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. 2010 Jan 15;21(2):219-31.
doi: 10.1091/mbc.e09-03-0193. Epub 2009 Nov 18.

Galectin-3, a novel centrosome-associated protein, required for epithelial morphogenesis

Annett Koch  1 , Francoise Poirier, Ralf Jacob, Delphine Delacour
Affiliations

Galectin-3, a novel centrosome-associated protein, required for epithelial morphogenesis

Annett Koch et al. Mol Biol Cell. .

Abstract

Galectin-3 is a beta-galactoside-binding protein widely expressed in all epithelia where it is involved in tissue homeostasis and cancer progression. We recently reported unique abnormalities in the identity of membrane domains in galectin-3 null mutant mice, suggesting that galectin-3 may participate in epithelial polarity program. We investigated the potential role of galectin-3 on early events in polarization of epithelial renal cells, using three-dimensional cultures of MDCK cells and also galectin-3 null mutant mouse kidneys. We show that depletion in galectin-3 systematically leads to severe perturbations of microtubular network associated with defects in membrane compartimentation, both in vitro and in vivo. Moreover, the absence of galectin-3 impinges on the morphology of the primary cilium, which is three times longer and unusually shaped. By immunological and biochemical approaches, we could demonstrate that endogenous galectin-3 is normally associated with basal bodies and centrosomes, where it closely interacts with core proteins, such as centrin-2. However, this association transiently occurs during the process of epithelial polarization. Interestingly, galectin-3-depleted cells contain numerous centrosome-like structures, demonstrating an unexpected function of this protein in the formation and/or stability of the centrosomes. Collectively, these data establish galectin-3 as a key determinant in epithelial morphogenesis via its effect on centrosome biology.

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Figures

Figure 1.
Figure 1.
Depletion of galectin-3 leads to abnormal cystogenesis in MDCK cells. MDCK cells were cultured in Matrigel 3D matrix for 5 d. (A) distribution of gp114 and gp135 in control (luciferase siRNA or Mock) or in cells transfected with galectin-3 siRNA (gal3 siRNA). (B) quantification. Controls, i.e., Mock (n = 206) and inefficient gal3 siRNA (n = 114), or galectin-3 siRNA (n = 137) cysts have been analyzed in three independent experiments. Bars, mean ± SD. Mann-Whitney tests have been performed, and statistical differences (p) are presented. Distribution of E-cadherin, ZO-1, aPKC (C), ezrin, and β-tubulin (D), in control (Mock) or in cells transfected with galectin-3 siRNA (gal3 siRNA). Nuclei were detected by Hoechst 33342 staining. Scale bars, 10 μm.
Figure 2.
Figure 2.
Defects in renal epithelial cell organization in galectin-3 null mutant mice. Confocal microscopy analysis of mouse kidney sections. Distribution of Na+/K+-ATPase (A), gp114 (B), aquaporin-2 (AQ-2; C), villin (D), and α-tubulin (E), in wild-type (wt) or galectin-3 null mutant (gal3−/−) mouse kidneys. Nuclei were detected by Hoechst 33342 staining. Renal tubules were delimited with a white line along basal membranes. The borders of a single cell are indicated with a dotted line in E. Scale bars, 10 μm.
Figure 3.
Figure 3.
Galectin-3 is required for ciliogenesis in renal epithelial cells. Confocal analysis of ciliogenesis in wt or gal3−/− mouse kidneys (A). Renal tubules were delimited with a white line along basal membranes. Confocal analyses of MDCK cysts after 5 d in Matrigel (B) or MDCK cells seeded in monolayer for 4 d (C). MDCK cells were transfected with control siRNAs (Mock and Ineff.gal3 siRNA) or galectin-3 siRNA (gal3 siRNA). Primary cilia were stained with antibody directed against acetylated-α-tubulin. Nuclei were detected by Hoechst 33342 staining. Scale bars, (A) 20 μm; (B) 10 μm; (C) lower magnifications, 10 μm. Higher magnifications (panels on the right side), 3 μm. (D) Primary cilium length was measured in 20 fields (20× objective) of postconfluent cells (day 4) from three independent experiments. Bars, mean ± SD. Statistical differences are presented (Mann-Whitney tests).
Figure 4.
Figure 4.
Galectin-3 is present at the centrosome of postconfluent MDCK cells. Double-immunostaining experiments with antibody directed against galectin-3 and against acetylated α-tubulin, centrin-2 (A), or γ-tubulin and pericentrin (B) MDCK cells after 3 d in monolayer cultures. Scale bars, 10 μm. (C) centrosomes were purified from day 3 MDCK cell cultures by density gradient centrifugation. Centrosomal fractions were identified based on the distribution of γ-tubulin and centrin-2. To test the purity of the preparations, the distribution of noncentrosomal proteins, such as flotillin-1, was also analyzed. PNS, postnuclear supernatant.
Figure 5.
Figure 5.
Galectin-3 transiently associates with the centrosome in MDCK cells. (A) Postnuclear supernatants (PNS) of MDCK cells, maintained in culture for different time points, were subjected to immunoprecipitation with centrin-2 antibody. The precipitated material was analyzed by immunoblot with anti-galectin-3 antibody. Crude lysates were loaded as controls for the expression level of galectin-3. Densitometric quantification of three representative immunoblot scans of galectin-3 copurified with centrin-2 are presented. (B) After centrosomal fractionation at different time points (days 1, 3, and 6), each gradient fraction was subjected to immunoprecipitation against centrin-2. Precipitates were analyzed by immunoblot with antibodies against centrin-2 and galectin-3.
Figure 6.
Figure 6.
Depletion in galectin-3 leads to centrosomal abnormalities in vitro and in vivo. (A) Confocal analysis of the distribution of centrin-2 and pericentrin on day 3 MDCK cells transfected with control siRNAs (Mock and Ineff.gal3 siRNA) or galectin-3 siRNA (gal3 siRNA). Nuclei were detected by Hoechst 33342 staining. Arrowheads and arrows, centrin-2– or pericentrin-positive dots or intracellular filaments, respectively. Scale bars, 6 μm. Results were normalized. Centrin-2–positive foci were counted (B), and their surface was measured (C), in 20 40× fields of three independent experiments. White, black, and gray bars, Mock, gal3 siRNA and Ineff.gal3 siRNA cells, respectively. Bars, mean ± SD. Statistical differences are presented (Mann-Whitney tests). (D) confocal analysis of centrin-2 distribution in mouse collecting ducts from wt (a) or mutant tissue (b and c). Nuclei were detected by Hoechst 33342 staining. Tubules were delimited with a white line along basal membranes. Arrowheads and arrows, centrin-2– or pericentrin-positive dots or intracellular filaments, respectively. Bars, 10 μm.
Figure 7.
Figure 7.
Loss of galectin-3 leads to the formation of aberrant centriolar-like structures in mouse gal3−/− kidneys. Ultrastructural analysis of centrioles in wt (A) or galectin-3 null mutant (gal3−/−) (B) mouse collecting ducts. Bars, 1 μm. The insets show high magnifications of the regions in black boxes in A and B; bars, 0.5 μm. (C) Immunostaining for centrin-2 on wt or gal3−/− ultrathin sections. Antibody binding was visualized by incubation with a polyclonal anti-centrin-2 and 10-nm immunogold conjugated goat anti-rabbit IgG antibody; bars, 0.5 μm.
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