Erratum Homophilic and heterophilic interactions regulate E-cadherin recruitment and junction assembly in MDCK cells Andrew J. Streets, Bart E. Wagner, Peter C. Harris, Christopher J. Ward and Albert C. M. Ong

Journal of Cell Science 122, 1702 (2009) doi:10.1242/jcs.053603

There was an error in the e-press version of the article published in J. Cell Sci. 122, 1410-1417.

The e-press version was incorrectly given page numbers 1360-1367.

We apologise for this mistake. 1410 Research Article Homophilic and heterophilic polycystin 1 interactions regulate E-cadherin recruitment and junction assembly in MDCK cells

Andrew J. Streets1, Bart E. Wagner2, Peter C. Harris3, Christopher J. Ward3 and Albert C. M. Ong1,* 1Kidney Genetics Group, Academic Nephrology Unit, Sheffield Institute, University of Sheffield Medical School, Sheffield S10 2RX, UK 2Electron Microscopy Unit, Histopathology Department, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK 3Division of Nephrology, Mayo Clinic and Foundation, Rochester, MN 55905, USA *Author for correspondence (e-mail: [email protected])

Accepted 19 January 2009 Journal of Cell Science 122, 1410-1417 Published by The Company of Biologists 2009 doi:10.1242/jcs.045021

Summary Autosomal dominant polycystic kidney disease (ADPKD) is the recruited to the lateral membranes coincident with E-cadherin most common inherited renal disease and is caused by within 30 minutes after a ‘ switch’. Recruitment of both mutations in two , PKD1 (85%) and PKD2 (15%). Cyst was significantly delayed when cells were treated with epithelial cells are characterised by a complex cellular a PC1 blocking antibody raised to the PKD domains. Finally, phenotype including changes in proliferation, apoptosis, PC1 and E-cadherin could be coimmunoprecipitated together basement membrane composition and apicobasal polarity. Since from MDCK cells. We conclude that PC1 has a key role in polycystin 1 (PC1), the PKD1 , has been located in the initiating junction formation via initial homophilic interactions basolateral membrane of kidney epithelial cells, we hypothesised and facilitates junction assembly and the establishment of that it might have a key role in mediating or stabilising cell-cell apicobasal polarity by E-cadherin recruitment. interactions. In non-ciliated L929 cells, stable or transient surface expression of the PC1 extracellular domain was sufficient to confer an adhesive phenotype and stimulate Key words: Polycystin 1, E-cadherin, Cell adhesion, MDCK, junction formation. In MDCK cells, we found that PC1 was ADPKD

Introduction PC1 is a large (>460 kDa) heavily glycosylated integral

Journal of Cell Science Autosomal dominant polycystic kidney disease (ADPKD) is the membrane protein with a predicted large N-terminal extracellular most common inherited human renal disease (incidence 1 in 1000 domain (~2500 aa), 11 transmembrane domains and a short live births) and is caused by mutations in two genes, PKD1 (85%) C-terminal cytoplasmic tail (Hughes et al., 1995). The extracellular and PKD2 (15%) (Ong and Harris, 2005; Torres and Harris, 2006). region appears to have a modular structure suggesting the presence ADPKD is an important cause of end-stage renal failure, accounting of potential functional motifs. These include two leucine-rich for ~10% of patients on renal replacement therapy. The disease is repeats (LRR), a C-type lectin, a LDL-A receptor motif and a large characterised by the formation of fluid filled cysts in both kidneys region (~1000 residues) with strong homology to the sea urchin of affected individuals, which ultimately result in end-stage renal receptor for egg jelly (REJ) protein. The major part of the N-terminal failure. Other extrarenal manifestations of the disease include region, however, consists of 16 novel repeats (80-90 aa) with low hypertension, cardiac valve abnormalities and cerebral aneurysms to immunoglobulin domains. These so called (Calvet and Grantham, 2001; Wilson, 2004). PKD domains or repeats are arranged in tandem (II-XVI) except The two proteins involved in ADPKD, polycystin 1 (PC1; also for domain I, which is present between the LRR and lectin modules. known as PKD1) and (PC2; also known as PKD2) The extracellular domain of PC1 has been shown to be cleaved at have been shown to function as a heterodimeric complex (Hanaoka a conserved G-protein-coupled receptor (GPCR) proteolytic site et al., 2000; Newby et al., 2002), activating a number of key (GPS) (position T3049) resulting in N-terminal and C-terminal signalling pathways, which in turn regulate diverse cellular fragments tethered to each other at the cell surface (Qian et al., functions including proliferation, apoptosis, tubulogenesis and 2002) This proteolytic event is thought to be essential for the fluid secretion. This is consistent with the largely overlapping renal function of PC1 in the mature kidney (Yu et al., 2007). and extrarenal phenotypes of PKD1 and PKD2 patients. PC1 and Antibodies to the PKD domains of PC1 have been shown to PC2 are likely to function together in many systems but there is disrupt cell-cell adhesion in subconfluent canine, murine and evidence to suggest they can also function independently (Ong human kidney epithelial cells (Ibraghimov-Beskrovnaya et al., 2000; and Harris, 2005). Both proteins have been located in several Streets et al., 2003). These results support a key role for PC1 in the subcellular structures including primary cilia and the basolateral regulation of cell adhesion. However, it is possible that the role of membrane. Functional evidence that the polycystins can transduce PC1 is dependent on the function of other adhesion molecules such a mechanosensitive Ca2+ current and mediate cell adhesion has as E-cadherin or desmosomal cadherins. In this regard, a role for been reported (Ibraghimov-Beskrovnaya et al., 2000; Nauli et al., PC1 in E-cadherin recruitment has also been reported (Charron et 2003; Streets et al., 2003). al., 2000). PC1 has also been shown to colocalise and PC1 and junction assembly 1411

coimmunoprecipitate with E-cadherin and the catenins in human pancreatic adenocarcinoma cells (Huan and van Adelsberg, 1999). In human cystic cells, the absence of surface PC1 is associated with concomitant loss of surface E-cadherin expression and its replacement by N-cadherin (Streets et al., 2003; Roitbak et al., 2004; Russo et al., 2005). A putative role for PC1 in function has also been postulated because of its immunolocalisation to desmosomal junctions, the mislocalisation of desmosome junction proteins in cystic cells and the reported interaction of the PC1 C-terminus with intermediate filaments (Scheffers et al., 2000; Xu et al., 2001; Russo et al., 2005; Silberberg et al., 2005). However, two studies reporting the timing of PC1 and desmoplakin recruitment after a ‘calcium switch’ conflict with one study showing that PC1 precedes desmoplakin and another showing that it follows desmoplakin (Scheffers et al., 2000; Silberberg et al., 2005). In this study, we present data demonstrating that expression of the extracellular domain of PC1 is sufficient to induce cell aggregation and junction formation in L929 cells and that it is involved in E-cadherin recruitment during MDCK junction reassembly. Our data support a proximal role for PC1 in the establishment of cell junctions and the subsequent acquisition of apicobasal polarity.

Results Cell surface expression of the extracellular domain of PC1 as a GPI fusion protein (NT1-GPI) To investigate whether PC1 is sufficient to mediate cell-cell adhesion, we expressed PC1 in L929 mouse fibroblasts which lack classical cell adhesion molecules. Previous studies have utilised this non- adhesive cell line to demonstrate the function of exogenous cell adhesion molecules including E-cadherin (Nagafuchi et al., 1987), and desmosome proteins (Marcozzi et al., 1998). In addition, we confirmed that these cells do not express primary cilia or detectable PC1 (not shown). Therefore we reasoned that this model system

Journal of Cell Science would also enable us to determine whether PC1 could function as a cell adhesion molecule independently of ciliary expression. Because our initial attempts to generate stable L929 clones Fig. 1. A PC1 N-terminus-GPI anchor fusion protein is expressed at the cell expressing full length PC1 were unsuccessful, an alternative strategy surface in transfected HEK293 and L929 cells. (A) The extracellular domain utilising a GPI anchor signal sequence (Bernasconi et al., 1996) to of PC1 (M1-R2961) was fused in frame with a Thy-1 GPI anchor signal direct the expression of the extracellular domain of PC1 (aa M1- sequence (NT1-GPI) and cloned into a modified PCR-3 vector. The regions to which the different antibodies used in this study (7e12, LRR, IgPKD) bind, are R2961) to the cell surface was devised (Fig. 1A). This site is indicated. (B) Surface expression of the N-terminus of PC1 was shown by cell N-terminal of the GPS cleavage site (located at T3049) and was surface biotinylation in transiently transfected HEK cells. Western blotting chosen so as to exclude the possibility of proteolytic cleavage of with a specific anti-PC1 N-terminus antibody (7e12) was carried out on the ectodomain (Qian et al., 2002). Surface expression of the NT1- biotinylated protein bound to and eluted from streptavidin beads. The blots GPI fusion protein was easily detectable in transiently transfected show that NT1 is present at the cell surface. Immunoblotting with antibodies directed to an ER resident protein, calnexin and a cell surface marker, HEK293 cells by cell surface biotinylation (Fig. 1B) and Na+/K+ATPase show that only cell surface proteins have been biotinylated. immunofluorescence (not shown). Of note, transient expression of (C) Immunofluoresence staining was carried out on L929 cells transiently NT1-GPI was sufficient to induce the formation of cell-cell contacts transfected with either (i-iii) NT-1/GPI or control empty vector (iv). NT1 was in L929 cell with the protein concentrated at these regions (Fig. detected with 7e12 at points of cell-cell contact (arrows). These areas of cell- cell contact were absent in mock-transfected control L929 cells. (D) L929 cells 1C). By contrast, L929 cells transfected with the empty vector were stably transfected with the NT1-GPI construct. Following G418 showed no cell-cell contacts and no PC1 staining (Fig. 1C). We selection, a number of resistant clones were tested for expression of the NT1- proceeded to generate a total of 20 stable L929 clones expressing GPI construct by RT-PCR using primers specific to the GPI signal sequence as NT1-GPI or the empty vector by screening G418-resistant cells by well as NT1. Clones with high expression (c13) and low expression (c2) as RT-PCR. Examples of two such clones are shown in Fig. 1D: c2 well as mock-transfected wild-type clones were identified. Actin was used as a loading control. Western blotting with 7e12 shows that c13 expresses the NT1- (a low expressor) and c13 (a high expressor). Using a previously GPI fusion protein. The protein is weakly expressed in c2 and is not detectable characterised N-terminal PC1 antibody (7e12), we confirmed the in mock-transfected control cells. Equal loading is shown by re-probing the expression of NT1 in both clones by immunoblotting (Fig. 1D) and same blot with an antibody to calnexin. (E) FACS was carried out on non- surface expression by FACS analysis (Fig. 1E). Of interest, NT1- permeabilised L929 clones. Cells were stained with 7e12. There was a significant shift in fluorescence intensity in c13 compared with the negative GPI was expressed as a doublet band possibly because of post- control. c13 had the highest expression (fivefold increase over wild type) of translational modification such as glycosylation. The absence of NT1 at the cell surface, confirming the results seen by RT-PCR and western the GPS cleavage site excludes this as a possible cause. blotting. 1412 Journal of Cell Science 122 (9) Journal of Cell Science

Fig. 2. Expression of NT1-GPI in L929 cells causes cell aggregation, which is inhibited by an anti-PC1 antibody. (A) Cell aggregation assays were carried out on the highest expressing clone (c13) and mock-transfected control cells. Images were taken and particle numbers quantified from six random fields using ImageJ (NIH). Results were expressed as Nt/N0 (where Nt is the number of particles after 4 hours incubation and N0 is the number of particles at the start) and are representative of three separate experiments. Each bar represent the mean ± s.e.m. *A significant difference (P<0.05) between c13 and mock transfectant. Expression of NT1-GPI resulted in significant cell aggregation after 4 hour with an Nt/N0 ratio of 0.14. By contrast, mock-transfected cells showed no aggregation after the 4-hour incubation and remained as a single cell suspension with an Nt/N0 ratio of 1.04. To confirm that cell aggregation was as a direct result of the expression of NT1 at the cell surface, cells were incubated with a blocking antibody (IgPKD) raised to the IgPKD domains (residues 843-2145) of the N-terminus of PC1 or non-immune rabbit serum. The antibody significantly disrupted cell aggregation in the NT1-GPI-expressing L929 cells (Nt/N0 ratio of 0.24) compared with non-immune serum (Nt/N0 ratio 1.01). (B) The NT1-GPI construct was sub-cloned into a pEBTetD-inducible vector and transiently expressed in L929 cells. Protein expression was induced by addition of 2 μg/ml tetracycline to the culture media 16 hours prior to the cell aggregation assay. Mock-transfected cells and uninduced NT1-GPI-expressing cells remained as a single cell suspension after a 4-hour incubation. Tetracycline induced NT1-GPI-expressing cells showed significant cell aggregation (*P<0.05 versus no tetracycline). Western blotting with 7e12 confirmed expression of NT1-GPI in the tetracycline-induced cells alone. Equal loading was confirmed by probing the same blot with an anti-calnexin antibody. (C) Electron microscopy shows cell-cell junction formation in L929 c13 cells stably expressing the NT1-GPI fusion protein. Sites of cell-cell contact are visible (i, arrows). Higher magnification of an area of cell-cell contact (ii) shows the appearance of cell adhesion plaques characteristic of clustering of cell adhesion molecules (arrows).

Stable expression of NT1-GPI in L929 cells causes cell IgPKD, a previously characterised PC1 inhibitory antibody. As aggregation inhibitable by an anti-PC1 antibody shown in Fig. 2A, this PC1 antibody completely inhibited cell To extend the findings seen in transiently transfected L929 cells, aggregation whereas non-immune serum had no effect at the same we carried out aggregation assays on c13, the highest NT1-GPI- dilution. These results confirm that PC1 can specifically confer a expressing L929 clone. After a 4 hour incubation, cells expressing cell-cell adhesive phenotype mediated by homophilic interactions the NT1-GPI fusion protein formed visible large aggregates in of the IgPKD domains in the absence of cell-matrix cues. In addition, suspension. By contrast, control mock-transfected cells remained we found no difference between c13 and mock-transfected L929 as a single cell suspension after the same treatment (Fig. 2A). To clones in adhesion, spreading or migration assays (not shown), confirm that PC1 was directly responsible for the increase in cell confirming that expression of NT1-GPI confers an increase in cell- aggregation, we incubated the cells simultaneously with anti- cell but not cell-matrix adhesion. PC1 and junction assembly 1413

Fig. 3. PC1 is involved in E-cadherin recruitment during junction reassembly following a calcium switch. (A) MDCK II cells were grown to confluence and subjected to a calcium switch assay. Data is representative of four separate experiments. Cells were fixed at various time points after restoring normal Ca2+ levels to the growth media. Cells were incubated either with a blocking antibody (IgPKD) or non-immune rabbit serum for the period of the calcium switch experiment. Cells were stained for E-cadherin, PC1, desmoplakin and ZO-1 expression. (B) Z section analysis of PC1 (i) and E-cadherin (ii) showing that the two proteins colocalised (iii) during junction reassembly. The presence of the IgPKD blocking antibody resulted in delayed recruitment of PC1 and E-cadherin compared with untreated cells. This effect is transient, as 2 hour after normal Ca2+ is restored, both proteins have relocalised to the lateral membrane. (C) Colocalisation and recruitment of PC1, E-cadherin and desmoplakin following a calcium switch was confirmed using a previously described antibody raised to the C-terminal of PC1 (BD3) in paraformaldehyde-fixed MDCK cells. PC1 and E-cadherin were recruited to the after 30 minutes. At this time point, no surface recruitment of desmoplakin was observed. (D) Recruitment of E-cadherin to the cell surface following a calcium switch was detected by cell surface biotinylation. E-cadherin is absent from the cell surface at the moment when Ca2+ is restored to the culture (time 0). 30 minutes after the calcium switch, there is reduced E-cadherin cell surface expression in cells treated with IgPKD. Total E- cadherin levels in the cell lysate remain unchanged. Immunoblotting for calnexin show that only cell surface proteins have been biotinylated.

PC1 regulates E-cadherin recruitment to intercellular junctions The finding that NT1-GPI could induce structural junctions led us to study the role of PC1 during junction reassembly in polarised epithelial cells. To do this, we exploited the well-established ‘calcium switch’ assay in MDCK cells (Nigam et al., 1992). In fully polarised cells grown to confluence, typical cell junctional proteins, E-cadherin (adherens junctions), desmoplakin () and ZO-1 (tight junctions) were appropriately located by immunofluorescence (Fig. 3A). In addition, endogenous PC1 detected with a previously well characterised antibody (anti-LRR)

Journal of Cell Science (Ibraghimov-Beskrovnaya et al., 1997; Ibraghimov-Beskrovnaya et al., 2000) could be clearly seen at primary cilia (not shown) and the lateral plasma membrane where it colocalised with E-cadherin and desmoplakin. Following overnight incubation in low Ca2+ medium (<5 μM Ca2+), cell-cell contacts were lost and cells appeared to round up. Inducible expression of NT1-GPI in L929 cells causes cell Immunofluorescence staining revealed that E-cadherin, aggregation desmoplakin, ZO-1 and PC1 were all redistributed from the cell To further demonstrate that PC1 can directly mediate cell adhesion, surface into cytoplasmic vesicles (Fig. 3A). When normal Ca2+ we subcloned the NT1-GPI fusion protein into a tetracycline- levels (1.8 mM Ca2+) were restored, intercellular junctions reformed inducible vector, pEBTetD (Bach et al., 2007) and expressed it over a period of 2 hours. Of the junctional proteins, E-cadherin was transiently in L929 cells. Following a 16 hour induction with recruited earliest to the lateral membrane by 30 minutes as shown tetracycline, but not in its absence, expression of the NT1-GPI fusion by immunofluorescence (Fig. 3A) and cell surface biotinylation (Fig. protein was detectable by immunoblotting (Fig. 2B). As shown for 3D). Lateral staining of ZO-1 and desmoplakin was only detectable the c13 line, induced cells showed significant cell aggregation after after 2 hours (Fig. 3A). Interestingly PC1 was also recruited to the 4 hours compared with non-induced or wild-type cells (Fig. 2B). lateral membrane within 30 minutes of the calcium switch where analysis of Z-sections showed that it colocalised with E-cadherin Formation of cell junctions in NT1-GPI expressing L929 cells for the 2-hour duration of the assay. The temporal and spatial by electron microscopy coexpression of PC1 with E-cadherin indicated that PC1 is likely The structural nature of the cell-cell contacts formed in C13 NT1- to be crucial to E-cadherin recruitment and the formation of GPI-expressing L929 cells following cell aggregation was adherens junctions (Fig. 3B). These findings were confirmed in investigated further by electron microscopy. Under lower paraformaldehyde-fixed MDCK cells using a second PC1 antibody magnification, cells can be seen to adhere to each other (Fig. 2Ci). (BD3) raised to the PC1 C-terminus (Fig. 3C). PC1 and E-cadherin With higher magnification, adhesion plaques, visible as regions of were recruited to the cell membrane after 30 minutes. At this increased membrane density, could be seen (Fig. 2Cii). By contrast, timepoint, no recruitment of desmoplakin was observed. wild-type L929 cells did not adhere and showed no signs of plaque To investigate the possibility that polycystin 1 was crucial for formation (not shown). E-cadherin recruitment to the plasma membrane and formation of 1414 Journal of Cell Science 122 (9)

Fig. 4. The C terminus of PC1 associates with the C-terminus of E-cadherin to form a protein complex independent of PC2 and β-catenin. (A) Reciprocal immunoprecipitation of native PC1 and E-cadherin from MDCK cell lysates show that the two proteins form a complex. Non-immune rabbit IgG and mouse IgG2a control antibodies show that the IP is specific. Data shown is from a representative experiment of two. (B) Diagrams showing (i) E-cadherin with the serine-rich domain sequence highlighted at which HA-tagged E-cadherin truncations were made at amino acids S840X and S855X; (ii) full length Myc-tagged PC1; (iii) HA-tagged PC1- R4227X; and (iv) the Myc-tagged CT1-CD8 fusion. (C) Myc-tagged PC1 (>500 kDa) coimmunoprecipitated with E-cadherin (120 kDa) from HEK cell lysates. In addition, PC1-R4227X (>500 kDa) could still be coimmunoprecipitated with E-cadherin. Data from a representative experiment of three. (D) Wild-type E-cadherin and the S855X mutant were still able to form a complex with endogenous β-catenin but the E-cadherin S840X mutant showed almost no binding. (E) Full length HA-tagged E-cadherin as well as HA-tagged C-terminal truncations are still able to form a complex with 45 kDa Myc-tagged CT1-CD8-Myc but the E-cadherin S840X mutant showed reduced binding. (F) In vitro binding assay of purified MBP-CT1 with His-Thio-tagged E-cadherin C-terminus (CT) demonstrated the absence of a direct interaction between the two proteins. Bacterially expressed MBP (43kDa), MBP-CT1 (65 kDa) or MBP-CT1R4227X (56 kDa) protein were incubated with amylose beads and mixed with His-Thio-tagged E-cadherin CT (50 kDa) or His-Thio-tagged PC2 C-terminus (50 kDa). Bead- associated proteins were eluted by boiling and

Journal of Cell Science immunoblotted with antibodies to MBP or thioredoxin. As a positive control, a direct interaction between MBP-CT1 and His-Thio-CT2 (CT2) was clearly detected.

adherens junctions, after the calcium switch we treated MDCK cells R4227X, in which the coiled-coil domain (responsible for with the PC1 antibody, anti-IgPKD. As shown in Fig. 3B, antibody interacting with PC2) has been deleted, could still bind to E-cadherin treatment following calcium switch resulted in a delay in the in this assay. Transfected E-cadherin was detectable as a prominent recruitment of both PC1 and E-cadherin to the lateral membrane at doublet band as previous reported (Lickert et al., 2000). The upper 30 minutes. This was confirmed by cell surface biotinylation band represents an immature precursor form which is also detectable experiments showing a reduction in surface E-cadherin expression for the endogenous protein (Fig. 3D). in antibody-treated cells compared with cells treated with non-immune To further define the interacting domains between the two serum (Fig. 3D). The effect was, however, transient since by 120 proteins, we expressed the entire PC1 C-terminus as an epitope- minutes after the reintroduction of Ca2+, PC1 and E-cadherin could tagged fusion with CD8 (CT1-CD8-Myc) and found that it was be seen at the lateral membranes in both antibody treated and untreated sufficient to pull-down E-cadherin. Previous studies had shown that cells. Cilia expression was unchanged by antibody treatment (data phosphorylation of a serine-rich domain (840-855) in the C-terminus not shown). These findings support the idea that PC1 regulates of E-cadherin is important for β-catenin binding (Lickert et al., E-cadherin surface expression during junctional assembly. 2000). Indeed, an E-cadherin mutant with a deletion in this region (S840X) lost the ability to bind endogenous β-catenin (Fig. 4E) but Mapping the interaction domains between PC1 and E-cadherin was still capable of binding CT1, albeit with a weaker signal than We next investigated the possibility that PC1 and E-cadherin form a mutant (S855X) retaining this domain (Fig. 4D). These findings a complex by studying their association in native and heterologous indicate that the binding domain for PC1 in the C-terminus of cells. In MDCK cells, endogenous PC1 can be E-cadherin must lie upstream of S840. Nevertheless, it is possible coimmunoprecipitated with E-cadherin (Fig. 4A). We confirmed that the serine-rich domain might act as a regulatory site for binding this interaction by reciprocal immunoprecipitation of transiently affinity, probably through phosphorylation at specific residues. It transfected heterologous PC1 and E-cadherin in HEK293 cells (Fig. is apparent however that PC1 can bind to E-cadherin independently 4C). Surprisingly we found that a naturally occurring PKD1 mutant, of PC2 or β-catenin. PC1 and junction assembly 1415

In vitro binding assays of purified bacterial fusion proteins and the establishment of apicobasal polarity (Gumbiner et al., 1988; containing the C-termini of PC1 (MBP-CT1) and E-cadherin were Yin and Green, 2004). performed to investigate whether the two proteins could interact Our results suggest that PC1 plays a proximal role in the process directly. Using this assay, we were unable to demonstrate a direct of junction assembly in MDCK cells both by mediating initial cell- interaction between PC1 and E-cadherin (Fig. 4F). By contrast, a cell association and by regulating E-cadherin recruitment. In direct interaction between the C-termini of PC1 and PC2 was clearly previous studies, we and others have shown that cell-cell association seen. Our findings indicate that PC1–E-cadherin interaction is likely in a number of renal epithelial cells including MDCK can be to be indirect and mediated through other, yet to be identified, disrupted by an antibody to the PKD domains (IgPKD) or soluble adapter proteins. Alternatively, their direct interaction may require GST recombinant protein (Ibraghimov-Beskrovnaya et al., 2000; an essential post-translational modification absent in the bacterial Streets et al., 2003). Of interest, the cell-cell dissociating effect of proteins. the antibody was reduced by the transgenic expression of PC1 and was absent in the PKD1 null cystic line (Streets et al., 2003). Taken Discussion together with our data from L929 cells, it seems highly probable In this paper, we provide the first direct evidence that PC1 can act that PC1 mediates cell-cell associations in the earliest phase of as a cell adhesion molecule by expressing the entire extracellular junction assembly. region of PC1 as a GPI-anchored protein (NT1-GPI) in L929 cells. During the calcium switch, PC1 and E-cadherin membrane Using a specific inhibitory antibody, we confirm that this is localisation overlap temporally and spatially and can be delayed by dependent on trans-homophilic interactions of the PKD extracellular pre-incubation with an antibody to PC1. The delay is, however, domains. Previous studies had demonstrated the importance of this transient and can be overcome 2 hours after the reintroduction of region for PC1 mediated cell-cell adhesion but were unable to Ca2+. These findings are corroborated by a recent study in which distinguish between its homophilic and heterophilic interactions the rate of E-cadherin recruitment was reported to be enhanced in (with other cadherins) in renal epithelial cells (Ibraghimov- MDCK cells over-expressing PC1 following calcium switch Beskrovnaya et al., 2000; Streets et al., 2003). In L929 cells, NT1- (Markoff et al., 2007). Our results are very similar to those GPI also induced the formation of structural junctions when the described for E-cadherin-depleted MDCK cells where junction cells were induced to aggregate in suspension implying that PC1 formation was delayed but not prevented (Capaldo and Macara, might play a crucial role in junction formation. 2007). These data indicate that PC1 and E-cadherin are important Our results also confirm that PC1 localisation is dynamic in in the establishment of junctions but dispensable for their MDCK cells and alters temporally with junctional assembly and maintenance. Human cystic epithelial cells have been reported with the reacquisition of cell polarity. Using the well established calcium variable levels of E-cadherin but with neo-expression of N-cadherin switch assay, we demonstrate that PC1 is localised best with (Streets et al., 2003; Roitbak et al., 2004; Russo et al., 2005). Since desmoplakin in fully polarised cells but overlaps more with these cells are primarily derived from large surface cortical cysts, E-cadherin during repolarisation. This may explain some of the we suggest that the loss of E-cadherin expression (rather than its differences observed in previous work in which PC1 expression early mislocalisation) and concomitant N-cadherin transcription are was found to colocalise either to adherens junctions or desmosomes likely to occur as later events in cystogenesis.

Journal of Cell Science (Scheffers et al., 2000; van Adelsberg, 2000). Differences in We extended these findings by first showing that PC1 and confluency, differentiation, polarity or cell type could have E-cadherin can be coimmunoprecipitated from MDCK cells contributed to some of the observed differences (Ong, 2000). confirming previous findings in human pancreatic adenocarcinoma The formation of a polarised kidney tubule relies on a series of (HPAC) cells and normal human kidney cells (Huan and van sequential events including cell-cell association, the formation of Adelsberg, 1999; Roitbak et al., 2004). We show for the first time epithelial junctions, the acquisition of apicobasal polarity and finally that both proteins can associate via their C-termini independently the imposition of planar polarity. A large body of work has shown of previously described binding domains. In particular the PC1 that the assembly of epithelial junctions is a hierarchical process mutant R4227X, which lacks a coiled-coil domain essential for PC2 in which, E-cadherin plays a primary role (Adams and Nelson, 1998; binding (Qian et al., 1997) was still able to coimmunoprecipitate Braga, 2002; Jamora and Fuchs, 2002). For instance, MDCK cells E-cadherin. For E-cadherin, it had been shown that a serine-rich depleted of E-cadherin by siRNA show a marked delay in re- domain between amino acids 840 and 855 is essential for β-catenin establishing cell polarity in a calcium switch assay (Capaldo and binding (Lickert et al., 2000). Significantly, binding affinity between Macara, 2007). However, once formed, cell junctions are maintained E-cadherin and β-catenin could be altered by casein kinase II (CKII) even when E-cadherin was subsequently depleted, indicating that and glycogen synthase kinase-3 β (GSK-3β) phosphorylation at the role of E-cadherin is likely to be in the initial phase of junction specific serine residues (Lickert et al., 2000). Although deletion of assembly but is not essential for their maintenance. During junction this domain abolished the binding of endogenous β-catenin, PC1 assembly, E-cadherin is recruited early to cell-cell contact sites, association was still present, albeit reduced. We found no evidence forming a scaffold for other proteins such as the α-catenin and of direct binding between PC1 and E-cadherin using in vitro binding β-catenin, other actin-binding proteins and signalling molecules assays. This could indicate the importance of key mediatory adapter (Vasioukhin and Fuchs, 2001; Perez-Moreno et al., 2003; proteins or the crucial role of post-translational modifications Bershadsky, 2004). The ectodomains of E-cadherin also mediate (including phosphorylation) in mediating and regulating this Ca2+-dependent homophilic interactions between adjacent cells thus process. Future work will seek to address these questions. strengthening cell-cell associations. Tight junction (TJ) proteins are In summary, we have demonstrated the ability of PC1 to promote initially recruited to cadherin contacts possibly through α-catenin- strong aggregating ability in non-adherent L929 cells via homophilic mediated recruitment of ZO-1 and only later move away to form interactions of the PKD domains. Moreover, its temporal, spatial the TJ (Muller et al., 2005). The desmosomal cadherin complex is and biochemical association with E-cadherin in MDCK cells then recruited to complete the formation of the intercellular junctions indicates a probable crucial role in junction initiation rather than 1416 Journal of Cell Science 122 (9)

maintenance. This model could explain why the mislocalisation of UK) or a non-immune rabbit IgG fraction (Dako, Ely, UK). Antibody binding was + + visualised using FITC-conjugated goat ant-mouse IgG and Alexa-Fluor-568-labelled some basolateral proteins such as Na -K -ATPase and epidermal goat anti-rabbit secondary antibodies. Slides were viewed using an Olympus Imaging growth factor receptor (EGFR) are often but not universally Systems inverted IX71 microscope configured for multi-fluorescence image capture. observed in cysts (Wilson et al., 1991; Du and Wilson, 1995) and Images were acquired and analysed using SimplePCI imaging software (Compix). why the deletion of Pkd1 in adult kidney tubules has a mild Z sections were acquired and analysed using a Zeiss LSM 510 META Axiovert 200M confocal microscope and Zeiss LSM image analysis software. phenotype compared with that in the foetus (Lantinga-van Leeuwen et al., 2007). If the major role of PC1 is in junction initiation, a Cell surface biotinylation disturbance in its function (leading to cyst initiation) is most likely Cell surface biotinylation was performed as previously described (Streets et al., 2006). to be manifest under conditions of high junctional turnover such as Samples were analysed by SDS-PAGE and western blotting using the antibodies described. during tubular elongation in the developing kidney or during tissue repair following injury in the mature kidney. Fluorescence-activated cell sorting (FACS) Cells cultured to confluency in 10 cm culture dishes were detached using cell dissociation buffer (Invitrogen) and resuspended in ice cold 3% BSA-PBS at 1ϫ107 Materials and Methods cells/ml. They were incubated with 1/50 dilution of anti-PC1 7e12 antibody or mIgG1 Materials negative control antibody (Serotec, Kidlington, UK) for 30 minutes and washed three All chemicals were purchased from Sigma (Poole, UK) unless otherwise stated. times by centrifugation at 400 g for 5 minutes in PBS. Secondary antibody incubation MDCK II cells were a gift from N. Simmons (University of Newcastle, Newcastle, was with a 1/300 dilution of goat anti-mouse FITC for 30 minutes. After washing UK) and L929 cells were purchased from the ECACC (Salisbury, UK). Antibodies cells were resuspended in PBS and analysed by flow cytometry. to PC1 (IgPKD, LRR and BD3) and desmoplakin were obtained from Oxana Ibraghimov-Beskrovnaya (Genzyme) and D. Garrod (University of Manchester, Cell aggregation assay Manchester, UK), respectively. A rat monoclonal antibody to ZO-1 was obtained Cells grown to confluence were detached using cell dissociation buffer (Invitrogen, from the Developmental Studies Hybridoma Bank (University of Iowa, USA) and UK). A single cell suspension was generated by gently passing the detached cells antibodies to E-cadherin and calnexin were purchased from BD Transduction Labs through a 21 gauge needle and this was verified by light microscopy. DNaseI (Cowley, Oxford, UK). The following cDNA expression constructs were kind gifts (1 μg/ml) was added to prevent clumping as a result of release of DNA from dead α from the following: PCMUIV-CD8 (S. Ponnambalam, University of Leeds, Leeds, cells. Cells were gently mixed at 100 r.p.m. in 12-well dishes pre-coated with 1.5% UK), pBAT-EM2 (D. Garrod), PCR-3/GPI (N. Fasel, University of Lausanne, BSA for 4 hours. To test the effect of the anti-PC1 blocking antibody IgPKD on Lausanne, Switzerland) and pEBTetD (D. Grundemann, University of Cologne, cell aggregation, IgPKD or non-immune rabbit serum was added at 1/50 for the Cologne, Germany). same period of time. Images were taken and particle numbers quantified from six random fields using ImageJ (NIH). Results were expressed as Nt/N0 where Nt is the Plasmid construction number of particles at 4 hours and N0 is the number of particles at the start (Hordijk A full-length PKD1 FLAG-tagged cDNA was generated by exon-linking (C.J.W., et al., 1997). unpublished data). We modified this cDNA by replacing the C-terminus FLAG epitope with a Myc epitope (PKD1-Myc) using PCR. Similarly, an epitope-tagged PKD1 Calcium switch assay truncation mutant (PKD1-R4227X-HA) was generated by introducing a premature Calcium switch assay was carried out essentially as described previously (Nigam et stop codon preceded by an HA-epitope tag sequence. To create NT1-GPI, the entire al., 1992) with the following modifications. Cells were grown on filters to full PKD1 extracellular domain of PC1 (M1-R2961) was removed from full-length cDNA confluence in complete medium. They were rinsed three times and then incubated Eco Bam by restriction digestion with RI and HI and ligated in frame with a Thy-1 overnight in Spinner’s modified Eagle’s medium (SMEM; low Ca2+ media: <5 μM GPI anchor signal sequence in PCR-3/GPI. A tetracycline inducible NT1-GPI Ca2+) with 5% dialysed FCS. The cells were then switched to complete medium for construct was generated by subcloning the NT1-GPI insert into the pEBTetD vector different lengths of time ranging from 0 minutes to 2 hours either in the presence of (Bach et al., 2007). Full length HA-tagged E-cadherin was generated in pcDNA3 by non-immune rabbit sera or 1/50 IgPKD antibody, after which cells were fixed as PCR from the plasmid pBATEM2, which contains the entire coding region of described above. Journal of Cell Science E-cadherin (Nose et al., 1988). HA-tagged E-cadherin truncation mutants were similarly constructed with stop codons replacing S840 and S855, respectively. The Electron microscopy PKD1 C-terminus (aa 4107-4303) containing a Myc epitope tag replacing the CD8 α Following an aggregation assay, cells in suspension were fixed with an equal volume cytoplasmic tail was cloned in frame with the preceding CD8 sequence to create of 3% phosphate-buffered glutaraldehyde in PBS for 1 hour and then gently the CT1-CD8-Myc fusion construct (Ponnambalam et al., 1994). All constructs were sedimented by centrifugation at 1000 g for 5 minutes. The supernatant was then verified by sequencing. removed and the cell pellet stored in 3% glutaraldehyde for a further 24 hours. The cells were then resuspended in molten 2% agarose. On setting, the agarose block was Cell culture and transfection chopped into 1 mm cubes and further fixed in 3% phosphate-buffered glutaraldehyde. HEK293, MDCK and L929 cells were cultured in Dulbecco’s modified Eagle’s This was followed by further fixation in 1% aqueous osmium tetroxide, processing medium (DMEM) supplemented with 10% fetal bovine serum. Transient transfection through graded alcohols, propylene oxide and into medium grade epoxy resin. was carried out on cells cultured to 90% confluency using Lipofectamine 2000 Following heat polymerisation, resin blocks were sectioned using a Reichert ultracut (Invitrogen, Paisley, UK) according to the manufacturer’s instructions. L929 cells E ultramicrotome at 0.6 μm, stained using 1% Toluidine blue in 1% sodium tetraborate stably expressing NT1-GPI were generated by selecting resistant clones with 800 and the sections examined by light microscopy. The most appropriate block was μ g/ml G418 for a period of 3 weeks. Protein expression in cells expressing a pEBTetD selected and thin-sectioned using a Diatome diamond knife at 85 nm. Thin sections μ NT1-GPI inducible vector was induced by addition of 2 g/ml tetracycline to the were then stained in saturated uranyl acetate in 99% ethanol and Reynold’s lead citrate. culture media for 16 hours. Sections were examined on a Philips 400 transmission electron microscope and photographed using large format Kodak EM negatives. RT-PCR RNA was extracted from stable clones using Trizol reagent, cDNA was synthesised In vitro binding assays using a reverse transcriptase kit (Ambion, Applied Biosystems, UK) and expression Constructs encoding the PC1 C-terminal domain and the truncated PC1 C-terminal of the NT1-GPI construct was determined by RT-PCR. A forward primer specific to domain (CT1R4227X) as maltose-binding protein (MBP) fusions or MBP alone were the N-terminus of PC1 and a reverse primer specific to the Thy-1 signal sequence made in pMAL-c2 (New England Biolabs). The C-terminus of E-cadherin or PC2 were used. (CT2) were cloned in-frame with histidine (His) and thioredoxin (Thio) in the pet32a+ vector (Novagen) and the constructs verified by sequencing. Fusion proteins were Immunoprecipitation and immunoblotting induced in BL21-DE3 with 1 mM isopropyl β-D-thiogalactopyranoside for 2 hours Total cell lysates were prepared and processed for immunoprecipitation and western and purified from cleared bacterial lysates using amylose (New England Biolabs) or blotting as previously described (Newby et al., 2002). Nickel His-Select beads (Sigma) as described by the manufacturer. Affinity-purified recombinant proteins were visualized on Coomassie blue stained gels and Immunofluorescence immunoblotting with MBP and thioredoxin (Invitrogen) antibodies. Binding assays Cells were grown on filters and fixed with acid-ethanol at 4°C for 1 hour or 4% were performed by mixing 2 μg of MBP, MBP-CT1, MBP-CT1R4227X fusion protein paraformaldehyde for 5 minutes followed by permeabilisation with PBS, 0.1% Triton attached to amylose beads with either 2 μg of His-Thio E-cadherin CT or His-Thio X-100. Blocking was carried out for 1 hour with 5% milk powder in PBS, and primary CT2 in binding buffer (5 mM Hepes, 150 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, antibodies were incubated overnight at 4°C in 3% BSA-PBS. Controls included cells 0.1% NP-40). Samples were rotated for 1.5 hours at 4°C, pelleted by centrifugation stained without the primary antibody, an irrelevant IgG1 mAb (Serotec, Kidlington, and washed three times with bead-binding buffer prior to elution. PC1 and junction assembly 1417

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