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First published online 13 March 2008
doi: 10.1242/dev.019646

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Shroom3-mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling

RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.

View larger version (50K): [in this window] [in a new window]   Fig. 1. Shroom3 binds ROCKs via its ASD2 domain. (A) Schematic representation of Shroom3 and its deletion mutants. (B) MDCK-Tet-Off cell lines inducibly expressing FLAG-tagged, full-length Shroom3 (Shrm-Full) or Shroom3-ASD2 (Shrm-ASD2) were double-immunostained for FLAG and ZO-1. Expression of these molecules is suppressed in the presence of doxycycline [Dox(+)], but induced by its removal (Dox[-]) from the culture medium. The apical cell junctions, delineated by ZO-1 signals, are tightened by expression of Shrm-Full but not of Shrm-ASD2. Scale bars: 10 µm. (C) In vitro interaction of ASD2 with endogenous Rock1 or Rock2. GST-tagged Shrm-ASD2 or GST was incubated with a MDCK cell lysate, and then pulled down with Glutathione-Sepharose 4B beads, followed by immunoblotting with antibodies against Rock1, Rock2, -tubulin and GST. (D) In situ interaction of ASD2 with Rock1 or Rock2. COS7 cells were co-transfected with FLAG-tagged Shrm-ASD2 and HA-tagged Rock1 or Rock2. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with anti-HA or anti-FLAG antibody.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Shroom3 recruits endogenous ROCK to the apical cell junctions in an ASD2-dependent way. (A) Localization of endogenous Rock1 in MDCK-Tet-Off cells expressing FLAG-tagged Shrm-Full or Shrm-ASD2. Endogenous Rock1 is visible along the apical cell junctions only in the presence of Shrm-Full [Dox(-)]. Scale bars: 10 µm. (B) Interaction of endogenous Rock1 or Rock2 with Shroom3 in cells. Lysates of the above cells were subjected to immunoprecipitation with anti-FLAG antibody, and then immunoblotted with antibodies against Rock1, Rock2, Lamin B1 and FLAG.

View larger version (53K): [in this window] [in a new window]   Fig. 3. A central fragment of ROCK interacts with ASD2, and can antagonize the endogenous ROCK-Shroom3 interaction. (A) (Top) Schematic representation of Rock2 and its deletion mutants. (Bottom) Interaction of ASD2 with Rock2 deletion mutants. COS7 cells were co-transfected with FLAG-tagged ASD2 and HA-tagged Rock2 (RII-Full) or its deletion mutants. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with anti-HA or anti-FLAG antibody. Only the constructs containing the region from amino acids 698 to 957 can bind ASD2. See Fig. S2 in the supplementary material for the corresponding experiments for Rock1. (B) Expression of the central region of Rock2 (RII-C1) interferes with the action of Shroom3. EGFP-tagged RII-C1 or RII-C2 was expressed in MDCK-Tet-Off Shroom3 transfectants. The apical junctions were visualized with anti-ZO1 antibody (red). RII-C1 (green) is localized at the junctions only when Shroom3 expression has been induced by Dox(-), as indicated by arrows. Bar graphs show quantitative measurement of the length of apical junctions and apical surface areas in the same cultures. A total of 100-200 of GFP-positive cells were counted. Histograms are the average of three independent experiments, and bars represent the standard deviation. *P<0.01 against the EGFP-transfected cells. P-values were analyzed by Student's t-test throughout the manuscript. (C) RII-C1 interferes with the ROCK-Shroom3 interaction. (Left) MDCK-Tet-Off FLAG-Shrm-Full transfectants were further transfected with either EGFP-tagged RII-C1 or EGFP. From their lysates, Shroom3 was immunoprecipitated with anti-FLAG antibody, which was followed by detection of endogenous Rock1 or EGFP. Black triangle, EGFP-RII-C1; white triangle, EGFP. (Right) Localization of endogenous Rock1 or pMLC in Shroom3-expressing cells, in which restricted cells had been transfected with EGFP-RII-C1. Junctional localization of those molecules is inhibited in the RII-C1-positive cells. Scale bars: 10 µm.

View larger version (86K): [in this window] [in a new window]   Fig. 4. Depletion of Shroom3 abolishes the apical localization of Rock1 in the neural tube. (A) Co-localization of Shroom3 and Rock1 at the apical junctions of chicken neural tubes. A transverse section of stage 9 chicken embryo at the hindbrain level was double-immunostained for these proteins. (B) Test for RNAi-mediated depletion of chicken Shroom3. MDCK cells were co-transfected with RNAi vectors for the chicken Shroom3 (Shrm RNAi), scrambled sequences (Control RNAi) or empty vector (Vec), and with either an EGFP-tagged middle region of chicken Shroom3 (EGFP-ch-Shrm/M) or EGFP alone. The cell lysates were immunoblotted with anti-GFP or anti--tubulin antibodies. There is effective and specific depletion of Shroom3 by the RNAi vector. (C) Effects of Shroom3 knockdown on the localization of Rock1 in the neural tube. Chicken embryos were co-electroporated with EGFP and RNAi vectors, as shown in Fig. S3 in the supplementary material, and fixed at stage 9. A section of an embryo at the forebrain-midbrain level was double-immunostained for EGFP and Shroom3, and adjacent sections were immunostained for Rock1, pMLC and ZO-1 in each experiment. Rock1 and pMLC levels are reduced at the apical junctions in the Shroom3-depleted tube (Shrm RNAi), the closure of which is also perturbed. Scale bars: 20 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Expression of RII-C1 in the neural tube antagonizes the apical localization of Rock1 as well as tube closure. Chicken embryos were electroporated with the expression vectors for EGFP, EGFP-tagged RII-C1 or EGFP-tagged RII-C2, and fixed at stage 9. A section of an embryo at the forebrain-midbrain level was double-immunostained for EGFP and Rock1, and adjacent sections were immunostained for pMLC and ZO-1 in each experiment. The neural tube fails to close at the side where the transfection of RII-C1 was more efficient. In this region, the apical levels of Rock1 and pMLC are downregulated. Scale bars: 20 µm.

View larger version (71K): [in this window] [in a new window]   Fig. 6. Cell arrangement and pMLC distribution at the apical surface of the neural tube. (A) Preparation of flat-mount samples to observe the inner (apical) surface of the neural tube in stage 8-9 chicken embryos. The midbrain areas, marked with dotted squares, were selected for the observations. DM, dorsal midline; VM, ventral midline. In all the images shown below, the dorsal side is at the top and the anterior side is towards the left. (B) Double-immunostaining for ZO-1 and pMLC at the apical surface of the neural tube in stage 8 embryos. Representative cell clusters with rosette-like arrangement, where pMLC is concentrated at the central regions, are encircled; and those with arch-type arrangement are marked by an oval. Arrows indicate examples of linearly arranged pMLC signals. A-P, anteroposterior axis; D-V, dorsoventral axis. Scale bar: 10 µm. (C) The angular distribution of linear pMLC signals on the apical surface of neural tubes. Linear pMLC signals of more than 10 µm (68-89 signals/field) from four different fields from four embryos at stage 8-9 were analyzed for length and orientation. Horizontal and vertical axes represent the anterior-posterior and dorsal-ventral orientations, respectively. Percentages of the signals oriented at the angles between 0° and 30°, 30° and 60°, and 60° and 90° to the anterior-posterior axis were calculated. pMLC signals are more abundant along the dorsoventral axis. (D) Double immunostaining for Shroom3 and ZO-1 or pMLC at the apical surface of the neural tube in stage 9 embryos. ZO-1 colocalizes with Shroom3, whereas pMLC colocalizes only with restricted Shroom3 signals. (E) Effects of Y27632 on the distribution of pMLC and cell morphology. Stage 8(-) embryos (with two or three somites) were incubated in the presence of Y27632 (100 µM) or 1% DMSO (control) in the culture medium for 6 hours prior to fixation, and their neural tubes were double-immunostained for pMLC and ZO-1. Examples of rosette-like and arched cell clusters are marked with a circle and oval, respectively. When rosette is defined as a cluster comprising six cells or more, in which the cells radially converged on a constricted point, cell clusters with such configuration are undetectable in the Y27632-treated sample shown. The bar graphs show the length of apical junctions, apical surface areas and percent of cells with pMLC signals. All the cells in randomly selected fields (105-250 cells/field) were measured. Histograms are the average of three different fields from three embryos, and bars represent the standard deviation. *P<0.05; **P<0.01 against 1% DMSO-treated embryos. Scale bars: 10 µm.

View larger version (99K): [in this window] [in a new window]   Fig. 7. Expression of RII-C1 disturbs cell arrangement at the apical surface of the neural tube. Confocal views of triple-immunostaining for EGFP, pMLC and ZO-1 at the apical surface of the neural tubes electroporated with EGFP, EGFP-tagged RII-C1 or RII-C2 and incubated as in the experiments for Fig. 5. Cells displaying the rosette configuration, which were defined as in Fig. 6E, are encircled. No such rosettes are detectable in the RII-C1-positive layer. Arrows indicate residual pMLC signals on which some cells converge, but not assuming the complete rosette configuration as defined above. The bar graph shows results of quantification of pMLC-positive cells. All EGFP-positive cells in randomly selected fields (30-102, 72-132 and 95-178 cells/field for RII-C1, RII-C2 and EGFP, respectively) were counted. Histogram is the average of three different fields from three chicken embryos, and bars represent the standard deviation. **P<0.01 against EGFP-transfected embryos. Scale bars: 10 µm.

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