half

ghi C-terminal half

jkl Leptomycin B: untreated B: Leptomycin

mno Leptomycin B: treated B: Leptomycin

Figure 5 | Nucleo-cytoplasmic shuttling of palladin in podocytes. Myc-tagged full-length palladin of podocytes exhibited the same staining pattern as endogenous palladin in F-actin structures (a–c). Expression of the N-terminal half of palladin reproduced the localization pattern of full-length palladin (d–f). In contrast, expression of the C-terminal half of palladin yielded a faint and diffuse cytoplasmic distribution but a strong signal in the nucleus (g–i). Overnight treatment of podocytes with leptomycin B resulted in a nuclear localization of endogenously expressed palladin (m–o) as compared with untreated control cells (j–l). Panel width represents 100 mm.

transfected podocytes. Besides the localization of palladin to palladin antisense expression. This result supports the dynamic structures, we demonstrate that palladin is required hypothesis that palladin is involved in dynamic processes of for the formation of RiLiS in cultured podocytes using actin organization in podocytes.

220 Kidney International (2009) 75, 214–226 N Endlich et al.: Role of palladin in actin dynamics original article

0 min 3 min 5 min 11 min

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Figure 6 | Localization of palladin in highly dynamic structures of living podocytes. GFP-palladin-transfected, living podocytes were studied by time-lapse microscopy. GFP-palladin is localized in highly dynamic focal adhesion-like structures (a–d and Supplementary Movie 2) advancing toward the cell margin (arrow in b), in fast-moving dorsal ruffles (e–h and Supplementary Movie 3), and in RiLiS (i–l). In panel e the focus plane was set on stress fibers; for observation of the dorsal membrane ruffle the focus was set on a higher plane (f–h). Panel width represents 30 mm.

Colocalization studies in podocytes revealed that, in dense somewhat decreased compared to that of full-length palladin, regions of stress fibers, palladin was found not only together suggesting additional functions of the three immunoglobulin with a-actinin-1, as described previously,4,7 but also with a- (Ig) domains in the C-terminal half of palladin in stress fiber actinin-4 and synaptopodin, an actin-associated protein of localization. Mykkanen et al.5 reported that the C-terminal podocyte foot processes and dendritic spines.15 In agreement half of palladin localized along stress fibers in HeLa cells. with earlier observations in fibroblasts,4 palladin was detected Moreover Dixon et al.23 recently reported that the Ig3 in focal adhesions of cultured podocytes. It has been shown domain is responsible for binding of actin. However, by Boukhelifa et al.12 recently that proline-rich regions of expression of the C-terminal half of palladin resulted only palladin binds to the EVH1 domain of VASP. Interestingly, in a diffuse cytosolic distribution in podocytes, indicating we observed that the staining intensity for palladin and VASP that the three Ig domains of palladin are not sufficient to are highly correlated in focal adhesions, whereas the staining target palladin to the different F-actin structures in intensity of focal adhesions varied considerably between podocytes. Given that podocytes in vivo express a-actinin-1 palladin and vinculin. This finding probably reflects the and a-actinin-424 as well as VASP (N. Endlich et al., molecular diversity of cell–matrix adhesions,21,22 suggesting unpublished data), palladin might participate in the targeting that palladin preferentially associates with a specific subset of of a-actinins and VASP in podocytes. Ronty et al.7 have focal adhesions. In confluent permanent as well as in primary shown that palladin expressed on the mitochondrial surface podocytes, palladin accumulated at areas of cell–cell contact. recruits a-actinin-1. The same may hold true for a-actinin-4, Our podocyte cell lines form simplified interlacing processes which shares high homology of the palladin-binding region expressing nephrin,14 an essential transmembrane glyco- around the EF-hand domain with a-actinin-1. protein of the slit diaphragm.17 Palladin colocalized with The C-terminal half of palladin accumulated in the nephrin at areas of cell–cell contact in cultured confluent nucleus of podocytes, which has also been observed by podocytes as well as in primary cells. Mykkanen et al.5 in HeLa cells. Although Mykkanen and co- Expression of the N-terminal half of palladin, containing workers could not exclude nuclear localization to be an the proline-rich regions for VASP binding12 and the a- artifact of transient overexpression, we now demonstrate actinin-1 binding site,7 was sufficient to target palladin to that, after addition of leptomycin B an inhibitor of Crm-1/ focal adhesions and to the cell margin. However, the intensity exportin 1-mediated nuclear export,25 endogenous palladin of the N-terminal half of palladin along stress fibers was does not only localize to stress fibers and focal adhesions in

Kidney International (2009) 75, 214–226 221 original article N Endlich et al.: Role of palladin in actin dynamics

Control a b c Palladin 60 Antisense Palladin

50

40

30

20 *

10 Cells with action rings, % 0

RFP-Actin Merge def Antisense Palladin

ghi GFP-Palladin

Figure 7 | Localization of RiLiS and effect of an antisense-palladin construct. By confocal laser microscopy it could be seen that the highest intensity of fluorescence of actin spots in RiLiS is found apically in podocytes (a). These data were confirmed by scanning electron microscopy where RiLiS (asterisk in b) are also at the dorsal side of podocytes. Transfection of podocytes with a GFP-antisense palladin construct resulted in a decrease of stress fibers (e, f) in comparison with podocytes transfected with GFP-palladin (h, i). Furthermore, podocytes expressing the antisense construct showed a reduction of RiLiS in response to stimulation with 30 nM EGF as compared with GFP-palladin-transfected cells (c). Panel width represents 100 mm(a, b, d–i). Data are mean±s.e.m.; *Po0.05.

cultured podocytes but also strongly accumulates in the The presence of palladin in highly motile actin structures nucleus. These findings suggest that the N-terminal half of such as dorsal ruffles already indicated that palladin is palladin contains a nuclear export sequence, or that the N- rapidly turned over in some actin structures. However, it terminal half of palladin binds to protein(s) being subject to was absolutely unexpected that palladin is exchanged in nuclear export. Concerning the import of palladin into the focal adhesion and dense regions of stress fibers, which are nucleus, our findings indicate that the C-terminal half of rather stable actin structures, with a half-time of 10 s as palladin contains a nuclear localization signal or translocates revealed by FRAP analysis. In contrast, FRAP analysis of GFP- into the nucleus by binding to proteins that are imported a-actinin-1 in fibroblasts yielded half-times of about into the nucleus. Indeed, we identified two putative nuclear 2.5–5min for focal adhesions and dense regions of stress localization signal in the C-terminal half of palladin using fibers.29 Likewise, actin is exchanged in filaments with a half- PSORT II:26 PHARRPR between the first and the second Ig time of about 3–5min as determined by FRAP and domain, and PKKVRPS after the third Ig domain. At present, photoactivation experiments in endothelial cells.30 Similar the specific conditions are unknown that enhance nuclear values for the turnover of a-actinin-1 as well as actin in focal localization of palladin. Mechanical stress has been reported adhesions and stress fibers are also observed in podocytes.31 to result in nuclear accumulation of zyxin in vascular smooth Thus, palladin is exchanged 15- to 30-fold more rapidly than muscle cells.27,28 However, palladin does not translocate into a-actinin-1 and actin in focal adhesions and dense regions of the nucleus in response to mechanical stress in cultured stress fibers. Bundling of actin filaments via a-actinin is podocytes. Further investigations have shown that basal crucial for stress fiber formation. As a-actinin and actin epithelial cells of human prostate tissue and Caki-2 renal possess similar turnover rates, both proteins form a carcinoma cells are strongly positive for palladin in the mechanical unit being stable for several minutes. It is nucleus under baseline conditions (Endlich et al., unpub- difficult to envisage how palladin could directly contribute lished data). to mechanical stability of stress fibers if half of the palladin

222 Kidney International (2009) 75, 214–226 N Endlich et al.: Role of palladin in actin dynamics original article

a bc

Pre-bleach 2 s 7 s 17 s 32 s 52 s d

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60.0 0 102030405060 Time (s) Figure 8 | Analysis of palladin turnover by FRAP. Rectangular areas (outlined rectangles in panels a–c) were bleached in living podocytes, which had been transiently transfected with a GFP-palladin construct. Time series of regions of interest are shown in panels d and e (overview in panel a), in panel f (overview in panel b), and in panel g (overview in panel c). More than half of the fluorescence intensity in the bleached area was recovered within 17 s as shown in panel d. The fluorescence intensity in the reference area (dashed rectangle in panel a) decreases slightly over time due to imaging (panel e). Recovery rates of fluorescence in focal adhesions (panel f) and in dense bodies (panel g, corresponding to the dense body marked by an arrow in panel c) are similar to the recovery rates of the total bleached areas. Note that only two-thirds of the focal adhesion in panels b and f were bleached, but the fluorescence intensity significantly dropped also in the unbleached part as well as in a neighboring focal adhesion (arrows in panel f) due to turnover during bleaching. The exponential recovery of GFP-palladin fluorescence intensity in bleached areas is depicted in panel h (n ¼ 25 cells). Photobleaching of GFP-transfected podocytes with an identical protocol did not result in time-dependent changes of fluorescence intensity in the bleached areas (n ¼ 6 cells). Scale bars are 5 mm(a–c), 3 mm(d, e), 6 mm(f), and 1 mm(g). Data are mean±s.e.m. molecules is exchanged every 10 s. Thus, our FRAP data assembly. As palladin is a phosphoprotein,4 its binding strongly speak against a role for palladin as a bundling interactions might be modified by kinases and phosphatases protein; rather, palladin acts as a scaffolding protein that enabling palladin to participate in complex regulatory brings several proteins together thereby regulating stress fiber networks.

Kidney International (2009) 75, 214–226 223 original article N Endlich et al.: Role of palladin in actin dynamics

The singularity of the actin cytoskeleton in podocytes and RT–PCR on human biopsies its critical role for podocyte function are highlighted by Total RNA was isolated with silica gel columns (RNeasy mini; several findings.32 First, podocytes express unique actin- Qiagen, Hilden, Germany) and was reversed transcribed using associated proteins such as synaptopodin.15 Second, muta- Superscript reverse transcriptase (Life Technologies). Real-time PCR tion or absence of a-actinin-4 in men or mice, results in was performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) using heat-activated TaqDNA polymerase glomerulosclerosis due to podocyte loss by unknown 2,24,33 (Amplitaq Gold; Applied Biosystems). Cycle conditions were as reasons. Third, several stimuli, for example polycations follows: after an initial hold of 2 min at 50 1C and 10 min at 95 1C, and mechanical stress, are known to induce rapid and the samples were cycled 40 times at 95 1C for 15 s and at 60 1C for profound reorganization of the actin cytoskeleton in 60 s. Commercially available predeveloped TaqMan reagents were 34,35 podocytes. In this study we demonstrate that palladin used for human palladin and 18S rRNA as reference . mRNA is expressed and localized to various actin structures in expression was analyzed by standard curve quantification. podocytes. We further show that palladin is significantly downregulated in patients with FSGS. As palladin has been Plasmids and transfection shown to induce and to be required for stress fiber The following plasmid expression vectors were used: GFP-palladin, formation,4,6,7 to interact with several proteins expressed in GFP-antisense palladin, myc-tagged full-length palladin, myc-tagged podocytes (a-actinin-4, VASP, and ), and to play a N-terminal palladin (1–416), myc-tagged C-terminal palladin (414–794), RFP-actin (a gift from Dr S Lindner, Munich, Germany). crucial role for the formation of dynamic actin structures, Differentiated cells were transiently transfected using PerFectin palladin may play an important role for the organization of reagent (Peqlab, Erlangen, Germany), according to the manufac- the actin cytoskeleton in podocytes and thus for the proper turer’s instructions. Cells were taken for experiments 1–3 days after filtration of the kidney. transfection.

MATERIALS AND METHODS Immunofluorescence Cell culture Immunofluorescence procedures were performed as described Cells of a well-established podocyte cell line were used and cultured as previously.35 For immunofluorescence studies cells were cultured described previously.14 Briefly, podocytes were maintained in RPMI on collagen IV-coated glass cover slips. Cells were fixed (2% 1640 (Life Technologies, Karlsruhe, Germany) supplemented with 10% paraformaldehyde, 4% sucrose, phosphate-buffered saline (PBS)) at fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. room temperature for 10 min, permeabilized (0.3% Triton X-100 in To propagate podocytes (permissive conditions), cells were cultivated PBS) for 8 min, and blocked in blocking solution (2% fetal at 33 1C in the presence of 10 U/ml mouse recombinant g-interferon bovine serum, 2% bovine serum albumin, 0.2% gelatine, PBS) for (Life Technologies). To induce differentiation (nonpermissive condi- 45 min. Antibodies for the following proteins were used and were tions), podocytes were maintained at 38 1Cwithoutg-interferon for at incubated for 60 min: mouse anti-palladin 1E6, mouse anti-a- least 2 weeks. For primary podocyte culture, glomeruli were isolated actinin-1 (Sigma), anti-a-actinin-4 (kindly provided by Dr CR from mouse kidneys by sieving.14 After outgrowth of cells, podocyte Kennedy, Ottawa, Canada), mouse anti-synaptopodin (Progen, phenotype was verified by positive staining for synaptopodin and WT- Heidelberg, Germany), mouse anti-cortactin (Upstate Biotechnol- 1 (C-19; Santa Cruz Biotechnology, Heidelberg, Germany). Nuclear ogy, Lake Placid, NY, USA), mouse anti-vinculin (Sigma), rabbit export was blocked by overnight treatment with 20 nM leptomycin B anti-CD2AP (H-290; Santa Cruz), rabbit anti-VASP (kindly (LC Laboratories, Woburn, MA, USA). provided by Dr Frank Gertler, Cambridge, MA, USA), guinea pig anti-nephrin (Progen). Antigen–antibody complex was visualized Human biopsies with Cy2- or Cy3-conjugated secondary antibodies (Dianova, Human kidney biopsies were obtained in the framework of the Hamburg, Germany). F-actin was stained using Alexa 488- European Renal cDNA Bank-Kro¨ner Fresenius Biopsy Bank phalloidin and Alexa 546-phalloidin (Molecular Probes, Eugene, consortium. All European Renal cDNA Bank-Kro¨ner Fresenius OR, USA). Biopsy Bank biopsies were approved by local ethic committees and For immunohistochemistry, mice were anaesthetized and informed consent of patients was obtained. Material was submerged perfused with 2% paraformaldehyde. Mice were used according to in an RNAse inhibitor (RNAlater; Ambion, Austin, TX, USA) and national animal protection guidelines. After perfusion, kidneys were microdissected under standardized conditions at the core facility as removed and stored in 4% sucrose overnight at 4 1C. Tissue was previously described.35 Pretransplant biopsies from living donors frozen at 80 1C and thin kidney sections (3–7 mm) were cut. served as controls. Sections were washed in PBS for 15 min and incubated with blocking solution for 1 h at room temperature in a humidity RT–PCR on cell culture and mouse kidneys chamber. The sections were then incubated with anti-palladin Total RNA of cultured podocytes or mouse kidneys was isolated antibody and Alexa-488-phalloidin for 90 min. with TriReagent (Sigma, Deisenhofen, Germany) according the For immunoelectron microscopy, fixed kidneys (pieces of manufacturer’s protocol. RT was performed as described in detail 1mm3) were washed with PBS, dehydrated in a graded ethanol previously.14 The following primers were used: 1 mmol/l reverse series, and embedded in LR White (Plano, Wetzlar, Germany). primer (GGGCAGTGCAGGACACAATG) and 1 mmol/l forward Immunogold staining was done on ultrathin sections of 70 nm, primer (GGACGCCGGCATCTACACAT). PCR was performed as a incubated with anti-palladin antibody overnight. After intensive hot-start PCR with Ampliwax (Applied Biosystems, Darmstadt, washing with PBS-Tween, sections were incubated with secondary Germany) and the products were separated on 1% agarose gels, antibody (anti-rabbit IgG; Dianova) conjugated with 10 nm gold stained with ethidium bromide. (1:50) for 1 h. After washing four times with Tris-buffered saline-

224 Kidney International (2009) 75, 214–226 N Endlich et al.: Role of palladin in actin dynamics original article

Tween and finally with water the sections were poststained with DISCLOSURE uranyl acetate for 5 min. We viewed the sections with an EM 910 All the authors declare no competing interests. microscope (Zeiss, Oberkochen, Germany) and photographs were done with a Kodak 4489 macro EM film. ACKNOWLEDGMENTS We thank Claudia Kocksch and Regina Maciejewski for skilled technical assistance, Thomas Berger for continuous technical support Induction of RiLiS regarding our in vivo microscopy setup, Rolf Nonnenmacher and Cultured podocytes were double transfected with the plasmid Ilona Dirks for graphical work. This study was supported by grants of coding for RFP-actin together with the plasmid for GFP-antisense the German Research Foundation (DFG, En 327/7-1) to KE, by grant palladin and GFP-palladin, respectively. Podocytes were stimulated GM61743 to CAO, by a Marie Curie fellowship of the European Union 48 h after transfection with 30 nM EGF for 7 min at 38 1C to induce to ES and by a grant from the Else Kro¨ner-Fresenius Foundation to the formation of RiLiS and fixed with 2% paraformaldehyde CDC. We thank all the members of the European Renal cDNA Bank- immediately. Finally, transfected cells were counted dependent on Kro¨ner Fresenius Biopsy Bank (ERCB-KFB) and their patients for their the protein they expressed and compared to the total amount of cells support (see www.research-projects.unizh.ch/p9291.htm for with RiLiS. participating centers at time of study).

Live cell microscopy SUPPLEMENTARY MATERIAL A custom-built Plexiglas chamber was filled with 400 ml observation Figure S1. Palladin expression in human glomerular disease. medium (RPMI 1640 w/o phenol red supplemented with 10% fetal Figure S2. Localization of palladin in primary podocyte cell culture. bovine serum). Cell-containing cover slips coated with mouse Figure S3. Distribution of GFP-palladin in cultured podocytes. Figure S4. Photobleaching of GFP-palladin in fixed and living collagen IV (0.1 mg/ml; BD Biosciences, Heidelberg, Germany) were podocytes. attached to the bottom of the chamber. The chamber was sealed and Movie S1. GFP-palladin-transfected podocyte imaged by confocal mounted on the stage of an inverted microscope (IRBE; Leica laser scanning microscopy over 30 min. Microsystems, Bensheim, Germany). The temperature of the Movie S2. GFP-palladin-transfected podocyte imaged by widefield chamber and of the objective was kept at 37 1C with the aid of an microscopy over 14.3 min. airstream incubator (ASI 400; Nevtek, Burnsville, VA, USA). Movie S3. GFP-palladin-transfected podocyte imaged by widefield Immersion objectives ( 63, NA 1.32) were used. Images were microscopy over 13.5 min. acquired either by widefield epifluorescence microscopy or by confocal laser scanning microscopy (Leica TCS-SP). In widefield Supplementary material is linked to the online version of the paper at microscopy, the cooled CCD camera (Photonic Science, Roberts- http://www.nature.com/ki bridge, UK) and the shutter were controlled with Openlab software (Improvision, Coventry, UK), and images were collected in 45 s REFERENCES 1. Kaplan J, Pollak MR. Familial focal segmental glomerulosclerosis. Curr intervals. Opin Nephrol Hypertens 2001; 10: 183–187. 2. Michaud JL, Lemieux LI, Dube M et al. Focal and segmental FRAP analysis glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4. J Am Soc Nephrol 2003; 14: 1200–1211. FRAP analysis was performed by confocal laser scanning microscopy 3. Kriz W. Podocyte is the major culprit accounting for the progression of (Leica TCS-SP). Images of 512 512 pixel were taken with a chronic renal disease. Microsc Res Tech 2002; 57: 189–195. resolution of 12.9 pixel/mm. A reference image was taken 4. Parast MM, Otey CA. Characterization of palladin, a novel protein immediately before photobleaching. Rectangular areas of 100, 52, localized to stress fibers and cell adhesions. J Cell Biol 2000; 150: 2 643–656. and 25 mm were bleached by means of increasing the zoom by a 5. Mykkanen OM, Gronholm M, Ronty M et al. Characterization of human factor of 4, 5.5, and 8, respectively, and by repeated scanning (10, 8, palladin, a microfilament-associated protein. Mol Biol Cell 2001; 12: and 4 times corresponding to a duration of 17, 14, and 7 s) with 3060–3073. full intensity of the 488 nm laser line. Applied intensities during 6. Boukhelifa M, Hwang SJ, Valtschanoff JG et al. A critical role for palladin in photobleaching were approximately 300- to 500-fold higher astrocyte morphology and response to injury. Mol Cell Neurosci 2003; 23: 661–668. than those applied during acquisition of one image. Immediately 7. Ronty M, Taivainen A, Moza M et al. Molecular analysis of the interaction after photobleaching, images were collected in 5 s intervals. between palladin and alpha-actinin. FEBS Lett 2004; 566: 30–34. Fluorescence intensities were determined in defined regions of 8. Hwang SJ, Pagliardini S, Boukhelifa M et al. Palladin is expressed interest using ImageJ (Wayne Rusband, NIH). Intensities of the preferentially in excitatory terminals in the rat central nervous system. J Comp Neurol 2001; 436: 211–224. pre-bleaching image were taken as 100%. Intensities in the 9. Boukhelifa M, Parast MM, Valtschanoff JG et al. A role for the bleached areas were corrected for fluorescence loss due to repeated cytoskeleton-associated protein palladin in neurite outgrowth. Mol Biol imaging by measuring the intensities in nonbleached reference Cell 2001; 12: 2721–2729. areas. 10. Gammill LS, Bronner-Fraser M. Genomic analysis of neural crest induction. Development 2002; 129: 5731–5741. 11. Luo H, Liu X, Wang F et al. Disruption of palladin results in neural tube Data analysis and statistic closure defects in mice. Mol Cell Neurosci 2005; 29: 507–515. ± 12. Boukhelifa M, Parast MM, Bear JE et al. Palladin is a novel binding Data are presented as mean s.e.m. Student’s t-test, Kruskal–Wallis partner for Ena/VASP family members. Cell Motil Cytoskeleton 2004; 58: test, or Mann–Whitney test were used as appropriate to assess 17–29. statistical significance (SigmaStat, Systat Software GmbH, Erkrath, 13. Goicoechea S, Arneman D, Disanza A et al. Palladin binds to Eps8 and Germany; SPSS, SPSS Inc., Chicago, IL, USA). A P-value below 0.05 enhances the formation of dorsal ruffles and podosomes in vascular was regarded as being significant. In FRAP analysis, fluorescence smooth muscle cells. J Cell Sci 2006; 119: 3316–3324. 14. Schiwek D, Endlich N, Holzman L et al. Stable expression of nephrin and intensities over time were fitted to a mono-exponential recovery localization to cell-cell contacts in novel murine podocyte cell lines. function using SigmaPlot (Systat Software GmbH). Kidney Int 2004; 66: 91–101.

Kidney International (2009) 75, 214–226 225 original article N Endlich et al.: Role of palladin in actin dynamics

15. Mundel P, Heid HW, Mundel TM et al. Synaptopodin: an actin-associated 26. Nakai K, Horton P. PSORT: a program for detecting sorting signals in protein in telencephalic dendrites and renal podocytes. J Cell Biol 1997; proteins and predicting their subcellular localization. Trends Biochem Sci 139: 193–204. 1999; 24: 34–36. 16. Welsch T, Endlich N, Kriz W et al. CD2AP and p130Cas localize to different 27. Wang Y, Gilmore TD. Zyxin and paxillin proteins: focal adhesion plaque LIM F-actin structures in podocytes. Am J Physiol Renal Physiol 2001; 281: domain proteins go nuclear. Biochim Biophys Acta 2003; 1593: 115–120. F769–F777. 28. Cattaruzza M, Lattrich C, Hecker M. Focal adhesion protein zyxin is a 17. Kestila M, Lenkkeri U, Mannikko M et al. Positionally cloned gene for a mechanosensitive modulator of gene expression in vascular smooth novel glomerular protein—nephrin—is mutated in congenital nephrotic muscle cells. Hypertension 2004; 43: 726–730. syndrome. Mol Cell 1998; 1: 575–582. 29. Edlund M, Lotano MA, Otey CA. Dynamics of alpha-actinin in focal 18. Axelrod D, Koppel DE, Schlessinger J et al. Mobility measurement by adhesions and stress fibers visualized with alpha-actinin-green analysis of fluorescence photobleaching recovery kinetics. Biophys J 1976; fluorescent protein. Cell Motil Cytoskeleton 2001; 48: 190–200. 16: 1055–1069. 30. McGrath JL, Tardy Y, Dewey Jr CF et al. Simultaneous measurements of 19. Swaminathan R, Hoang CP, Verkman AS. Photobleaching recovery and actin filament turnover, filament fraction, and monomer diffusion in anisotropy decay of green fluorescent protein GFP-S65T in solution and endothelial cells. Biophys J 1998; 75: 2070–2078. cells: cytoplasmic viscosity probed by green fluorescent protein 31. Endlich N, Otey CA, Kriz W et al. Movement of stress fibers away translational and rotational diffusion. Biophys J 1997; 72: 1900–1907. from focal adhesions identifies focal adhesions as sites of stress 20. Pavenstadt H, Kriz W, Kretzler M. Cell biology of the glomerular podocyte. fiber assembly in stationary cells. Cell Motil Cytoskeleton 2007; 64: Physiol Rev 2003; 83: 253–307. 966–976. 21. Rottner K, Krause M, Gimona M et al. Zyxin is not colocalized with 32. Endlich K, Kriz W, Witzgall R. Update in podocyte biology. Curr Opin vasodilator-stimulated phosphoprotein (VASP) at lamellipodial tips and Nephrol Hypertens 2001; 10: 331–340. exhibits different dynamics to vinculin, paxillin, and VASP in focal 33. Kaplan JM, Kim SH, North KN et al. Mutations in ACTN4, encoding alpha- adhesions. Mol Biol Cell 2001; 12: 3103–3113. actinin-4, cause familial focal segmental glomerulosclerosis. Nat Genet 22. Zamir E, Katz BZ, Aota S et al. Molecular diversity of cell-matrix adhesions. 2000; 24: 251–256. J Cell Sci 1999; 112: 1655–1669. 34. Kerjaschki D. Polycation-induced dislocation of slit diaphragms and 23. Dixon RD, Arneman DK, Rachlin AS et al. Palladin is an actin cross-linking formation of cell junctions in rat kidney glomeruli: the effects of low protein that uses immunoglobulin-like domains to bind filamentous temperature, divalent cations, colchicine, and cytochalasin B. Lab Invest actin. J Biol Chem 2008; 283: 6222–6231. 1978; 39: 430–440. 24. Kos CH, Le TC, Sinha S et al. Mice deficient in alpha-actinin-4 have severe 35. Endlich N, Kress KR, Reiser J et al. Podocytes respond to mechanical stress glomerular disease. J Clin Invest 2003; 111: 1683–1690. in vitro. J Am Soc Nephrol 2001; 12: 413–422. 25. Kudo N, Matsumori N, Taoka H et al. Leptomycin B inactivates CRM1/ 36. Cohen CD, Frach K, Schlo¨ndorff D et al. Quantitative gene expression exportin 1 by covalent modification at a cysteine residue in the central analysis in renal biopsies: a novel protocol for a high-throughput conserved region. Proc Natl Acad Sci USA 1999; 96: 9112–9117. multicenter application. Kidney Int 2002; 61: 133–140.

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