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. 2012 Oct;139(20):3775-85.
doi: 10.1242/dev.074229.

Gipc1 Has a Dual Role in Vangl2 Trafficking and Hair Bundle Integrity in the Inner Ear

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Free PMC article

Gipc1 Has a Dual Role in Vangl2 Trafficking and Hair Bundle Integrity in the Inner Ear

Arnaud P Giese et al. Development. .
Free PMC article

Abstract

Vangl2 is one of the central proteins controlling the establishment of planar cell polarity in multiple tissues of different species. Previous studies suggest that the localization of the Vangl2 protein to specific intracellular microdomains is crucial for its function. However, the molecular mechanisms that control Vangl2 trafficking within a cell are largely unknown. Here, we identify Gipc1 (GAIP C-terminus interacting protein 1) as a new interactor for Vangl2, and we show that a myosin VI-Gipc1 protein complex can regulate Vangl2 traffic in heterologous cells. Furthermore, we show that in the cochlea of MyoVI mutant mice, Vangl2 presence at the membrane is increased, and that a disruption of Gipc1 function in hair cells leads to maturation defects, including defects in hair bundle orientation and integrity. Finally, stimulated emission depletion microscopy and overexpression of GFP-Vangl2 show an enrichment of Vangl2 on the supporting cell side, adjacent to the proximal membrane of hair cells. Altogether, these results indicate a broad role for Gipc1 in the development of both stereociliary bundles and cell polarization, and suggest that the strong asymmetry of Vangl2 observed in early postnatal cochlear epithelium is mostly a 'tissue' polarity readout.

Figures

Fig. 1.
Fig. 1.
Gipc1 is a binding partner for Vangl2. (A) Vangl2 is a four transmembrane-domain (TMD) protein, with intracellular N-terminus (N-ter) and C-terminus (C-ter) ending with a PDZ binding motif (-ETSV, orange). The bait used to screen the embryonic cochlear library is indicated (aa 438-521). (B) Gipc1 is a cytosolic protein of 333 aa with a central PDZ domain, an N-ter proline-rich domain (PRD) and a C-ter acyl carrier protein domain (ACP). (C,D) Yeast-two-hybrid assay validating the interaction between Gipc1 and Vangl2. (C) The bait of Vangl2 positively interacts with two of the clones (GIPC1C1 and GIPC1C15) from the yeast-two-hybrid screen. Deletion of the PDZ-BM (last four aa, Δ4) leads to a strong reduction of the interaction, which is completely inhibited by deletion of the last 12 aa of the C-ter (Δ12). The interaction with Scrib1 was used as a positive control. (D) Only the PDZ domain of Gipc1 interacts with Vangl2. (E,F) Co-immunoprecipitation (co-IP) of GFP-Vangl2 with myc-Gipc1. (E) The interaction is strongly impaired by deletion of the PDZ-BM and abolished by removal of the last 12 aa of Vangl2. (F) The interaction is disrupted by mutation of the PDZ domain of Gipc1.
Fig. 2.
Fig. 2.
Vangl2 and Gipc1 colocalize in endosomal vesicles that are redistributed peripherally by MyoVI. (A-F) Immunofluorescence microscopy of COS-7 cells transiently transfected with DsRed-Vangl2, myc-Gipc1 or a combination of the constructs, and labeled with phalloidin (blue), with a line scan corresponding to the dashed line for each condition. The arrows on the line scans represent cell boundaries labeled by phalloidin (A-D). Single expression of DsRed-Vangl2 (red) or myc-Gipc1 (green) led to vesicular, membrane and perinuclear staining. (E-F) DsRed-Vangl2 and myc-Gipc1 colocalize in cytoplasmic vesicles, clusters and at the plasma membrane. (G) Schematic of the localization of transferrin and Eea1 in early endosomes containing Vangl2. (H) Sixty percent of GFP-Vangl2 vesicles colocalized with Eea1-labeled vesicles. This colocalization is increased in the presence of myc-Gipc1, and severely impaired by removal of the PDZ-BM of Vangl2 (Vangl2Δ4). (I) Similar profiles are observed when we measured colocalization of Vangl2-positive vesicles with fluorescent transferrin after a 3-minute treatment. (J-K) DsRed-Vangl2, GFP-MyoVI and myc-Gipc1 co-expression leads to an almost complete colocalization of the three proteins, with a re-localization of these clusters close to, but not at, the plasma membrane (magenta). Note on the line scan (K) that the peaks with high Vangl2 intensity (asterisks) do not correspond to the plasma membrane peaks (arrows). (L-M) DsRed-Vangl2 and GFP-MyoVI are expressed in the cytoplasm, in vesicle-like cytoplasmic punctae, weakly at the membrane and in the perinuclear region of the cell. (N-O) A mutation in the PDZ domain of Gipc1 (myc-Gipc1PDZdead) prevents the formation of the clusters and their re-localization. Note the presence of Vangl2 at the membrane (inset). (P-Q) A short treatment (3 minutes) with Alexa Fluor568-conjugated transferrin (red) reveals colocalization with the clusters. Insets show magnifications of boxed area within the same panel. *P≤0.05, **P≤0.01, ***P≤0.001. Error bars represent the standard error of the mean of triplicates. Scale bar: 15 μm.
Fig. 3.
Fig. 3.
Gipc1 and MyoVI form a protein complex that participates in Vangl2 removal from the plasma membrane. (A,B) Co-IP of Gipc1 and MyoVI with Vangl2. HEK293T lysates were subjected to IP with an anti-Vangl2 antibody and probed with anti-myc (for Gipc1) and anti-GFP (for MyoVI), or with an anti-GFP antibody and probed with anti-Vangl2 and anti-myc (for Gipc1). The three proteins are present in a complex that is disrupted when the PDZ domain of Gipc1 is mutated (Gipc1PDZdead). (C) Endogenous Gipc1, Vangl2 and MyoVI co-immunoprecipitate in lysates of mouse olfactory bulbs, but not with Gad65/67. Asterisks denote IgG heavy chains. (D) Overexpression of Gipc1-YFP (green) in two Ohc1 cells leads to the formation of clusters containing endogenous Vangl2 (red). The image was taken just below the apical surface of the inner hair cell (Ihc) and outer hair cell (Ohc1); phalloidin labeling of the F-actin is in blue. (E) Vangl2 interacts with its PDZ-BM with the central PDZ domain of Gipc1, which can interact with MyoVI. The vesicular complex can be translocated along F-actin filaments by the motor function of MyoVI. (F) Schematic of the fractionation protocol. Fractions are as follows: H, total homogenate; S1, cell soma; P1, dense nuclei-associated membrane; S2, supernatant; P2, membrane fraction; S3, cytosolic; P3, microsomes. (G) Cellular fractionation assay. Antibodies against pan-cadherin and Gapdh show the segregation of the cytosolic (Cyto) and membrane (Mb) fractions from homogenate (H), and the antibody against myc reveals the presence of Gipc1 in the cytosolic fraction (with a weaker expression of the mutated form). Vangl2 is present in the membrane fraction, with higher levels when the PDZ of Gipc1 is mutated (Gipc1PDZdead).
Fig. 4.
Fig. 4.
A MyoVI mutation in sv/sv mice leads to increased levels of Vangl2 at the plasma membrane. (A) Schematic of the various cell types composing the organ of Corti as indicated in the text. (B-E′) Surface view of a P0 rat cochlea in the basal (B-C′) and the apical (D-E′) regions, showing the localization of Gipc1 (green) and β-catenin (red). Gipc1 is expressed at the pericuticular necklace of Ihc and Ohc, with a preferential accumulation on the proximal side of the cells (Fig. 4B, arrows). (C,C′) xz view of the stack at the level indicated by the dotted line in B′. Gipc1 is apical (star) and more basolateral (bracket). (D-E′) In a more apical and less differentiated region of the same cochlea, Gipc1 localization extends more basolaterally (E,E′, bracket). Brackets in B,B′,D,D′ indicate the row of Ipcs. (F-G″) Surface views of sv/+ (F-F″) and sv/sv (G-G″) P1 mouse cochleae labeled with phalloidin (blue), β-catenin (red) and Vangl2 (green). (H) Quantification of the mean pixel intensity of Vangl2 at the junction between a HC and a SC (corresponding zones illustrated by boxes in G″) shows an increase in intensity at an Ohc2-Opc junction (mean=47.2 for sv/+, n=55; and mean=74.3, n=49 for sv/sv; P<0.001) and at an Ohc3-DC1 junction (mean=34.9 for sv/+, n=54; and mean=61.7, n=44 for sv/sv; P<0.001). Scale bar: 8 μm.
Fig. 5.
Fig. 5.
In utero downregulation of Gipc1 leads to PCP phenotype and HC maturation defects, including hair bundle integrity. (A,A′) Surface view of a mouse cochlea electroporated at E11.5 with sh-GFP (green) and labeled seven days later with phalloidin (red). HC development was not affected. (B-E′) The expression of shGIPC1b-GFP (green) leads to a PCP phenotype in HCs (B″). We observed a reduction of the apical surface area of the HC, and reduced pericuticular MyoVI expression (B′,C′,D′, green stars), and an impairment in hair bundle orientation and integrity (C′,D′). When downregulation of Gipc1 did not completely disrupt the HC-SC junction, Vangl2 expression is present (C″, green arrow), but when a strong HC phenotype is observed, there is a disruption of the HC-SC junction (E,E′). (F) Pie charts illustrating the variation in distribution of the HC phenotypes. Scale bar: 8 μm.
Fig. 6.
Fig. 6.
Upregulation of Gipc1 in vitro leads to a hair bundle phenotype. (A-D) The expression of the protein leads to a disruption of the orientation of the hair bundles in 23.8% of electroporated HCs (A-A″,D), a reduced apical surface area (B′, star; D) or a disrupted hair bundle phenotype (C″,D) in 14.2% of electroporated HCs. Scale bar: 8 μm.
Fig. 7.
Fig. 7.
Vangl2 is enriched on the SC side at P0. (A ,B) Vangl2 (green) is asymmetrically accumulated at the junction between SC and HC (phalloidin, red). Arrows point to zones where SC membranes that are not in contact with a HC with strong Vangl2 enrichment. B shows a magnification of the boxed region in A. (C,D) STED microscopy (single plane view, XY) reveals numerous foci of Vangl2 expression and shows a strong enrichment of Vangl2 on the SC side. Asterisks indicate Vangl2 enrichment in SCs. D is the same image as in C after 3D deconvolution. (E-H′) Surface view projection (Proj XY), single plane view (single XY) and z-stack series (XZ) of the organ of Corti from rat cochlear cultures electroporated with GFP and GFP-Vangl2 constructs (left panels), and labeled with anti-GFP antibody (green) and phalloidin (red). (E-F) The empty vector expressing GFP alone fills the cytoplasm of transfected HCs and SCs (E′, asterisk). (G-H′) Full-length GFP-Vangl2 accumulates distally at the membrane of a SC. (H,H′) z-stack series along the dotted line in G′ and G″, respectively, showing the basolateral plasma membrane localization of GFP-Vangl2. (I) The corresponding line scan for GFP-Vangl2 expression along the dotted line in H′. Scale bars: in A,E-H′, 4 μm; in B, 0.5 μm; in C,D, 1 μm.

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