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Rho1 interacts with p120^ctn and -catenin, and regulates cadherin-based adherens junction components in Drosophila

¹ Division of Basic Sciences and Program in Developmental Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
² Department of Zoology, University of Washington, Seattle, WA 98195, USA

View larger version (93K): [in a new window]   Fig. 1. P1D9 monoclonal antibody is specific for Rho1 protein in vitro and in vivo. (A) Western analysis of Drosophila Rho family GST fusion proteins hybridized with -GST (top) and P1D9 monoclonal (bottom) antiserum. (B) Western analysis of whole cell (wc) and nuclear (n) lysates prepared from 0- to 2-hour and 0- to 12-hour embryos, respectively. P1D9 recognizes a single major band in whole cell but not nuclear lysates. This band is not detected when primary antibody is omitted. (C,D) P1D9 staining of a stage 14 embryo homozygous for a deficiency that includes the Rho1 locus (C) relative to a sibling control (D). The relative intensity of the staining in these embryos can be directly compared, as both embryos were photographed in the same visual field. (E-J) P1D9 recognizes ectopically expressed Rho1 (E), but not Rac1 (G) or Cdc42 (I). Each protein was overexpressed in the Engrailed domain, as highlighted by green fluorescent protein expression (F,H,J), and embryos were examined at stage 14. For all embryos, anterior is left and dorsal is up. Scale bars: 50 µm.

View larger version (90K): [in a new window]   Fig. 2. Rho1 protein is ubiquitously expressed, but concentrated apically, in blastoderm embryos. (A,B) Confocal micrograph showing Rho1 localization at interphase (A) and metaphase (B) in an early syncytial blastoderm stage. Note that the accumulation of Rho protein (arrows) relative to nuclei (arrowheads) changes with cell cycle phase. (C-D') Confocal micrograph showing P1D9 (C,C') and DE-cadherin (D,D') staining of embryos at the cellular blastoderm stage. Note apical accumulation of Rho1 protein at this stage (arrow in C'). (E-F) Rho protein is localized cytoplasmically. (E) Double staining with P1D9 (green) and propidium iodide (red) to label nuclei. (F) Double staining with P1D9 (green) and an antibody against Lava lamp (red), a protein present in the cytoplasm. (G) P1D9 staining of Rho1E3.10 homozygous embryos at the cellular blastoderm stage. Note the lack of apical Rho1 accumulation (arrow, compare with C'). (H) Close-up of an embryo at the cellular blastoderm stage showing basal accumulation of Rho1 protein in cells underlying the pole cells (arrow). (I-J') P1D9 staining in stage 11 (I,I') and stage 14 (J,J') embryos. (I',J') Higher magnification views of the portion of the embryos boxed in I and J, respectively. Note subcellular punctate spots of Rho1 accumulation (arrows in I'), and that Rho1 protein does not accumulate in cells at the leading edge during dorsal closure (J'). (K) Neural commissures of a stage 14 embryo stained with P1D9. (L) Grazing section of a cellular blastoderm stage embryo showing accumulation of Rho1 protein at a puncture wound site. In all images except (L), anterior is left and dorsal is up. Scale bars: A,C',I': 10 µm; C, I: 50 µm; H,J',K,L: 25 µm.

View larger version (123K): [in a new window]   Fig. 3. Rho1 accumulates at sites of DE-cadherin localization in the embryo and ovary. (A-C) Confocal image of a hindgut tube from a stage 14 embryo showing double labeling for both DE-cadherin (A) and Rho1 (B). A higher magnification view of the merged image is shown in C. In all merged images, Rho1 staining is red and the staining for other molecules is green. Apical is up in all images. Note the apical accumulation of Rho1 protein that coincides with DE-cadherin expression (arrow in C). (D-F) A stage 14 hindgut tube double labeled for the septate junction protein neurexin (D), Rho1 (E), and the merged image (F). Note that the neurexin staining (green) is largely exclusive of Rho1 apical accumulation (red). (G-I) Control staining documenting DE-cadherin (green) localization relative to neurexin (red). (J) A schematic diagram of the gut showing the relative locations of the adherens junctions (AJ) and septate junctions (SJ). (K-M',T-U) Expression of Rho1 protein during oogenesis. Rho1 protein is expressed in all stages, including the germarium (K). Note Rho1 accumulation in the apical regions of follicle cells (L',M'; arrows in L,M). Rho1 expression is upregulated in border cells (T') and accumulates at lateral follicle cell contacts (cross section, T''; grazing section, U) and in the cortex of the oocyte (arrows in T,T''). (N-P',V-W) Localization of DE-cadherin during oogenesis. Cadherin is upregulated in the border cells (P', arrowhead in V;V') and also localized to lateral follicle cell contacts (arrow in V; V'', W). (Q-S',X-Y) Merged images of Rho1 and DE-cadherin staining (red: Rho1, green: DE-cadherin). Stages of oogenesis shown are the germarium (K,N,Q), stage 3 and 5 (L,O,R), stage 8 (M,P,S) and stage 10a (T,V,X). Scale bars: A,D,G: 0.6 µm; C,F,I: 0.2 µm; Q,R,S,X: 30 µm; X',X'',Y: 10 µm.

View larger version (138K): [in a new window]   Fig. 4. Embryos mutant for Rho1 show aberrant DE-cadherin and catenin localization. Confocal micrographs showing junction protein expression in wild-type and Rho1 mutant embryos. (A-B,E-F) DE-cadherin expression in stage 14 (A,B) and stage 15 (E,F) wild-type embryos. (C-D,G-H) DE-cadherin expression in stage 14 (C,D) and stage 15 (G,H) Rho1 mutant embryos. Note the disruption of DE-cadherin localization near the leading edge (arrow in H), but not in more lateral regions (arrowhead in H). (I-L) No difference in neurexin expression (septate junctions) is observed in the stage 15 Rho1 mutant embryo (K,L) compared to wild type (I,J). DE-cadherin and neurexin were simultaneously imaged in the same wild-type and Rho1 mutant embryos. Brackets in D indicate the leading edge, and in H,L,P,T, the dorsal midline. (M-P) ß-catenin expression is disrupted in stage 15 Rho1 mutants (O,P) compared to wild-type embryos (M,N). (Q-T) -catenin expression is disrupted in stage 15 Rho1 mutants (S,T) compared to wild-type embryos (Q,R). In all images, anterior is left. Dorsal is up in A-D. E-T are dorsal views. Boxes in A,C,E,G,I,K,M,O,Q,S indicate the region of the embryo shown in B,D,F,H,J,L,N,P,R,T, respectively. Scale bars: A: 50 µm, B: 10 µm.

View larger version (36K): [in a new window]   Fig. 5. Rho1 interacts directly with p120ctn and -catenin. (A) GST pulldown experiments assessing binding among Rho1, the DE-cadherin intracellular domain, p120ctn, ß-catenin and -catenin. 35S-labeled in vitro translated (IVT) -catenin (third panel from top) binds GST-Rho1 independently of the phosphorylation state of its associated nucleotide, IVT-p120ctn binds preferentially to GST-Rho1GDP (top panel) and IVT-ß-catenin does not interact with either form of GST-Rho1 (second panel from top). Rok and a putative RhoGDI are included as binding controls. 5% input is shown. (B) GST pulldown experiment utilizing purified bacterially-expressed His-p120ctn. His-p120ctn also binds preferentially to GST-Rho1GDP. (C) Immunoprecipitations showing in vivo interaction between Rho1 and -catenin, and Rho1 and p120ctn. ß-catenin immunoprecipitations were performed as a positive control. 5% input is shown. (D) Diagram of the protein fragments, substitutions and point mutants used to map interaction domains on Rho1. (E) Computer model of GDP-bound RhoA crystal structure. Residues required for -catenin binding are shownin yellow and those required for p120ctn binding are shown in red. For reference the effector domain is highlighted in green. (F) GST pulldown experiments demonstrating the regions of Rho1 required for binding of -catenin (top panel) and p120ctn (second panel from top). -catenin binds to region A and its binding is disrupted by replacing aa 27-29 (KDQ) with alanines, whereas p120ctn binds to region C and its binding is disrupted by replacing aa 51-54 (KQVE) with alanines. Rok and RhoGDI bound preferentially to constitutively active (V14A) and dominant negative (N17A) forms of Rho1, respectively, but equally well to all other forms of Rho1 tested. V14A protein was exchanged with GTP and N17A with GDP in all experiments. All other forms of Rho1 in the -catenin, p120ctn and RhoGDI binding experiments were exchanged with GDP, while the Rho1 proteins used to test Rok binding were exchanged with GTP.

View larger version (169K): [in a new window]   Fig. 6. Rho1 localization is aberrant in catenin mutants. (A-B') Rho1 expression in stage 15 embryos homozygous for a deficiency that removes the p120ctn locus. (C-F') Rho1 (C-F) and ß-galactosidase (E',F') expression in stage 15 puc-lacZ/TM3 embryos injected with p120ctn dsRNA. Note the accumulation of Rho1 protein at the leading edge in both deficiency and RNAi embryos (arrows in A',B',E,F), not observed in either ftz RNAi embryos (G-H') or other dorsal closure mutants basket (I,K) and hemipterous (J,L) (arrows in K, L; compare with A',B'). (M-O') Rho1 (M,N,O) and -spectrin (N',O') expression in stage 6 embryos injected with -catenin dsRNA. (P,Q) Rho1 (P) and DE-cadherin (Q) expression in embryos injected with -catenin dsRNA. Note apical localization of DE-cadherin protein in some cells (arrow in Q). In all images, anterior is left. Scale bars: A,C,G,I,M: 50 µm; A',E,H,K,N,P: 25 µm.

View larger version (52K): [in a new window]   Fig. 7. Overexpression of p120ctn or -catenin enhances the Rho1 phenotype. The graph shows percentage total embryos (y-axis) with segmental patterning in each phenotypic class (w,wild type; m, mild; s, severe) for the following genotypes: Rho1 homozygous mutants (¬Rho), Rho1 mutants with one copy of UAS-p120ctn overexpressed with the actin-Gal4 driver (¬Rho + p120ctn), Rho1 mutants with one copy of UAS--catenin overexpressed with the actin Gal4 driver (¬Rho + -ctn), and Rho1 mutants with one copy of UAS-actin-GFP overexpressed with the actin Gal4 driver (¬Rho + actinGFP). The number of embryos scored is indicated beneath each genotype. (B-D) Cuticles depicting representative examples of each phenotypic class: (B) homozygous Rho1 mutant phenotype exhibiting an anterior dorsal hole but relatively normal anterior-posterior (AP) segmentation, (C) mild disruption of AP segmentation resulting from overexpressed -catenin in the Rho1 mutant background, (D) severe disruption of AP segmentation resulting from overexpressed -catenin in the Rho1 mutant background. (E) Model depicting the relationship of Rho1 to components of adherens junctions. p120ctn can cycle between the cytoplasm and AJs. In the cytoplasm p120ctn inhibits Rho by preventing the exchange of GDP for GTP. At AJs it binds to the JMD of cadherin and can no longer inhibit Rho. p120ctn and/or -catenin may be involved in recruiting Rho1 to AJs, allowing it to be activated by GEFs and carry out its downstream functions. Rho1 could be anchored at the AJ through its interaction with -catenin or by insertion into the PM mediated by its isoprenylation modification. (PM: plasma membrane, CBD: catenin binding domain, JMD: juxtamembrane domain). For cuticles, anterior is left. Scale bar: 50 µm.

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