Ouabain Modulates Ciliogenesis in Epithelial Cells

Ouabain modulates ciliogenesis in epithelial cells

Isabel Larrea, Aida Castilloa, Catalina Flores-Maldonadoa, Ruben G. Contrerasa, Ivan Galvanb, Jesus Muñoz-Estradaa, and Marcelino Cereijidoa,1

aDepartment of Physiology, Biophysics and Neurosciences, and bCentral Laboratories, Center for Research and Advanced Studies of the National Polytechnic Institute, Mexico City, DF 07300, Mexico

Edited* by Lutz Birnbaumer, National Institute of Environmental Health Sciences, Research Triangle Park, NC, and approved October 27, 2011 (received for review February 24, 2011)

The exchange of substances between higher organisms and the Results environment occurs across transporting epithelia whose basic Ouabain Accelerates Ciliogenesis. MDCK cells display procilia features are tight junctions (TJs) that seal the intercellular space, some 12 h after reaching conﬂuence (Fig. 1A). Procilia pro- and polarity, which enables cells to transport substances vecto- gressively lengthen until they become mature cilia. At the third rially. In a previous study, we demonstrated that 10 nM ouabain day almost all cells have a cilium (Fig. 1B). Ouabain increases modulates TJs, and we now show that it controls polarity as well. the length of the cilium (Fig. 1C) but not its thickness (Fig. 1D). We gauge polarity through the development of a cilium at the We also followed ciliogenesis by staining the cells with an anti- apical domain of Madin-Darby canine kidney cells (MDCK, epithe- body against acetylated α-tubulin and counting the number of lial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2- E F + cells at stages without cilium (Fig. 1 ), with procilium (Fig. 1 ), dependent manner. Claudin-2, a molecule responsible for the Na and with a mature cilium (Fig. 1G). Fig. 1H summarizes the and H2O permeability of the TJs, is also present at the cilium, as it progressive increase in the percentage of prociliated and ciliated α colocalizes and coprecipitates with acetylated -tubulin. Ouabain cells that tend to reach 100% (open circles). Ouabain accelerates modulates claudin-2 localization at the cilium through ERK1/2. the kinetics of procilia and cilia formation (Fig. 1H, red ﬁlled Comparing wild-type and ouabain-resistant MDCK cells, we show circles), an effect that reaches a 400% increase at 24 h. that ouabain acts through Na+,K+-ATPase. Taken together, our previous and present results support the possibility that ouabain Role of Homo- and Heterotypic Cell-Cell Contacts. To study the role

constitutes a hormone that modulates the transporting epithelial of cell-cell contacts on the effect played by ouabain on cilio- CELL BIOLOGY phenotype, thereby playing a crucial role in metazoan life. genesis, we plated cells at confluency, in mixed populations of 1% MDCK and 99% NRK cells (epithelial rat kidney). Under this E-cadherin | occludin | cell adhesion | cardiotonic steroids condition, most single MDCK cells (Fig. 2A, red) surrounded by NRK cells do not exhibit a cilium (Fig. 2 A and C). Nevertheless, he high affinity and specificity of Na+,K+-ATPase for the ouabain is able to stimulate cilliogenesis even in these MDCK Tplant-derived inhibitor ouabain suggested the possibility that cells totally surrounded by NRK cells (Fig. 2 B and C). there might exist endogenous analogs. Hamlyn et al. (1) dem- In keeping with our previous observations (19, 20) that the TJ onstrated that there is a substance in plasma that cannot yet be is a promiscuous structure that can be established by epithelial distinguished from ouabain, even by electrospray ionization-mass cells from different organs and even from different animal spe- 1 spectrometry, H-NMR, and liquid chromatography (2–4). Fur- cies, we found that monolayers of mixed MDCK (Fig. 2, red) and thermore, the observation that this endogenous ouabain and its NRK cells develop a transepithelial electrical resistance (TER). analogs increase during exercise (5), salty meals (6), and path- This parameter is proportional to the TER in the monolayer of ological conditions [such as arterial hypertension, eclampsia (7), a single-cell type and their proportion in the mixture (Fig. 2D). and myocardial infarction (8)] raised the possibility that it may Mixed cells also express ZO-2 at the TJs (Fig. 2E). Nagafuchi function as a hormone (9). This theory prompted efforts to un- et al. (21) showed that the expression of E-cadherin requires that ravel its physiological role (10). The present work stems from our the two neighboring cells belong to the same animal species previous observations that toxic levels of ouabain invariably af- (homotypic contact). In accordance with this observation, Fig. 2F fect cell-cell and cell-substrate adhesion molecules in Madin- shows that monolayers of pure MDCK (Fig. 2F, red), as well as F Darby canine kidney (MDCK) cells (11), suggesting that low-dose small groups of MDCK cells (Fig. 2 , red), express E-cadherin F G ouabain may be able to modulate cell contacts without causing (Fig. 2 and , green) at homotypic cell-cell borders. However, irreversible damage (12). In keeping with such possibility, we have this molecule is not observed at heterotypic MDCK/NRK con- G previously shown that ouabain promotes cell-cell contacts as well tacts (Fig. 2 ). Accordingly, a quiescent single MDCK cell (Fig. H as contact-dependent phenomena, such as increases in cell com- 2 ) expresses no E-cadherin at its borders. We conclude that TJ munication, the expression of connexin-32 (13), and the molec- sealing and ouabain stimulation of ciliogenesis occur in cells that ular structure and hermeticity of the tight junction (TJ). This establish homo- and heterotypic borders, even if these express no process occurs by regulating the specific expression and distri- E-cadherin at the plasma membrane. bution of claudin-1, -2, and -4 (12) during differentiation toward Ouabain and Na+,K+-ATPase. Ouabain promotes ciliogenesis when the so-called “epithelial transporting phenotype.” added from the basolateral (Fig. 3 A, B, and J, first and second Pursuing our exploration of the effect of low-dose ouabain on columns) but not from the apical side (Fig. 3C), in accordance cell contacts, we now study whether 10 nM ouabain can modulate polarity, one of the two basic cell features of the transepithelial transporting phenotype (14, 15). We gauge polarity Author contributions: I.L., A.C., C.F.-M., R.G.C., and M.C. designed research; I.L., A.C., C.F.-M., through the expression of a single cilium at the center of the R.G.C., I.G., and J.M. performed research; I.L., A.C., C.F.-M., R.G.C., and M.C. analyzed data; apical domain, a process that relays on the polarized delivery of and I.L., R.G.C., and M.C. wrote the paper. many proteins to this apical compartment (16, 17). At 10 nM, The authors declare no conflict of interest. ouabain causes none of its well-known toxic effects; it does not *This Direct Submission article had a prearranged editor. + + + inhibit the unidirectional transport of K by Na ,K -ATPase 1To whom correspondence should be addressed. E-mail: Cereijido@fisio.cinvestav.mx. + nor distorts the K balance of the cell, and it does not cause cell This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. death (12, 18). 1073/pnas.1102617108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1102617108 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 Fig. 1. Ouabain, at a concentration of 10 nM, accelerates ciliogenesis. Scanning electron micrographs of a cilium (between arrows) in a monolayer that has been confluent for 12 h (A), or 3 d (B) under control conditions. Cilia length (C) and thickness (D) as measured in scanning electron microscopy micrographs of wild-type MDCK cells, under control (white bars) or 24 h of ouabain treatment (red bars); ***P < 0.001. Monolayers of MDCK cells stained with antiacetylated α-tubulin at zero (E), 24 h (F), or 72 h (G)in confluence. Ciliogenesis as observed in confluent monolayers under control (open circles) and ouabain (red circles) treatment as a function of time (H).

with the fact that the Na+,K+-ATPase located at the intercellular cell borders is only accessible from the basal side (20, 22– fi 24). This nding suggests that this enzyme may act as receptor Fig. 2. Ciliogenesis in proliferation-arrested MDCK cells does not depend for low concentrations of ouabain acting on cilliogenesis. This on homotypic cell contacts. Stable red color MDCK cells were produced by possibility was further tested using ouabain-resistant R-MDCK transfection of red fluorescent protein. To produce MDCK cells with arrested cells, a stable line produced by Soderberg et al. (25) with very low proliferation, we plated them at confluence in a 1/99 ratio with NRK cells. affinity for ouabain. Procilia and cilia in R-MDCK cells are ru- Forty-two hours later cells were fixed and stained with the indicated anti- fi dimentary (Fig. 3 D and E), and their length (Fig. 3F) and bodies (green) and to detect nucleus with DAPI (blue, NUC). The gure G shows a single MDCK completely surrounded by NRK cells, under control thickness (Fig. 3 ), as well as the number of ciliated and pro- condition (A), and treated with 10 nM ouabain for the last 30 h (B). The ciliated cells (Fig. 3 H, I, and J. third and fourth columns), are cilium is stained with a primary antibody against antiacetylated α-tubulin (Ac not stimulated by ouabain, as observed by scanning electron Tub) and a fluoresceinated secondary one (green). Statistical analysis of microscopy and immunodetection of acetylated α-tubulin. single MDCK cells, either under control (open circles) or ouabain treatment Zampar et al. (26) have clearly demonstrated that acetylated conditions (red circles) (C). Establishment of tight junctions as revealed by fi α-tubulin binds and inhibits Na+,K+-ATPase in cells of rat brain the value of TER (D) in monolayers of pure MDCK cells ( rst column), pure fi ouabain-resistant NRK cells (second column), and a 50/50 mixture of both cell and COS (monkey kidney broblasts), and Menco et al. (27) types (third column) cultured for 48 h; ***P < 0.001. In mixed monolayers of have found this enzyme in the cilium of olfactory receptor cells. MDCK (red) and NRK cells, the cytoplasmic protein ZO-2 is detected by im- In MDCK cells instead, this enzyme only occupies its well-known munofluorescence (green) (E). Immunodetection of E-cadherin (green) in specific position at lateral homotypic cell borders (20, 23, 24), a monolayer of pure MDCK cells (3 d old) (F), or in mixed monolayers and does not colocalize with ciliar acetylated α-tubulin (Fig. 3K). (48 h old) showing a small group (G) or a single (H) MDCK cell surrounded by μ Small groups of MDCK cells expressing red fluorescent protein NRK cells. (Scale bars, 10 m.) and surrounded by NRK cells only express Na+,K+-ATPase at homotypic MDCK-MDCK cell borders (Fig. 3L, arrow). When Cell Signaling. Na+,K+-ATPase forms a receptor complex by as- a single MDCK cell is completely surrounded by NRK cells, sociating with signaling molecules, such as c-Src and IP3-re- Na+,K+-ATPase cannot be observed at the plasma membrane ceptor, which in turn may activate other proteins, such as ERK1/ (Fig. 3M). 2. These associations may be modulated by ouabain (28, 29). In

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1102617108 Larre et al. Downloaded by guest on September 25, 2021 Fig. 3. Role of Na+,K+-ATPase in the effect of ouabain. Monolayers of wild MDCK cells, under control (A)and48-h treatment with ouabain (B and C), added from the basolateral (A and B) or the apical side (C). Ouabain-resistant MDCK cells (MDCK-R) display very short cilia (D)thatdonot become larger (E and F) nor thicker (E and G), nor increase the number of ciliated cells (H vs. I and J)inresponseto2d of incubation with ouabain. Cilia were stained with an antibody against acetylated α-tubulin. Statistical analysis of ciliogenesis in wild-type MDCK cells under control (J, ﬁrst column) and ouabain (J, second column). Third and fourth CELL BIOLOGY columns indicate that ouabain-resistant MDCK cells do not increase the number of ciliated cells when treated with ouabain for 48 h (J); ***P < 0.001. In a 4-d old monolayer of pure MDCK cells (the last 3 d at conﬂuence), Na+,K+-ATPase (green) is localized at all cell borders, and it was never observed at the cilium (red) (K). In 1.5-d mixed monolayers of MDCK (red) with NRK (unstained), the enzyme is only observed at homotypic MDCK-MDCK borders (L,arrow). In keeping with this observation, a single MDCK cell does not express the protein because all its borders contact NRK cells (M). (Scale bars, 20 μm, except for D and E,whichare1μm.)

previous work, we observed that 10 nM ouabain increases the induces the delivery of claudin-2 to the cilium through ERK1/2. cell content of claudin-1 and -4 through ERK1/2, and c-Src Ouabain does not modify the content of acetylated α-tubulin participates in the regulation of claudin-1 but not -4 (12). This (Fig. 5N, open squares), but it does increase the synthesis of finding suggests that in principle ERK1/2 may play a significant claudin-2, as first observed by Larre et al. (12) (Fig. 5N, open role in ouabain-modulated ciliogenesis. Fig. 4A shows that under control conditions, there are only a few ciliated cells, as observed with an antiacetylated α-tubulin antibody, and 10 nM ouabain enhances this number (Fig. 4 B and E, first two columns). Twenty-five micromolars of PD98059 (PD), a known inhibitor of ERK1/2, is able to promote ciliogenesis by itself (Fig. 4 C and E, third column), yet it clearly prevents a full effect of ouabain (Fig. 4 D and E, fourth column). These results indicate that ouabain regulates the localization of acetylated α-tubulin at the cilium although ERK1/2.

Claudin-2 as a Ciliary Protein. Larre et al. (12) showed that 10 nM ouabain modulates the amount and distribution pattern of claudin-1, -2, and -4. The staining of claudin-2 and -1 in green (Fig. 5 A, D, G, and J) and acetylated α-tubulin in red (Fig. 5 B, E, H, and K) indicates that claudin-2 is present at the cilium. Claudin-2 and acetylated α-tubulin are observed to colocalize in transversal optical section (Fig. 5 D, E, and F), as well as x/y projections (Fig. 5 G, H, and I). Colocalization occurs along the Fig. 4. Ouabain induces ciliogenesis through ERK1/2. Cilia stained with an α whole cilium; this is speciﬁc for claudin-2, as claudin-4 cannot be antibody against acetylated -tubulin in a control (A) and a monolayer observed (Fig. 5 J, K, and L). Claudin-4 and occludin cannot be treated with ouabain for 48 h (B). Results corresponding to experiments similar to A and B, but in the presence of PD (25 μM), an inhibitor of ERK1/2 observed at the cilium either (Fig. S1). Ouabain increases ciliary (C and D). Statistical analysis of the percentage of ciliated cells under control, claudin-2 (Fig. 5M, ﬁrst two columns), and PD inhibits this effect ouabain-stimulated, control with PD, and ouabain treatment conditions in (Fig. 5M, fourth vs. third columns), indicating that ouabain the presence of PD (columns 1–4) (E); ***P < 0.001.

Larre et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 Fig. 5. Claudin-2, besides of being a typical TJ protein, is also a ciliary protein that is affected by ouabain via ERK1/2 signaling. Immunofluorescence pictures of monolayers incubated with ouabain for 48 h to stain claudin-2 (A, D, and G, green) and -1 (J, green) and acetylated α-tubulin (B, E, H,andK, red). C, F, I,and L correspond to the superposition of the corresponding images. D, E, and F are lateral views of monolayer showing claudin-2 at the cilium. (G, H,andI) x/y projections showing that claudin-2 and acetylated α-tubulin colocalize along the whole cilium at the apical domain. Statistical analysis of the percentage of cells with ciliar claudin-2 observed in images of immunofluorescent cells costained with antibodies against this protein and acetylated α-tubulin, under control, ouabain-stimulated, control with PD, and ouabain treatment in the presence of PD (columns 1–4) (M); ***P < 0.001. Total cell content of acetylated α-tubulin (N, open squares) and claudin-2 (N, open circles). The correspondent effect of PD is shown in gray circles for claudin-2 and gray squares for acetylated α-tubulin. To gain information on the association between acetylated α-tubulin and claudin-2, we coprecipitated the first and blotted for the second (O). Lane 1 corresponds to total homogenate (Total Ext), and the lanes 2–5 depict immunoprecipitation with an antibody against acetylated α-tubulin (IP: AcTub), blotted with an antibody against the same acetylated α-tubulin (WB: AcTub) or claudin-2 (WB: claudin-2). Ouabain effect is shown in the lane 3 (ouabain). Lane 4 shows the effect of PD. Interestingly, this inhibitor increases by itself the association. The effect of ouabain does not depend on ERK1/2 as PD does not block the effect (lane 5). Claudin-2 (green) in a MDCK clone that does not express this protein in the cilia, identified as usual with fluorescent red label, in a x/y (P) and z/x image (Q). Ciliary claudin-2 is not required for the development of the cilium. (Magnification: A–F and J –L,400×; G and H, 1,200×; P and Q, 600×.) (Scale bar in J,10μm.)

circles). Fig. 5N (gray circles) shows that this last effect does not work, we also observe that 10 nM ouabain accelerates ciliogenesis, require the activation of ERK1/2. Claudin-2 is associated to as well as increases the length of the cilium. Ouabain accelerates acetylated α-tubulin, as both coimmunoprecipitate with acety- the expression of acetylated α-tubulin at the cilium, yet it does lated α-tubulin antibodies (Fig. 5O, lane 2). Ouabain increases not change its cell content. This molecule also increases the total the extent of this association (Fig. 5O, lane 3). This association cell content of claudin-2, as well as its localization at the cilium, does not depend on ERK1/2, as PD does not block it (Fig. 5O, and promotes the association between these proteins. Cilium lanes 4 and 5). Interestingly, PD by itself elicits a similar effect as disassembly operates usually at a constant rate in different spe- ouabain. This coimmunoprecipitation of acetylated α-tubulin cies and conditions (16), therefore ouabain may accelerate the and claudin-2 further demonstrates that this TJ molecule is also polarized traffic to provide building blocks to the growing cilium, a ciliary protein. Ciliary claudin-2 is not necessary for ciliogenesis, as well as the anterograde transport. as different clones of MDCK cells that do not express this protein We demonstrated that ouabain requires the lateral ouabain- develop normal cilia (Fig. 5 P and Q). sensitive Na+,K+-ATPase to modulate the sealing of TJs (32). To test if Na+,K+-ATPase is the receptor occupied by ouabain Discussion to accelerate ciliogenesis, we used resistant (R)-MDCK cells At 10 nM, ouabain does not produce its well-known toxic effects, (25), whose resistance to ouabain results from a substitution of such as the inhibition of K+ and Na+ pumping, cell detachment a cysteine by tyrosine or a phenylalanine in the first trans- from its neighbors, and substrate or cell death (12, 13, 18, 20, 30, membrane segment of Na+,K+-ATPase (33). R-MDCK cells do 31). Nevertheless, at this concentration, oubain modulates the not respond to low concentrations of ouabain, a result that sealing of TJs by changing the cell content and localization of strengthens the possibility that Na+,K+-ATPase is the receptor. different claudins through specific mechanisms. In the present As in other cellular processes, such as cell proliferation (34),

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1102617108 Larre et al. Downloaded by guest on September 25, 2021 death (35), and TJ sealing (12), ouabain modulates the ciliary respect, we observed that the effect of ouabain is also mediated localization of acetylated α-tubulin although ERK1/2. by ERK1/2. Chrystallographic analysis shows that there is a single ouabain We have previously showed that 10 nM ouabain modulates binding site per α-subunit (36), supporting the possibility that cell contacts, such as TJs (12), and communicating junctions despite being extremely different, toxic and hormone-like effects (13). Here we demonstrate that 10 nM ouabain also stimulates are triggered by binding to the same site of in the α-subunit. In cell polarity, the other fundamental feature of the transporting classic pharmacology, “intrinsic efficacy” is viewed as a constant epithelial phenotype. for each ligand at a given receptor, irrespective of where the receptor is expressed. This concept is somewhat insufficient to Materials and Methods provide a useful theoretic framework for the multitude of ligand- Antibodies and Chemicals. Antibodies against: claudin-1(Cat. no. 51–9000), -2 receptors known today. For this reason, Urban et al. (37) pro- (Cat. no 51-6100), -4 (Cat. no 32-9400), ZO-2 (Cat. no 71-1400), and occludin posed that ligands induce unique ligand-receptor conformations (Cat. no 71-1500), as well as secondary HRP-anti mouse (Cat. no 61-6620), HRP-anti rabbit (Cat. no 62-6120), FITC-anti rabbit (Cat. no F2765), TRITC-anti that frequently result in the differential activation of signal- “ mouse (Cat. no 62-6514), and FITC-goat anti-rat (Cat. no 62-9511) were transduction pathways, and coined the term functional selec- obtained from Invitrogen. Antiacetylated tubulin was obtained from Sigma tivity.” In this respect, the present work provides evidence that (T-7451) and α-subunit Na+,K+-ATPase from Thermo Scientific (MA3-929). the interaction of ouabain with Na+,K+-ATPase at the same Ouabain was obtained from Sigma (O-3125), and inhibitor PD98059 was receptor site, results in several drastically different cell respon- from Calbiochem-Novabiochem. ses. The situation is further complicated by several additional facts: (i) the expression of Na+,K+-ATPase at the cell mem- Cell Culture, Chemicals, and Antibodies. MDCK-II cells (canine renal; American brane depends on the interaction of two receptors present in Type Culture Collection, CCL-34) were grown at 36.5 °C in a 5% CO2 atmo- sphere in DMEM (GIBCO-Invitrogen) supplemented with penicillin-strepto- neighboring cells that anchor each other in such a position (22, ·μ · ii + mycin 10,000 U g mL (In Vitro) and 10% FBS (GIBCO-Invitrogen). This 23); ( ) the overall effect (e.g., vectorial Na transport) requires medium will be referred to as CDMEM. Cells were harvested with trypsin- that at least two cells collaborate (e.g., in the making of a TJ); EDTA (In Vitro) and plated on glass coverslips contained in 24-well multid- and (iii) the variety of responses is further compounded by the ishes (Costar 3524) and other supports specified below for each experiment. fact that the enzyme can be assembled by at least four different The medium were cultured at a ∼70% saturating density, maintained for α- and three different β-isoforms, which have different sensitiv- one day in CDMEM, followed by serum starvation (24 h, 1% FCS in CDMEM) ities to ouabain. The reasons why a given isoform predominates and then treatment with or without 10 nM ouabain. NRK cells (rat kidney; in certain tissues remain to be elucidated. American Type Culture Collection CRL 1571) were cultured in the same CELL BIOLOGY + + manner. Monolayers were exposed to PD 1 h before starting the challenge The exposure of Na -K -ATPase at a given cell border with ouabain. depends on the homotypic interaction between two identical β + + -subunits of Na -K -ATPases (22, 23). Failure to observe the Ciliogenesis in Mixed Monolayers. To obtain small groups (1–5 cells) of MDCK expression of this enzyme at heterotypic borders may not be cells, suspensions of MDCK cells expressing the red fluorescent protein (see because of its absence, but because of the fact that it only dwells below) were mixed at a ratio of 1:99 with nonstained NRK cells and plated to briefly at the plasma membrane. In this respect, we have pre- confluence. These cells were allowed 30 min to attach, and then the medium viously observed that subconfluent cells and confluent MDCK was changed to medium containing 10% FCS. This medium was replaced cells incubated in media without Ca2+ express up to a third of 12 h later by medium with 1% CDMEM with or without 10-nM ouabain and fi the Na+-K+-ATPases population at the membrane, according to cells xed at different times and stained as described below. the 3H-ouabain binding method (38). We have also made a sim- Immunoblot. Total cell extracts from monolayers in inserts were washed three ilar observation with the Shaker ion channel molecule in which we times with ice-cold PBS with Ca2+ and then incubated at 4 °C for 10 min with had deleted the retention domain: the deletion does not impair the lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, correct polarized expression of the channel, but results in transient 0.1% SDS, and 50 mM Tris pH 7.5) for protein extraction. Then cells were dwelling (39). However, a brief exposure of Na+-K+-ATPase to 10 centrifuged 10 min at 17,000 × g. The supernatant was recovered and the nM ouabain is sufficient to trigger ciliogenesis in single MDCK total protein content was measured by the BCA assay (Pierce), subsequently cells completely surrounded by NRK cells. boiled in Laemmli sample buffer (Bio-Rad; Cat. no. 1610737) and resolved by We observed that claudin-2, a typical molecular component of SDS-PAGE and transferred to PVDF sheets (Hybond-P; Amersham Bio- sciences). These sheets were blocked overnight with 5% BSA. Specific bands the TJ, localizes also at the cilium, and that this expression is fi fi were detected with speci c antibodies and chemiluminiscence (ECL and speci c, as neither claudin-1 nor -4, nor occludin were detected Hiperfilm; Amersham). Resolved bands were analyzed with the software (Fig. 5 and Fig. S1). These results show that the expression of Kodak 1D 3.5.4 (Eastman Kodak) and data were processed with GraphPad claudins at the cilium is not restricted to the human retinal Prism 4 (GraphPad Software). pigment epithelium, as previously suggested (40). Nevertheless, here we show that ciliary claudin-2 is not necessary for cilio- Immunoprecipation. After 48 h of 10 nM ouabain treatment, 100 cm2 mon- genesis. Septin forms a diffusion barrier that restricts the passage olayers were washed with PBS containing 0.1 mM phenylmethylsulfonyl of large molecules toward and away from the cilium (41), with fluoride (PMSF) and scraped into 1,500 μL of RIPA buffer (20 mM Tris pH 7.5, β a limit around 10 kDa (17). This barrier may prevent ciliary 2 mM EGTA, 5 mM EDTA, 30 mM NaF, 40 mM -glycerophosphate, 20 mM sodium orthovanadate, 3 mM benzamidine; 0.5% Nonidet Nonidet P-40, claudin-2 from diffusing toward the rest of the apical domain. Yu and a protease inhibitor mixture from Roche) and lisated with an insuline et al. (42) and Muto et al. (43) observed that claudin-2 has the syringe. Samples were centrifuged at 20,000 × g, 4 °C for 10 min, the super- ability to form pores in the TJs where the side-chain carboxyl natants were collected and 5.5 mg of protein were used for the immuno- group of aspartate-65 forms a site that specifically favors the precipitation. This assay was performed with the exacta Cruz Kit (Cat. no. passage of Na+ ions. However, given that the cilium does not sc-45053; Santa Cruz Biotechnology) following the recommendations of separate the two liquid compartments, it is highly unlikely that the manufacturer. ciliary claudin-2 would play a role in permeation. However, if claudin-2 contains a Na+-sensitive site, it may possibly bind to Immunofluorescence. Monolayers on coverslips were washed three times fi − this ion in a concentration-dependent manner and act as a sensor with ice-cold PBS xed and permeabilized with methanol for 8 min at 20 °C, + washed three times with PBS, blocked 1 h with 0.5% BSA, and incubated of Na concentration in the lumen of the nephron. This sensing for1hat37°Cwithaspecific primary antibody, followed by washes as role of cilia would be consistent with its participation in olfaction above, and an incubation with a second antibody against the primary and photoreception. Furthermore, cilia are connected to a series antibody. Monolayers were then rinsed six times with PBS, incubated with of other mechanisms, such as signaling routes (44). In this a FITC- or TRITC-labeled secondary antibody according to the animal

Larre et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 species used (1 h at room temperature), rinsed as indicated before, plasmid DNA. The mixture was incubated for 5 min at room temperature mounted in Fluorguard (Bio-Rad), and examined by confocal microscopy after the addition of the transfection reagent, and another 30 min after (SP2 Leica Microsystems). Captured images were processed with ImageJ addition of DNA. Lipofectamine 2000 complexes with DNA were added in (National Institutes of Health) and figures constructed with GIMP (GNU a volume of 0.5 mL per dish. Cells were incubated at 37 °C for 4 h and the image manipulation program). transfection complexes were removed. All transfectants were maintained in antibiotic-free complete medium. The stable cell line was acquired under Scanning Electron Microscopy. After 24 h of 10 nM ouabain treatment, cells stress condition with antibiotic G418. The fluorescent cells were viewed with fi were xed with 2.5% glutaraldehyde in saline buffer (100 mM KCl, 10 mM a confocal and Epi-fl microscopy. pDsRed2-N1 was used to express a DsRed2 CaCl2, 3.5 mM MgCl2, and 10 mM Hepes pH 7.4) for 1 h at 37 °C and post- protein in the MDCK cell line as a transfection marker (red-MDCK). pDsRed2- fixed with 1% osmium tetroxide in PBS for 1 h at room temperature. N1 was amplified and then purified by Wizard Plus Maxipreps DNA Purifi- Monolayers were gently washed three times with PBS, dehydrated in in- cation System (Promega; A7270). creasing concentrations of ethanol (from 50% to 100%), critical-point dried using a Sandri-780A apparatus (Tousimis), gold-coated with a Desk II Gold Statistical Analyses. Statistical analyses were performed with GraphPad sputter-etch unit (Denton Vacuum Inc.), and examined with a Jeol JSM- ± fi 6510LV scanning electron microscope. Prism 4. Results are expressed as the mean SE. Statistical signi cance was estimated with a one-way ANOVA followed by a Bonferroni’smul- < < < Tansepithelial Electrical Resistance. Cells were grown on Transwell permeable tiple comparison test (*P 0.05, **P 0.01, ***P 0.001). All experi- supports as described by Larre et al. (13). ments were repeated at least three times, and the data are presented as mean ± SE. Cell Transfection to Obtain Permanently Red-Stained MDCK Cell. MDCK-II (3 × 105 cells/mL, that affords roughly 80–90% confluence) were seeded in 2 mL ACKNOWLEDGMENTS. We thank Dr. A. Arias for his expert input; E. del Oso, of growing medium in 35-mm diameter dish 1 d before transfection. Cells Y. de Lorenz, J. Soriano, E. Estrada, and E. Méndez for technical and admin- istrative aid; S. González (Electron Microscopy Unit, Centro de Investigación were prewashed with Opti-MEM I Reduced Serum Medium (GIBCO; 31985– fi y de Estudios Avanzados del Instituto Politécnico Nacional) for her compe- 062). Lipofectamine 2000 (Invitrogen) was complexed with unmodi ed tent assistance in studies involving scanning electron microscopy; and pDsRed2-N1 expression vectors (BD Biosciences Clontech). Plasmid at reagent: R. Bonilla for plasmid manipulations. This work was supported by the Con- DNA ratio of 1.56 μL:3 μg. Complexes were prepared by mixing lipofect- sejo Nacional de Ciencia y Technología and the Instituto de Ciencia y Tecno- amine 2000, 6 μL with 250 μL of Opti- MEM I, followed by the addition of logía del DF (México City Research Council).

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