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Files in this Data Supplement:
Fig. S1. (A) The MBR region of shroom2 binds to the C-terminal MyTH4-FERM fragment of myosin VIIa. The left panel shows the biotin-tagged fusion proteins used in the in vitro binding experiment, i.e. the myosin VIIa C-terminal MyTH4/FERM fragment (1), the myosin VIIa-FERM domain (2) and thechloramphenicol acetyl transferase (CAT) (3), which used as a negative control. GST-mShrm2-MBR binds to the biotin-tagged myosin VIIa MyTH4/FERM fragment, but not to the biotin-tagged FERM domain, or CAT (right panel). (B) Western blot characterization of anti-shroom2 antibodies. The position of the two shroom2 peptides (mShrm2P1 and mShrm2P2) used in a mix to produce antibodies in two different rabbits (R1, R2), are indicated under the graphic of mShroom2. Both antibodies (anti-mShrm2R1 and anti-mShrm2R2) recognize the overexpressed Myc-mShrm2. The lower bands, absent in the blot probed with the anti-Myc antibody, are likely to be degradation products that lack the N-terminal Myc tag. Anti-mShrm2R1 was used for all further experiments. In bacterial cell lysates that contained either the GST-mShrm2MBR or a GST-tagged unrelated protein (GST-YAP65), this antibody only recognizes the mShroom2 fusion protein. (C, D) Immunoprecipitation experiments. Soluble extracts from P3 mouse cochleas were immunoprecipitated using either (C) anti-mShrm2R1 or (D) anti-myosin VIIa polyclonal antibody. Neither anti-mShrm2R1 nor anti-myosin VIIa antibody could immunoprecipitate the corresponding protein. T and S, total and soluble fractions of the cochlear extracts, respectively. (E) Pull-down assay. Extracts from HEK cells expressing untagged mShrm2 were incubated with either the GST-tagged ZO-1 prey or GST alone. Bound proteins were analyzed using the anti-mShrm2R1 antibody. The untagged full-length mShrm2 binds to the GST-tagged ZO-1 prey, but not to GST alone.
Fig. S2. Distribution of different shroom2 fragments in transfected Caco-2 cells. Cells were transfected with five different GFP-tagged shroom2 constructs (shown on top of each panel). GFP- mShroom2ΔPDZ correctly localizes to the AJC, suggesting the PDZ domain is not required for the junctional targeting of mShroom2. The shroom2 N-terminal region, composed of the PDZ domain and the SPR region (PSP), is efficiently targeted to the AJC too (construct GFP-mShrm2PSP). By contrast, the MBR region requires the ASD1 domain to localize to the junctional complex (construct GFP-mShrm2MBR-ASD1), whereas the C-terminal region up to the ASD2 domain displays a diffuse cytoplasmic staining (construct GFP-mShrm2ASD2).
Fig. S3. Shroom2 distribution in different mouse epithelia, brain, and vasculature cells. (A-H) The bulk of shroom2 immunoreactivity (green) is colocalized with that of ZO-1 in the apical region of epithelial cells, as shown in the pancreas and submandibular gland cells. (C, D) Distribution of shroom2, E-cadherin (red in D), and ZO-1 (red in C) in the stomach, kidney, lung, liver, intestine, pancreas, and submandibular gland. E-cadherin is distributed along the adherens junctions and does not significantly overlap with mShroom2. (E) Distribution of ZO-1, myosin VIIa, and shroom2 in the choroid plexus. (F) In the central nervous system, shroom2 is present in the ependymal cell layer (arrowheads in the hindbrain), brain capillaries, and some axons of Purkinje cells in the cerebellum or other neurons (arrowheads in the forebrain). Although neurons do not form tight junctions, in myelinated axons, mShroom2 might be associated with the TJ-like structures that form between the myelin sheath lamellae (Dermietzel and Kroczek, 1980). (G) In the embryonic head, endothelial cells of all vessels are labelled. (H) In some kidney epithelial cells, most of the shroom2 immunoreactivity (green) is present at the luminal apical surface (*) of the cells, even though shroom2 also localizes to the TJs (arrowheads in H). This is consistent with the results obtained by (Dietz et al., 2006). Nuclei are labelled with DAPI (blue).
Fig. S4. Myosin VIIa, shroom2, and ZO-1 in the mouse retina. (A-F) Sections of the mouse retina at E14 (A, B, E, F) and P90 (C, D). Intensive myosin VIIa labelling is detected in retinal pigment epithelium (RPE) cells at (A,B)bE14 and (C) P90. Myosin VIIa is present throughout the cells, including the ZO-1 labelled TJs. (D) Distribution of MyRIP (a ligand of myosin VIIa) in the apical microvilli of RPE cells at P90. At E14 (E), shroom2 is present mainly in RPE cells and in some neuronal processes in the inner region of the neuroretina. The shroom2 labelling is concentrated at the TJs of RPE cells, where it is co-distributed with ZO-1 (red). Nuclei are labelled with DAPI (blue).
Fig. S5. Shroom2, ZO-1, myosin VIIa and F-actin in the mouse inner ear. (A,B) Diagram of a cochlear cross-section illustrating the positions of shroom2 immunoreactive structures(A, middle panel), namely the cochlear ganglion (A, left panel), stria vascularis (A, right panel) and the (B) auditory sensory epithelium. In the P14 stria vascularis (sv), shroom2 is located at the AJC of marginal cells. Shroom2 labelling is also observed along neuronal processes of cochlear ganglion neurons (arrowheads). Nuclei are labelled with DAPI (blue).
(Upper panels, B) whole-mount preparation (left) or cross sections (right) of a P2 organ of Corti, showing shroom2 and myosin VIIa distributions in the inner (IHC) and outer (OHC) hair cells (asterisks over cell bodies). (Lower panels, B) whole-mount preparations of the organ of Corti at different postnatal stages (P2, P14, P90), showing both shroom2 and either ZO-1 or F-actin distributions. At P14 and P90, shroom2 (green) still persists at the apical hair cell surface. Note that there is no shroom2 staining within the F-actin-rich hair bundles (red, arrowheads). (C) Whole-mount preparation (left panel) or cross section (right panel) of an utricular macula (vestibule) at P2, showing shroom2 and ZO-1 distributions in the hair cells. Shroom2 is targeted to the AJC, where it colocalizes with ZO-1.
Fig. S6. Subcellular distribution of shroom2 and the MBR fragment in transfected MDCK cells. (A) In islands of MDCK cells, GFP-mShrm2 is present in some (arrowheads) but not all (arrows) junctional regions, whereas ZO-1 is recruited all along the nascent cell-cell contacts. Open arrowheads indicate the plasma membrane in the non-junctional regions. In confluent MDCK cells producing the GFP-tagged mShroom2, the protein is broadly distributed all along the cell-cell contacts, where it co-distributes with ZO-1. (B) MDCK cells analyzed at different times during the formation of contacts between cells (16, 24 and 48 hours after cell transfection). In subconfluent cells, overexpression of GFP-mShrm2MBR induces the formation of large extended F-actin bundles. In confluent cells, the ability of GFP-mShrm2MBR to bundle F-actin has decreased. The GFP-mShrm2MBR labelling becomes more diffuse, and is occasionally seen at the cell junctions (arrows). Overexpression of GFP-mShrm2MBR does not affect the formation of cell-cell contacts. (C) Ca2+-switch experiment on stablly transfected MDCK cells producing GFP. After the Ca2+ switch (t=0 hours), cells were analyzed at different times (t=1, 2 and 7 hours), which correspond to different stages of TJ reorganization. At all stages analyzed, GFP remains diffuse in the cytoplasm and does not seem to affect the cell-cell junction reassembly. Note that ZO-1 is not especially abundant at tricellular junctions, as illustrated in the fluorescence-intensity profiles. Bars, 20 μm.
Fig. S7. (A) Schematic diagrams showing the domains of molecular interaction between shroom2 and myosin VIIa, ZO-1 or F-actin. (B) Model illustrating the localization of shroom2, its binding partners, and shroom3 at the apical junctional complex. Shroom1, which lacks the PDZ domain, has been involved in a molecular complex with an amiloride-sensitive Na+ channel (ENaC) at the apical region of A6 kidney epithelial cells (Staub et al., 1992; Zuckerman et al., 1999).
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