DEV005074 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Alignments of the PH and GAP domains of RGA-2 and ARHGAP20. Protein sequences were aligned using the PipeAlign web interface for ClustalX (http://bips.u-strasbg.fr/PipeAlign/). Accession numbers: Ce, Caenorhabditis elegans Q9XW53; Cb, Caenorhabditis briggsae Q61AR9; Hs, Homo sapiens Q9P2F6; Rn, Rattus norvegicus Q6REY9. The PH and GAP domains are well conserved and predicted under Pfam; the RA domain is not predicted by Pfam, yet is partially conserved.

• Supplemental Figure S2 -

  Fig. S2. rga-2 is not required to assemble junctions or maintain actin cytoskeleton integrity. Primary antibodies were detected using Cy3-conjugated or FITC-conjugated secondary antibodies. Confocal images of wild-type (A,C,E) and rga-2(hd102) (B,D,F) embryos collected 4-6 hours after egg-laying for immunostaining using 1:1000-diluted VAB-10A antiserum (green) and 1:150-diluted AJM-1 monoclonal antibody (MH27, Developmental Studies Hybridoma Bank, University of Iowa; red). (A,B) VAB-10A, a component of fibrous organelles; (C,D) junctional protein AJM-1 (monoclonal antibody MH27); (E,F) merged images. Confocal images of wild-type (G) and rga-2(hd102) (H) embryos expressing a lin-26p::vab-10ABD::gfp construct revealing the actin pattern. This probe was modelled after the N-terminal region of MACF and Short-stop, two close VAB-10 homologues in mouse and Drosophila, respectively, which are known to interact with actin through their two calponin-homology domains (Lee and Kolodziej, 2002; Sun et al., 2001). We created a fusion between the equivalent region of VAB-10 (cDNA fragment encoding residues 1-290; denoted VAB-10ABD, or VAB-10 Actin-Binding Domain, below) and GFP, expressed it under the control of the epidermal promoter lin-26, and subsequently demonstrated that this construct specifically decorates actin filaments in the epidermis (F.L. and M.L., unpublished). For each marker, no apparent defects were observed, except where rga-2 embryos ruptured (arrows in B,D,F,H). The HMP-1 distribution was also normal (data not shown). At least 15 rga-2(hd102) embryos were examined and the results were confirmed on embryos obtained after RNAi by injection against rga-2. Scale bar: 10 μm.

• Supplemental Figure S3 -

  Fig. S3. RGA-2 is more active on RHO-1. Example of a GAP assay using purified recombinant GST-RGA-2_GAP and GTPase proteins. TLC plates were analysed using a phosphorimager. The spots of GDP (upper on TLC plate) result from the hydrolysis of GTP (lower on the plate).

• Movie 1 -

  Movie 1. DIC video of rga-2(hd102) embryo grown at 25.5°C shown between approximately 1.2-fold and 2-fold stages of elongation. Images were acquired every 3 minutes (see Fig. 1).

• Movie 2 -

  Movie 2. Fluorescent video of rga-2(hd102) embryo expressing dlg-1::RFP shown between approximately 1.5-fold and 2-fold stages. Stacks of ten focal planes were acquired every 10 seconds (see Fig. 4A), two of which were projected for the movies.

• Movie 3 -

  Movie 3. DIC video of wild-type embryo grown at 25.5°C shown between approximately 1.2-fold and 2-fold stages of elongation. Images were acquired every 3 minutes (see Fig. 1).

• Movie 4 -

  Movie 4. Fluorescent video of wild-type embryo expressing dlg-1::RFP shown between approximately 1.5-fold and 2-fold stages. Stacks of ten focal planes were acquired every 10 seconds (see Fig. 4A), two of which were projected for the movies.

• Movie 5 -

  Movie 5. DIC video of let-502(sb118ts) embryo grown at 25.5°C shown between approximately 1.2-fold and 2-fold stages of elongation. Images were acquired every 3 minutes (see Fig. 1).

• Movie 6 -

  Movie 6. DIC video of mel-11(it26) embryo grown at 25.5°C shown between approximately 1.2-fold and 2-fold stages of elongation. Images were acquired every 3 minutes (see Fig. 1).