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First published online 31 July 2007
doi: 10.1242/jcs.002568

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Shroom2, a myosin-VIIa- and actin-binding protein, directly interacts with ZO-1 at tight junctions

Raphaël Etournay^1,*, Ingrid Zwaenepoel^1,*,, Isabelle Perfettini¹, Pierre Legrain², Christine Petit¹ and Aziz El-Amraoui^1,

¹ INSERM UMRS 587, Unité de Génétique des Déficits Sensoriels, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France
² Département de Biologie Joliot-Curie, CEA, 91191 Gif-sur-Yvette, France

View larger version (95K): [in this window] [in a new window]   Fig. 1. Myosin VIIa binds to shroom2. (A,B) Domain structures of (A) myosin VIIa and (B) shroom2 (mShrm2). The domain composition and position of the yeast two-hybrid bait (myosin-VIIa–SH3-MyTH4-FERM) and of the prey clone (mShrm2MBR) are indicated. (B) Shroom2 consists of an N-terminal PDZ domain, an SPR, an MBR, and the ASD1 and ASD2 domains. (C) In transfected HeLa cells expressing GFP-tagged shroom2 MBR (GFP-mShrm2MBR; prey) alone, the protein exhibits a punctate cytoplasmic staining and is also particularly abundant in cytoplasmic extensions (arrowheads). (D) In co-transfected HeLa cells expressing both the prey GFP-mShrm2MBR (left panel) and Myosin VIIa tail (right panel), the two proteins colocalize in punctate structures (arrows) in the cytoplasm, arrowhead indicates cytoplasmic extension. (E) In transfected HeLa cells expressing GFP-tagged mShrm2MBRC (GFP-mShrm2MBRC, aa 350-563) alone, the fusion protein is diffusely distributed throughout the cytoplasm. (F) In co-transfected HeLa cells expressing both GFP-mShrm2MBRC (left panel) and Myosin VIIa tail (right panel), the two proteins colocalize in cytoplasmic punctate structures (arrows). Bars, 10 µm. (G) 35S-labeled myosin VIIa fragments (1-4 in upper panel) were incubated with either GST-mShrm2MBR or GST alone (see blots in bottom panels). The 35S-labeled myosin VIIa tail binds to GST-mShrm2MBR but not to GST (two lanes on left side (1) of right blot). Fragments 2 and 4 (Myosin VIIa SH3/MyTH4/FERM and Myosin VIIa MyTH4/FERM, respectively) bind to GST-mShrm2MBR (lanes 2 and 4 in right blot), whereas the SH3-MyTH4 fragment does not (lane 3 in right blot). (H) Co-immunoprecipitation assay. HEK293 cells were co-transfected with plasmids encoding either the full-length myosin VIIa and Myc-tagged shroom2 (Myc-mShrm2) or the full-length myosin VIIa and Myc tag alone (Myosin VIIa) (middle panels). The same experiment was carried out using the untagged myosin VIIa tail (Myosin VIIa tail) instead of the entire protein (top panels). Upon immunoprecipitation with the anti-Myc antibody, immunoblots were probed with antibodies against myosin VIIa or shroom2 as indicated. Both the Myosin VIIa and Myosin VIIa tail co-immunoprecipitate with Myc-mShrm2 (bottom panel).

View larger version (94K): [in this window] [in a new window]   Fig. 2. Shroom2 binds to F actin. (A,B) Using (A) the anti-mShrm2R1 antibody, faint shroom2 labeling is observed in some MDCK cells, especially at cell-cell contacts. Shroom2 colocalizes with ZO-1 (yellow in right panel) especially at tricellular contacts. In (B) transfected MDCK cells that express GFP-labeled full-length shroom2 (GFP-mShrm2) a similar distribution pattern is displayed. Both endogenous shroom2 (A) and GFP-mShm2 (B) labeling is more intense at tricellular contacts (arrowheads in A and B). The TJs are visualized by ZO-1 labeling (red in A,B). (C,D) In MDCK cells expressing GFP-mShrm2 that were treated with cytochalasin D, the GFP-mShrm2 (green) colocalizes with F-actin (red), along the cell membrane. (E,F) By contrast, in cells expressing GFP-mShrm2MBR (green) that were treated with cytochalasin D, the large F-actin bundles induced by overexpression of GFP-mShrm2MBR (red) are protected against depolymerization (arrowheads). (G) In MDCK cells that express GFP alone, a diffuse GFP staining is observed in cytochalasin-D-treated cells. (H) F-actin co-sedimentation assay. Lanes 1 and 2 contain the soluble and pellet fractions of GST-mShrm2MBR, respectively, obtained upon high-speed centrifugation. GST-mShrm2MBR is not recovered in the pellet fraction (lane 2). When the same amount of GST-mShrm2MBR is incubated with F-actin at 37°C for 30 minutes (lanes 3 and 4), almost all GST-mShrm2MBR is recovered with F-actin in the pellet fraction after ultracentrifugation (lane 4). GST alone was used as a negative control (lanes 5 and 6). Bars, 20 µm.

View larger version (101K): [in this window] [in a new window]   Fig. 3. Shroom2 in embryonic and adult mouse epithelia. (A,B) In embryonic mouse epithelia, at E15, the shroom2 staining (green) significantly overlaps with that of ZO-1 (red) in (A) the olfactory epithelium, and (B) the choroid plexus. (C-F) At adult stages (P20 and P90), in (C,D) the gastric glands and (E,F) small intestine, shroom2 colocalizes with ZO-1 at the TJs (yellow in C and E), but not with (D) -catenin or (F) E-cadherin. Bars, 10 µm.

View larger version (112K): [in this window] [in a new window]   Fig. 4. Shroom2, myosin VIIa and ZO-1 in the mouse retina. (A-F) Sections of the mouse retina at (A,B) E14 and P90 (C-F). Shroom2 is detected in the retinal pigment epithelium (RPE) cells, and some neuronal extensions in the inner region of the neuroretina. (C-F) Shroom2 is concentrated at the TJs, where it is co-distributed with ZO-1 (red). The neuronal labeling of shroom2 persists in adults, in the inner plexiform layer (IPL). (E,F) Adjacent tangential sections of the P90 mouse retina labeled for ZO-1 and either shroom2 or myosin VIIa. Note that shroom2 is co-distributed essentially with ZO-1 at the TJ zone, whereas myosin VIIa is present throughout the cytoplasm, and in the apical microvilli. Colocalization with ZO-1 is illustrated in the confocal orthogonal (YZ) reconstructions. ONL, outer nuclear layer; INL, inner nuclear layer, GCL, ganglion cell layer. Bars, 10 µm.

View larger version (101K): [in this window] [in a new window]   Fig. 5. Spatiotemporal shroom2 distribution in the mouse auditory epithelia. (A-D) At E12, shroom2 colocalizes with ZO-1 at the apical surface of all the epithelial cells delineating the cochlear duct (cd) (A). As development proceeds, the shroom2 labeling becomes more intense in the sensory epithelium (organ of Corti) (B). At birth, a transient diffuse labeling is observed throughout the cytoplasm of the sensory inner (IHC), and outer (OHC) hair cells (* over cell bodies), and pillar (p) cells (C). This cell body labeling decreases at later stages, and is not detected from P10 onwards (D). The bulk of shroom2 labeling is associated with the tightly joined apical domains of the hair cells and their adjacent supporting cells, forming the reticular lamina (D), and also at the apical cell-cell junctions of the marginal cells in the stria vascularis (arrowheads in E). The stria vascularis is a bilayer epithelium of the cochlear duct lateral wall, which secretes K+ in the endolymph (the fluid filing the cochlear duct) and produces the endocochlear potential. (F) Whole-mount preparation of a mouse organ of Corti at P2, illustrating the codistribution of shroom2 and myosin VIIa. (G) The absence of myosin VIIa in shaker-1 mutant mice does not alter the normal targeting of shroom2 in the hair cells. Stronger shroom2 labeling is observed in the reticular lamina, whereas weaker labeling is seen in other supporting cells (arrows). Bars, 10 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 6. Shroom2 targeting requires the presence of TJs. (A) In non-confluent MDCK cells, GFP-mShrm2 displays a punctate labeling, that perfectly colocalizes with F-actin (red) in the cytoplasm or beneath the plasma membrane. (B) In nascent cell-cell contacts, as shown in the peripheral cells of MDCK islands, GFP-mShrm2 is recruited to the plasma membrane at the AJC. GFP-mShrm2 is present in some (arrowheads) but not all junctional regions visualized by ZO-1 labeling (arrows). (C) Upon cell confluency, GFP-mShrm2 spreads to all junctional sites, as shown in Caco-2 cells. In these cells, GFP-tagged shroom2 colocalizes with occludin and ZO-1 but not with -catenin; see confocal orthogonal (YZ) reconstructions at the bottom.

View larger version (86K): [in this window] [in a new window]   Fig. 7. Shroom2 recruitment at the forming TJs after calcium switch in MDCK cells. The lines were used to calculate the fluorescence intensity in line scan. In micrographs, dots with numbers indicate fluorescence intensity at cell edges. Blue asterisks indicate nontransfected cells that do not express GFP-mShrm2. In the fluorescence intensity profiles, vertical blue lines delineate the emplacement of these cells that were used as an internal control for fluorescence quantification. After 20 hours of incubation in low-Ca2+ medium (t=0), cells were fixed and analyzed 1 hour (t=1 hour), 2 hours (t=2 hours) and 7 hours (t=7 hours) after Ca2+ repletion. At t=0, the fluorescence intensity profiles of transfected cells indicate that both ZO-1 (red) and GFP-mShrm2 (green) are distributed throughout the cytoplasm. At t=1 hour, ZO-1 is recruited at cell-cell junctions in a discontinuous pattern, whereas GFP-mShrm2 is not yet present at the junctions. At t=2 hours, GFP-mShrm2 is mainly detected at cell-cell junctions and the intensity of the labeling at the junctions correlates with that of ZO-1. At t=7 hours, ZO-1 and GFP-mShrm2 display continuous fluorescence patterns along the junctions, and both protein labelings are more intense at tricellular junctions. Bars, 20 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 8. Shroom2 in mouse L2071 fibroblasts stably transfected with human E-cadherin (LE cells). (A) In non-confluent LE cells that overexpress GFP-mShrm2, the protein is scattered within the cytoplasm or linked to the plasma membrane, associated with F-actin. (B-D) In confluent LE cells, GFP-mShrm2 is not recruited to the cadherin-mediated cell-cell contacts, visualized by (B) -catenin and (C,D) F-actin. Rather, the GFP-mShrm2 staining is more diffuse, and occasionally forms clusters in the (C, XZ plane) basal, or (B, XZ and YZ planes; C, YZ plane) lateral regions of LE cells. Such clusters were not observed in transfected MDCK or Caco-2 cells (see Fig. 6). XZ and YZ sections show coronal () and orthogonal () confocal reconstructions, respectively. Bars, 10 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 9. Shroom2 binds to ZO-1 in vitro and in vivo. (A) Domain structure of the shroom2 yeast two-hybrid bait (mShrm2PSP) and of the two ZO-1 preys isolated. (B) Several 35S-labeled shroom2 fragments (labeled 1 to 6) were incubated with the GST-tagged ZO-1 prey (GST-ZO1 prey) or GST alone. mShrm2, mShrm2PDZ, mShrm2PSP and mShrm2SPR bind to the GST-ZO1 prey, but not to GST. By contrast, mShrm2ASD2 and mShrm2PDZ do not bind to the GST-ZO1 prey. (C) Characterization of the shroom2-interaction domain of ZO-1. 35S-labeled shroom2 and 35S-labeled shroom2-SPR only bind to the original GST-ZO1 prey and to the GST-ZO1 SH3/GuK fragment. (D) Protein extracts from the mouse auditory organ (left panels) or brain (right panels) were used for the immunoprecipitation assays. The endogenous shroom2 protein is immunoprecipitated in the presence of the anti-ZO-1 antibody, but not with protein G alone. Under the conditions we used, ZO-1 immunoprecipitates do not contain myosin VIIa (bottom panels).