Abstract
In ciliated mammalian cells, the precise migration of the primary cilium at the apical surface of the cells, also referred to as translational polarity, defines planar cell polarity (PCP) in very early stages. Recent research has revealed a co-dependence between planar polarization of some cell types and cilium positioning at the surface of cells. This important role of the primary cilium in mammalian cells is in contrast with its absence from Drosophila melanogaster PCP establishment. Here, we show that deletion of GTP-binding protein alpha-i subunit 3 (Gαi3) and mammalian Partner of inscuteable (mPins) disrupts the migration of the kinocilium at the surface of cochlear hair cells and affects hair bundle orientation and shape. Inhibition of G-protein function in vitro leads to kinocilium migration defects, PCP phenotype and abnormal hair bundle morphology. We show that Gαi3/mPins are expressed in an apical and distal asymmetrical domain, which is opposite and complementary to an aPKC/Par-3/Par-6b expression domain, and non-overlapping with the core PCP protein Vangl2. Thus G-protein-dependent signalling controls the migration of the cilium cell autonomously, whereas core PCP signalling controls long-range tissue PCP.
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References
Simons, M. & Mlodzik, M. Planar cell polarity signaling: from fly development to human disease. Annu. Rev. Genet. 42, 517–540 (2008).
Vladar, E. K., Antic, D. & Axelrod, J. D. Planar cell polarity signaling: the developing cell’s compass. Cold Spring Harb. Perspect Biol. 1, a002964 (2009).
Jones, C. et al. Ciliary proteins link basal body polarization to planar cell polarity regulation. Nat. Genet. 40, 69–77 (2008).
Mirzadeh, Z., Han, Y. G., Soriano-Navarro, M., Garcia-Verdugo, J. M. & Alvarez-Buylla, A. Cilia organize ependymal planar polarity. J. Neurosci. 30, 2600–2610 (2010).
Song, H. et al. Planar cell polarity breaks bilateral symmetry by controlling ciliary positioning. Nature 466, 378–382 (2010).
Wallingford, J. B. Planar cell polarity signaling, cilia and polarized ciliary beating. Curr. Opin. Cell Biol. 22, 597–604 (2010).
Montcouquiol, M. et al. Identification of Vangl2 and Scrb1 as planar polarity genes in mammals. Nature 423, 173–177 (2003).
Morin, X. & Bellaiche, Y. Mitotic spindle orientation in asymmetric and symmetric cell divisions during animal development. Dev. Cell 21, 102–119 (2011).
Ezan, J. & Montcouquiol, M. Revisiting planar cell polarity in the inner ear. Semin. Cell Dev. Biol. 5, 499–506 (2013).
Goldstein, B. & Macara, I. G. The PAR proteins: fundamental players in animal cell polarization. Dev. Cell 13, 609–622 (2007).
Gonczy, P. Mechanisms of asymmetric cell division: flies and worms pave the way. Nat. Rev. Mol. Cell Biol. 9, 355–366 (2008).
Gohla, A. et al. An obligatory requirement for the heterotrimeric G protein Gi3 in the antiautophagic action of insulin in the liver. Proc. Natl Acad. Sci. USA 104, 3003–3008 (2007).
Ross, A. J. et al. Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates. Nat. Genet. 37, 1135–1140 (2005).
Sipe, C. W. & Lu, X. Kif3a regulates planar polarization of auditory hair cellsthrough both ciliary and non-ciliary mechanisms. Development 138, 3441–3449 (2011).
Mogensen, M. M., Mackie, J. B., Doxsey, S. J., Stearns, T. & Tucker, J. B. Centrosomal deployment of gamma-tubulin and pericentrin: evidence for a microtubule-nucleating domain and a minus-end docking domain in certain mouse epithelial cells. Cell. Motil. Cytoskeleton 36, 276–290 (1997).
Du, Q. & Macara, I. G. Mammalian Pins is a conformational switch that links NuMA to heterotrimeric G proteins. Cell 119, 503–516 (2004).
Nelson, W. J. Remodeling epithelial cell organization: transitions between front-rear and apical-basal polarity. Cold Spring Harb. Perspect Biol. 1, a000513 (2009).
Warchol, M. E. & Montcouquiol, M. Maintained expression of the planar cell polarity molecule Vangl2 and reformation of hair cell orientation in the regenerating inner ear. J. Assoc. Res. Otolaryngol. 11, 395–406 (2010).
Giese, A. P. et al. Gipc1 has a dual role in Vangl2 trafficking and hair bundle integrity in the inner ear. Development 139, 3775–3785 (2012).
Krumins, A. M. & Gilman, A. G. Targeted knockdown of G protein subunits selectively prevents receptor-mediated modulation of effectors and reveals complex changes in non-targeted signaling proteins. J. Biol. Chem. 281, 10250–10262 (2006).
Chacon-Heszele, M. F. & Chen, P. Mouse models for dissecting vertebrate planar cell polarity signaling in the inner ear. Brain Res. 1277, 130–140 (2009).
Kim, J. C. et al. MKKS/BBS6, a divergent chaperonin-like protein linked to the obesity disorder Bardet-Biedl syndrome, is a novel centrosomal component required for cytokinesis. J. Cell Sci. 118, 1007–1020 (2005).
Hallworth, R., McCoy, M. & Polan-Curtain, J. Tubulin expression in the developing and adult gerbil organ of Corti. Hear Res. 139, 31–41 (2000).
May-Simera, H. L. et al. Patterns of expression of Bardet-Biedl syndrome proteins in the mammalian cochlea suggest noncentrosomal functions. J. Comp. Neurol. 514, 174–188 (2009).
Montcouquiol, M. et al. Asymmetric localization of Vangl2 and Fz3 indicate novel mechanisms for planar cell polarity in mammals. J. Neurosci. 26, 5265–5275 (2006).
Deans, M. R. et al. Asymmetric distribution of prickle-like 2 reveals an early underlying polarization of vestibular sensory epithelia in the inner ear. J. Neurosci. 27, 3139–3147 (2007).
Labbe, J. C., Maddox, P. S., Salmon, E. D. & Goldstein, B. PAR proteins regulate microtubule dynamics at the cell cortex in C. elegans. Curr. Biol. 13, 707–714 (2003).
Galli, M. & van den Heuvel, S. Determination of the cleavage plane in early C. elegans embryos. Annu. Rev. Genet. 42, 389–411 (2008).
Dave, R. H., Saengsawang, W., Yu, J. Z., Donati, R. & Rasenick, M. M. Heterotrimeric G-proteins interact directly with cytoskeletal components to modify microtubule-dependent cellular processes. Neurosignals 17, 100–108 (2009).
Laan, L. et al. Cortical dynein controls microtubule dynamics to generate pulling forces that position microtubule asters. Cell 148, 502–514 (2012).
Seo, S. et al. BBS6, BBS10, and BBS12 form a complex with CCT/TRiC family chaperonins and mediate BBSome assembly. Proc. Natl Acad. Sci. USA 107, 1488–1493 (2010).
Sun, X. et al. Tubby is required for trafficking G protein-coupled receptors to neuronal cilia. Cilia 1, 21 (2012).
Jiang, M. et al. Mouse gene knockout and knockin strategies in application to alpha subunits of Gi/Go family of G proteins. Methods Enzymol. 344, 277–298 (2002).
Wiege, K. et al. Galphai2 is the essential Galphai protein in immune complex-induced lung disease. J. Immunol. 190, 324–333 (2013).
Lu, X. et al. PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates. Nature 430, 93–98 (2004).
Montcouquiol, M., Jones, J. M. & Sans, N. Detection of planar polarity proteins in mammalian cochlea. Methods Mol. Biol. 468, 207–219 (2008).
Exner, T., Jensen, O. N., Mann, M., Kleuss, C. & Nurnberg, B. Posttranslational modification of Galphao1 generates Galphao3, an abundant G protein in brain. Proc. Natl Acad. Sci. USA 96, 1327–1332 (1999).
Belotti, E. et al. Molecular characterisation of endogenous vangl2/vangl1 heteromeric protein complexes. PLoS ONE 7, e46213 (2012).
Sans, N. et al. mPins modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression. Nat. Cell Biol. 7, 1179–1190 (2005).
Acknowledgements
We thank the animal and genotyping facilities’ members of the Neurocentre for technical assistance, notably H. Doat and D. Gonzales. We also thank the entire team of the Bordeaux Imaging Center (BIC) for the constant technical assistance, notably P. Legros, S. Marais and C. Poujol. We thank P. Beales (UCL, UK) for the Mkks mutants. We thank L. Mays (Tubingen, Germany) for critical reading of the manuscript, and F. Schweisguth (Paris, France) and J. Raff (Oxford, UK) for thoughtful discussions. We apologize to all whose relevant work could not be cited.
This research was supported by an INSERM grant to M.M. and N.S., the Conseil Regional d’Aquitaine Neurocampus program, La Fondation pour la Recherche Medicale (M.M., N.S., J.E., A-C.L.), ANR-08-MNPS-040-01 (M.M.), the European Commission Coordination Action ENINET (LSHM-CT-2005-19063; N.S. and M.M.), Ligue Nationale Contre le Cancer (Label 2010, J-P.B.), EUCAAD (FP7 program, J-P.B.), Fondation ARC pour la Recherche sur le Cancer (B.N., E.B.), the Deutsche Forschungsgemeinschaft (DFG; B.N., S.B-H.), the Intramural Research Program of the NIH (Project Z01-ES-101643 to L.B.), the European FP7 program (HEALTH-F2-2008-200234, A.L.B.) and ANR (BLAN07-2-186738, A.L.B.).
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M.M., J.E. and N.S. designed and carried out experiments, analysed data and wrote the paper. A.G. and L.L. carried out immunocytochemistry on cochleae, cultures and western blots. L.B., B.N., S.B-H., A.N. and A.L.B. generated and provided the G-protein mutant and G-protein antibodies, and carried out the characterization of the Go-protein antibody. E.B., A-C.L. and J-P.B. generated the rat anti-Vangl2 monoclonal antibody and PTK7-deficient mice. J-P.B., S.B-H., B.N. and A.L.B. provided experimental and conceptual advice and edited the manuscript. All authors discussed the results and implications and commented on the manuscript.
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Supplementary Figure 1 Loss of protein expression in Gαi3 knockout.
Surface views of cochleae from Gnαi3 wild-type (Gαi3 WT) (a–a”) and Gnαi3 knockout mice (Gαi3 KO) (b–b”). The strong acetylated tubulin between IHCs and OHC1 rows (b) is due to labeling of the inner pillar cell heads. Labeling of (green) is absent from the cochlear epithelium of the knockouts. IHC (#) and OHC (*,**,***). Scale bar: 10 μm (a–b).
Supplementary Figure 2 Acetylated tubulin and pericentrin labeling correlates after G-protein signaling disruption.
(a-b’) Representative surface views of HCs from mPins Ctrl (a) and mPins KO mice (b) labeled with pericentrin (green), acetylated tubulin (red) and phalloidin (blue), and the corresponding diagram on the right. Kinocilium and pericentrin coordinates were determined according to a grid applied to each HC as depicted on the HC schemes. The Y axis on the histograms correspond to the pericentrin position (x or y coordinates), the X axis correspond to the kinocilium position (x and y coordinates). Scale bar: 2 μm (a-b). (c,d’) Plots illustrating the correlation between the x and y coordinates from the base of the kinocilium and the pericentrin labeling in (c, c’) Gαi3 WT (n = 102 OHCs, n = 42 IHCs) and (d, d’) Gαi3 KO (n = 108 OHCs, n = 35 IHCs) mice. (e,f’) Plots illustrating the correlation between the x and y coordinates in (e, e’) mPins Ctrl (n = 77 OHCs, n = 34 IHCs) and (f, f’) mPins KO (n = 77 OHCs, n = 33 IHCs) mice. (g,h’) Plots illustrating the correlation between the x and y coordinates in (g,g’) PTX-untreated (n = 101 OHCs, n = 30 IHCs) and (h, h’) PTX-treated (n = 110 OHCs, n = 47 IHCs) cochlear explants.
Supplementary Figure 3 Generation of mPins conditional mutant and localisation of apical asymmetrical complexes in the organ of Corti.
(a) Schematic representation of the mouse mPins gene targeting strategy. Exons 5, 6, and 7 of the gene were excised following Cre and Flpe-mediated recombination. Regions of homologous recombination are indicated by light gray shading. AseI (A) and SacI (S) and the locations of the probes (5’, 3’) used for Southern blotting analysis are indicated. (b) Southern blot of recombinant ES cells showing homologous integration. (c) PCR genotyping to detect wild-type (170 bp) and targeted (250 bp) alleles. Cre-mediated excision was confirmed in cochlea by the presence of a 300 bp product. (d) Western blotting analysis of the mPins protein (75 kDa) in control and mPins cKO inner ear. In the cKO, the protein levels are strongly reduced. (e-f”) Surface views of cochleae from mPins controls (Ctrl, e-e”) and mPinsconditional knockouts (mPins cKO, f-f”) from P0/P1 mice processed for immunocytochemistry. Labeling of mPins (green) is absent from the cochlear epithelium of the conditional knockouts. (g,g’) Surface view (g) and Z-stack (g’) confocal cross-section showing the apical localization of Par-6b (green) compared to β-catenin (red). (g’) Lateral view of (g) at the level indicated by the dashed line. (h, h’) Gαi3 (green) is located on the distal–abneural side of the HCs, opposite Vangl2 (red). (h’) High magnification of the inset in h. (i, i’) Gαo (green) labeling reveals weak cytosolic staining in HCs and in the non-sensory region, but no asymmetrical accumulation. Hair bundles and cell junctions were labeled with phalloidin (blue). (j) Representative immunoblot of brain (expressing Gαo) and liver (not expressing Gαo) membranes using Gαo antibody. Purified Gαo1 protein served as a positive control. IHC (#) and OHC (*,**,***). Scale bar: 10 μm (e-i).
Supplementary Figure 4 Gαi inhibition leads to loss-of-cilium-like morphological defects in the hair bundle.
(a) In cultures treated with 5 pg/ml PTX for 48 hours (T3) the distributions of the orientations follow a gradient of severity from OHC1 to OHC3. HCs numbers, out of 2 cochleae per condition, are as follows: PTX T3 (n = 65,68,64 respectively for OHC1, OHC2, OHC3) (b, b’) Vangl2 asymmetry (green) is maintained in mPins cKO. (c-d’) In cultures treated with 5 pg/ml PTX for 24 hours (T1), we observe hair bundles exhibiting round (c, c’) or flat shapes (d, d’). (e-f”’) The aPKC expression domain is more extended at the membrane of HCs in cultures treated with high doses (1 ng/ml) of PTX (f–f”’) compared to untreated (Unt) cultures (e–e”’). Note the long cilium in PTX-treated cultures. (g-i) High magnifications of a representative HC from (e) and (f). (h, j) Circular histograms of the distribution of aPKC staining in Unt and PTX-treated condition. Each histogram represent data from 30 HCs. Statistical analysis via a two-tailed t test (P*<0.0001). Error bars show s.e.m. Scale bars: 10 μm (in b, e-f), 2 μm (in b’-d’, g’-i”’).
Supplementary Figure 5 Gαi3 is recruited asymmetrically prior to kinocilium migration.
(a–c”’) Luminal surface views of a cochlear epithelium at E15.5 at different locations along the duct. In the apical region of the sensory epithelium, only a few scattered HCs (future IHCs) have started to undergo differentiation, as indicated by MyoVI expression (a’, arrowheads) and an apical actin ring (a’’, arrowheads). (b-b4) In a more basal region of the cochlea, and in the distal-abneural OHC region, Gαi3 starts to accumulate centrally in a few differentiating cells (b’, white arrow). In the absence of asymmetrical Gαi3 localization, the kinocilium does not migrate in these cells (b, b1, b2, white arrowhead). By comparison, a line of differentiated IHCs is present, expressing Gαi3 asymmetrically and showing a distal kinocilium (b’, b3-b4, yellow arrow and arrowhead). The white lines indicate the relative midline of every HC. (c–c”) Luminal surface views of the basal region of cochlear epithelia at E15.5. Gαi3 is asymmetrically localized at the distal-abneural side of IHCs but absent from the prospective OHC region. Insets: higher magnification view of IHCs (yellow arrow) and future OHCs (white arrow) from (c) revealing that the kinocilium is always localized distally in IHCs (yellow arrowhead) but not in future OHCs (white arrowhead). (d-e’) Luminal surface views of the mid-basal to apical region of cochlear epithelia at E16.5 labeled with aPKC (d-d’) and Par-6b (e,e’). The proteins are not asymmetrically distributed. (f-g) HC differentiation is completed by P0, with every HC expressing MyoVI (f, green), and Gαi3 is asymmetrically localized to the distal-abneural side along with the kinocilium (g and g1-g4, green). Scale bars: 10 μm (in a-f). (h-i’) The asymmetric distribution of mPins (h-h’, green) and Par-6B (i, i’) are inverted across the striola in the P0 utricular epithelium (dashed line). β2-spectrin immunostaining labels the cuticular plate (red). Arrows indicate HC polarity with respect to the lateral (Lat) or medial (Med) side of the utricle. (j) Scatter plots of the localization of the kinocilium relative to the center of the Gαi3 expression domain reveals a strong association between Gαi3 and the kinocilium position in wild type (n = 218 OHCs and 68 IHCs), V angl2Lp/Lp (n = 237 OHCs and 73 IHCs) and PTK7 mutants (n = 124 OHCs and 33 IHCs). The dashed line indicates a perfect correlation (r = 1). (k, k”) Ectopic HCs induced by Atoh1-IRES-GFP expression (green) in the non-sensory epithelium display asymmetric cortical aPKC (red) localization opposite to the kinocilium side. The dashed line indicates the boundary between the sensory and non-sensory epithelium. (l) Schematic representation of (k).
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Ezan, J., Lasvaux, L., Gezer, A. et al. Primary cilium migration depends on G-protein signalling control of subapical cytoskeleton. Nat Cell Biol 15, 1107–1115 (2013). https://doi.org/10.1038/ncb2819
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DOI: https://doi.org/10.1038/ncb2819
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