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. 2011 Sep;301(3):F622-33.
doi: 10.1152/ajprenal.00134.2011. Epub 2011 Jun 15.

Galectin-7 modulates the length of the primary cilia and wound repair in polarized kidney epithelial cells

Christine Rondanino  1 , Paul A Poland, Carol L Kinlough, Hui Li, Youssef Rbaibi, Michael M Myerburg, Mohammad M Al-bataineh, Ossama B Kashlan, Nuria M Pastor-Soler, Kenneth R Hallows, Ora A Weisz, Gerard Apodaca, Rebecca P Hughey
Affiliations

Galectin-7 modulates the length of the primary cilia and wound repair in polarized kidney epithelial cells

Christine Rondanino et al. Am J Physiol Renal Physiol. 2011 Sep.

Abstract

Galectins (Gal) are β-galactoside-binding proteins that function in epithelial development and homeostasis. An overlapping role for Gal-3 and Gal-7 in wound repair was reported in stratified epithelia. Although Gal-7 was thought absent in simple epithelia, it was reported in a proteomic analysis of cilia isolated from cultured human airway, and we recently identified Gal-7 transcripts in Madin-Darby canine kidney (MDCK) cells (Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP. J Biol Chem 286: 6780-6790, 2011). We now report that Gal-7 is localized exclusively on the primary cilium of MDCK, LLC-PK(1) (pig kidney), and mpkCCD(c14) (mouse kidney) cells as well as on cilia in the rat renal proximal tubule. Gal-7 is also present on most cilia of multiciliated cells in human airway epithelia primary cultures. Interestingly, exogenous glutathione S-transferase (GST)-Gal-7 bound the MDCK apical plasma membrane as well as the cilium, while the lectin Ulex europeaus agglutinin, with glycan preferences similar to Gal-7, bound the basolateral plasma membrane as well as the cilium. In pull-down assays, β1-integrin isolated from either the basolateral or apical/cilia membranes of MDCK cells was similarly bound by GST-Gal-7. Selective localization of Gal-7 to cilia despite the presence of binding sites on all cell surfaces suggests that intracellular Gal-7 is specifically delivered to cilia rather than simply binding to surface glycoconjugates after generalized secretion. Moreover, depletion of Gal-7 using tetracycline-induced short-hairpin RNA in mpkCCD(c14) cells significantly reduced cilia length and slowed wound healing in a scratch assay. We conclude that Gal-7 is selectively targeted to cilia and plays a key role in surface stabilization of glycoconjugates responsible for integrating cilia function with epithelial repair.

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Figures

Fig. 1.
Fig. 1.
Galectin-7 (Gal-7) localization in polarized Madin-Darby canine kidney (MDCK) cells. Polarized MDCK cells were fixed, blocked, permeabilized, and incubated with primary antibodies [mouse anti-Gal-7 and rabbit anti-polycystin-1 (PC-1)], then secondary antibodies, before fixation, staining with TOPRO, and mounting for confocal microscopy. A: distribution of Gal-7 (green) and the nucleus (blue) at the apical pole (a–c) and along the lateral surface (d–f) of MDCK cells. Arrows indicate the localization of Gal-7 on the primary cilia. B: distribution of Gal-7 (green), PC-1 (a marker of cilia; red), and the nucleus (blue) at the apical pole (a–d) of MDCK cells. Arrows indicate colocalization between Gal-7 and PC-1 on the primary cilia. C: distribution of Gal-7 (green) and the nucleus (blue) at the apical pole (a–c) of MDCK cells grown overnight with 100 mM lactose. Single representative merged images (Ac, Af, Bd, and Cc,) and XZ sections (Ag, Be, and Cd) are shown. Scale bar = 10 μm.
Fig. 2.
Fig. 2.
Recombinant glutathione S-transferase (GST)-Gal-7 binding to polarized MDCK cells. Polarized MDCK cells were incubated with recombinant GST-Gal-7 or GST on ice for 1 h before (B) or after (A and C) fixation, permeabilization, and incubation with goat anti-GST (green) or mouse anti-acetylated tubulin (red) primary antibodies. After incubation with secondary antibodies, cells were fixed, stained with TOPRO-3, and mounted for confocal microscopy. Endogenous galectins were released by growing cells overnight in 100 mM lactose before incubation with GST-Gal (C, f–j). A: distribution of GST-Gal-7 or GST at the apical pole (a and d) and along the lateral surface (b and e) of MDCK cells. XZ sections (c and f) are shown. Arrows indicate the localization of GST-Gal-7 on the primary cilia. B: distribution of GST-Gal-7 at the apical pole (a) and along the lateral surface (b) of nonpermeabilized MDCK cells. An XZ section (c) is shown. Arrows indicate the localization of GST-Gal-7 on the primary cilia. C: distribution of GST-Gal-7 (green), acetylated tubulin (red), and the nucleus (blue) at the apical pole of MDCK cells (a–e) and MDCK cells preincubated with 100 mM lactose (f–j). Merged images (d and i) and XZ sections (e and j) are shown. Arrows indicate colocalization between GST-Gal-7 and acetylated tubulin on the primary cilia. Scale bar = 10 μm.
Fig. 3.
Fig. 3.
Localization of H antigen-bearing glycoproteins in polarized MDCK cells. Polarized MDCK cells were fixed, permeabilized, and incubated with or without biotinylated Ulex europeaus agglutinin (UEA). Cells were incubated with FITC-conjugated streptavidin (green), fixed, stained with TOPRO-3 (blue), and mounted for confocal microscopy. Representative images are shown for the apical pole of MDCK cells incubated with (A–D) or without (E–H) UEA are shown. Merged images (C and G) and XZ sections (D and H) are presented. Arrows indicate the localization of UEA on the primary cilia. Scale bar = 10 μm.
Fig. 4.
Fig. 4.
β1-Integrin from the apical and basolateral surface of polarized MDCK cells binds Gal-7. Polarized MDCK cells were treated with sulfo-NHS-biotin on the apical (6 filters) or basolateral (2 filters) surface, and biotinylated proteins were recovered with avidin-conjugated beads. Proteins recovered from the apical (Ap) or basolateral (BL) surface were pooled and then split in half for incubation with either GST-Gal-7 (7) or galectin-like HSPC15 (control; C) prebound to glutathione-beads. Samples were analyzed by immunoblotting (IB) with rabbit anti-β1-integrin antibodies. A: aliquots of total detergent extract (5% total) and recovered biotinylated proteins (10% Avidin) were included as controls. B: after overnight incubation, the beads were incubated with sucrose (nonspecific binding), and then lactose (specific binding), and released proteins were recovered for immunoblotting. Numbers on the left indicate the mobility of the Bio-Rad protein standards at 100 and 150 kDa.
Fig. 5.
Fig. 5.
Gal-7 binds to cilia in rat kidney and cultured human airway cells (HAE). A: slices of rat kidney were fixed, embedded, and sectioned before immunostaining with mouse anti-acetylated tubulin (a, green) and mouse anti-Gal-7 (b, red) antibodies. The merged figure is shown in c. Scale bar = 5 μm. Details of the staining protocol for costaining with 2 mouse antibodies are described in materials and methods. Control images for protocol are presented in Supplemental Fig. S3. B: primary cultures of HAE were fixed, permeabilized, and incubated with rabbit anti-PC-1 antibody and mouse anti-Gal-7 antibody. Cells were incubated with secondary antibodies, fixed, stained with TOPRO-3, and mounted for confocal microscopy. 3-Dimensional reconstruction of optical sections taken from the apical to the basolateral surface of the cells shows the distribution of PC-1 (green) and the nucleus (blue; a), the distribution of Gal-7 (red) and the nucleus (blue; b), and the merged image (c). Each segment of the grid is equivalent to 3.76 μm.
Fig. 6.
Fig. 6.
Cilia length decreases upon knockdown of Gal-7. mpkCCDc14 cells stably transduced by lentiviral vectors to express doxycyclin (DOX)-inducible TurboRFP and either control shRNA (A and C) or short-hairpin (sh) RNA targeting Gal-7 (B and D) were plated on filter supports for 4 days, with (gray line in A and B, dashed line in C and D) or without (black line in A and B, solid line in C and D) DOX treatment. Cells were fixed and processed for indirect immunofluorescence using mouse anti-acetylated tubulin antibodies, and cilia lengths of at least 100 cells/condition were quantitated in RFP-positive cells as described in materials and methods. The cilia lengths were sorted in ascending order for each condition and plotted as either length vs. percentile (A and B, where each symbol represents a single cilium) or as a histogram with number of cilia (0.25-μm bin size) vs. cilia length (C and D). In aggregate data from 2 independent experiments, induction of Gal-7 shRNA resulted in a statistically significant decrease in cilia length compared with identical cells plated in the absence of DOX (mean cilia length was 1.30 and 1.77 μm, with and without DOX, respectively, P < 1 × 10−9 by Mann-Whitney U-test). Cilia length in cells transduced with control shRNA was slightly different in the presence or absence of DOX (mean cilia length was 1.42 and 1.61 μm, with and without DOX, respectively, P < 0.03 using the same analysis).
Fig. 7.
Fig. 7.
Wound healing is reduced in cells upon knockdown of Gal-7. mpkCCDc14 cells stably transduced by lentiviral vectors to express DOX-inducible TurboRFP and either control shRNA or shRNA targeting Gal-7 were plated on 35-mm dishes for 4 days, with or without DOX treatment, as indicated. Cells were scraped with a sterile pipette tip, and 20 random images of the wound were captured at 0 and 4 h (time for ∼50% recovery). Three random measurements of the wound width in each image were carried out using Adobe Photoshop CS2 software. Data from each experiment were normalized to control (−Dox) set as 1, and values of means and SE from 3 experiments are presented here (*P < 0.01 by Student's t-test; A). Representative pictures of the cultures are presented in B.
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