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First published online 30 May 2007
doi: 10.1242/dev.005074

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The RhoGAP RGA-2 and LET-502/ROCK achieve a balance of actomyosin-dependent forces in C. elegans epidermis to control morphogenesis

Marie Diogon^1,*, Frédéric Wissler^1,, Sophie Quintin^1,, Yasuko Nagamatsu¹, Satis Sookhareea¹, Frédéric Landmann¹, Harald Hutter^2,, Nicolas Vitale³ and Michel Labouesse^1,

¹ IGBMC, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP.10142, 67400 Illkirch, France.
² MPI for Medical Research, Jahnstr. 29, 69120 Heidelberg, Germany.
³ Institut des Neurosciences Cellulaires et Intégratives, UMR 7168 CNRS, 67084 Strasbourg, France.

View larger version (121K): [in this window] [in a new window]   Fig. 1. Loss of the RhoGAP RGA-2 or MEL-11/MYPT induce similar defects in C. elegans. (A-F) DIC micrographs of (A) wild-type 3-fold, (B) rga-2(RNAi) and (C) 2-fold and (D) 3-fold rga-2(hd102) embryos, (E) mel-11(it26) embryo and (F) let-502(sb118ts) hatchling at the non-permissive temperature. Arrows, ventral bulge. (G) Physical map of Y53C10A.4/rga-2 with position of the hd102 deletion indicated. Predicted domains of the putative 908-residue RGA-2 protein along with its closest mammalian homologue, ARHGAP20. PH, Pleckstrin Homology; RhoGAP, GTPase-activator protein for Rho-family GTPases; RA, Ras-association (RalGDS/AF-6); ANXL, annexin-like. The percentages of identity and similarity are indicated (for alignments, see Fig. S1 in the supplementary material). (H) Elongation rates for the mutants presented in I-L based on at least four embryos per genotype. (I-L) Still images from DIC videos for wild-type (I), rga-2(hd102) (J), mel-11(it26) (K) and let-502(sb118ts) (L) embryos maintained at 25.5°C. Timing (in minutes) is shown above each column. Arrows point to areas where rga-2 and mel-11 embryos appear to rupture; double-headed arrows, failure to constrict the head. Most rga-2(hd102) embryos (18/21) ruptured before the 2.3-fold stage (5/21 embryos ruptured before the 2-fold stage) in the anterior ventral midline (17/21) or less frequently posteriorly (3/21). Consistent with previous findings (Wissmann et al., 1999), 12/13 mel-11(it26) embryos ruptured at about the 1.6-fold stage. For originals, see Movies 1-4 in the supplementary material. Scale bar: 10 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 2. RGA-2 colocalises with actin microfilaments in the epidermis. (A-C) Confocal lateral images of C. elegans embryos carrying the rescuing rga-2::gfp fusion F1 (see Fig. 3). (A) 1.2-fold, (B) 1.5-fold and (C) 3-fold stage embryos. GFP expression was restricted to the epidermis; it initially highlighted cell-cell junctions (white arrows), plus thin and short filaments that progressively became organised into a circumferential filamentous network. (D) Confocal image of a small area of the body from a rga-2::gfp transgenic 3-fold embryo showing GFP fluorescence (green, left), bodipy-phallacidin staining to visualise actin (red, middle) and merged picture (right) showing colocalisation. Arrows, actin in underlying muscles; arrowheads, epidermal cell-cell junctions. (E-J) DIC (top) and GFP fluorescence (bottom) pictures of adults, illustrating expression in sensory support cells of the head (E,F), vulval cells (G,H) and rectal cells (I,J), which are epithelial. Scale bars: 10 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 3. Structural and functional requirement for actin colocalisation in C. elegans. (A) Schematic of the RGA-2 protein and deletion constructs analysed. All constructs were expressed under the control the rga-2 promoter. Black bars, constructs conferring strong colocalisation with actin. Right column, ability to rescue rga-2(hd102) lethality: +, rescue; -, no rescue; no sign, not tested. (B-G) Confocal lateral pictures of 3-fold embryos. (B) PH domain-containing F2 construct. Note the punctate localisation. (C) RhoGAP domain-containing constructs (construct P12 is shown). The distribution was mostly cytoplasmic, with regularly spaced and faint bands visible in areas where the epidermis is thinner (arrow). An enlargement of the boxed area is shown in C'. (D) Constructs lacking the final 46 residues (construct P6 is shown). The distribution is cytoplasmic. (E) Constructs containing the final 46 residues (construct P10 is shown). An enlargement of the boxed area is shown in E'. (F,G) P13 constructs with a CAAX or SAAX box, respectively, positioned after the GFP. The CAAX box confers disorganised membrane localisation; the SAAX box confers an apparently normal localisation; it is unclear why the presence of the SAAX box improves the faint actin localisation of construct P13 alone. Scale bar: 10 µm.

View larger version (103K): [in this window] [in a new window]   Fig. 4. Severe pulling on junctional complexes in rga-2 and mel-11 mutants. (A) Still projections from time-lapse videos of wild-type (top) and rga-2(hd102) (bottom) C. elegans embryos containing the mcIs46[dlg-1::rfp] transgene. Time is shown in seconds (bottom right corner). Insets in the rightmost images show single focal plane enlargements of boxed regions. For original wild-type and rga-2 videos, see Movies 5 and 6 in the supplementary material. (B) Additional still images for wild-type, mel-11(it26), rga-2(hd102) embryos and embryos laid by mel-11(it26) mothers after injecting rga-2(RNAi). Arrows, indentations; arrowheads, deformed seam cells. (C) Summary of the defects observed in rga-2 and mel-11 mutants. (D) The number of indentations (from videos such as in A) that were perpendicular to the plane of junctions and at least twice the width of a junction. Counting was performed only in the head (hyp6/hyp7 and H1/H2 area) on at least three embryos per genotype.

View larger version (7K): [in this window] [in a new window]   Fig. 5. The GAP domain of RGA-2 enhances GTP hydrolysis by RHO-1 and CDC-42. Results of GAP assays obtained by incubating purified GTPases and GST-RGA-2GAP fusion protein in the presence of [32P]GTP (see Materials and methods), resolving the reaction products by thin layer chromatography (see Fig. S3 in the supplementary material), and quantifying the GTP/GDP fraction with a phosphorimager. Each time point represents the fraction of GTP hydrolysed after 2 minutes as a function of RGA-2GAP concentration. The graph is representative of four independent experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 6. LET-502 and RGA-2 are dispensable beyond the 2-fold stage. (A) The percentage of lethality reflects larval and embryonic lethality. Temperature-shifts were performed at least three times as described in the Materials and methods. More than 60 individuals were sampled for each time point. Full genotypes: squares, let-502(sb118ts); circles, let-502(sb118ts) dpy-5(e61) rga-2(hd102). let-502(sb118ts) dpy-5(e61) behaved like let-502(sb118ts) but was omitted for clarity (data not shown). Black symbols, 25.5 to 20°C downshift; white symbols, 20 to 25.5°C upshift. LB, lima-bean stage (no elongation); C, comma/1.2-fold stage. (B) Genetic model for the position of rga-2 in the C. elegans embryonic elongation pathway, based on Tables 1 and 2 and on Fig. 5. (C) Developmental model for the role of RGA-2 in elongation (see Discussion).