DEV02588 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Inhibition of Rho1 in the spiracular chamber cells disrupts the pattern of myosin II distribution and apical cell-cell adhesion. (A) Antibody staining against the heavy chain of myosin II (Zip) (red) in wild-type spiracle cells expressing GFP-Actin (green). Notice the apical enrichment of both proteins. This pattern is partially lost upon Rho1 loss of function, by the expression of the dominant-negative form RhoN19. (B) Armadillo staining (red) in wild-type and in RhoN19- or RacN17- expressing spiracle cells (dorsal views). Green is GFP-Actin. The honeycomb pattern of the adherens junctions in wild-type spiracles is completely disrupted upon expression of RhoN19 (arrows), and only partially affected by the dominant-negative form RacN17. Notice that Rac inhibition does not block cell invagination or lumen formation, as opposed to Rho. Scale bars: 10 μm.

• Supplemental Figure 2 -

  Fig. S2. PKNG58AeGFP binds to Rho1-GTP. (A) PKNG58AeGFP is a C-terminal fusion of eGFP with the first 339 amino acids of Drosophila Pkn (Protein kinase N) containing three Rho-binding domains. It includes a point mutation, G58A, which reduces Pkn affinity for active Rac (Lu and Settleman, 1999). The construct produces a 65 kDa protein, detected by western blot with an anti-GFP antibody after transfection of S2 cells. Control and eGFP lanes: non-transfected and eGFP transfected cells, respectively. (B) PKNG58AeGFP distribution during cell cycle. The probe accumulates at sites of activated Rho: the centrosomes during metaphase and the contractile Actin ring during telophase, similar to another Rho probe, GFP-Rhotekin (Bement et al., 2005). (C) Co-transfection of S2 cells with PKNG58AeGFP and the constitutively active and dominant-negative forms of small RhoGTPases. PKNG58AeGFP recognizes the active conformation of Rho1 (RhoV14) at the cell membrane. A weak membrane recruitment is also observed in 27% of cells transfected with the constitutively active RacV12, n=290. No recruitment is seen with active Cdc42 (Cdc42V12), nor with any of the dominant-negative forms (RhoN19, RacN17 and Cdc42N17). (D) Treatment of S2 cells with dsRNA for Rho1 before transfection with RhoV14 abolishes membrane localization of PKNG58AeGFP. Western blot against Rho1 protein P1D9 antibody (Magie et al., 2002) in untreated S2 cells (control), Rho1 RNAi-treated cells (Rho1 RNAi) and RhoV14 transfected cells (RhoV14). (E) In vitro binding assays between purified GST fusions of Drosophila GTPases in their GTPγS/GDP bound forms and PKNG58AeGFP. This probe binds specifically to Rho1-GTPγS at ratios 5:1 to 10:1 (probe: small RhoGTPase; arbitrary mass units). Lysate represents 3% of PKNG58AeGFP input used in each binding assay. Higher ratios (e.g. 20:1) may lead to binding of PKNG58AeGFP to active Rac1 and Rac2 (data not shown), which reflects the weak affinity of the probe to activated Rac. The apical RhoGTPase activity detectable during spiracle invagination using our probe is likely to be mainly Rho1 derived, as the role of Rac is secondary for spiracle cell movement (see also Fig. S1B).

• Supplemental Figure 3 -

  Fig. S3. RhoGEF2 is apically localized and required for correct spiracular chamber formation. (A) Antibody staining against RhoGEF2 (red) in a stage 11 embryo showing its ubiquitous expression in the A8 segment, including the spiracular chamber cells before invagination (in green, expressing GFP-Actin). At stage 13, apical enrichment of RhoGEF2 is seen in the invaginated spiracle cells, overlapping with apical GFP-Actin. Left image, dorsal view; right images, lateral view. (B) Posterior spiracle defects in germline clones for the null allele DRhoGef2^I(2)04291 (DRhoGEF2 MZ). Two major classes of phenotypes are found (arrows): lumen defects, with broader and shorter Filzkörpers (i) and invagination defects, with Filzkörpers on the surface of the embryo (ii). (C) Quantification of spiracle defects in DRhoGEF2 MZ (n=201) and DRhoGEF2MZ embryos carrying one or two copies of the null allele RhoGEF64C¹ (1:1, n=86). Scale bar: 10 μm.

• Movie 1 -

  Movie 1. Time-lapse confocal movie showing the posterior region of a Drosophila embryo expressing DE-Cadherin-GFP between stages 11 and 13. As germ band retracts, apical constriction and invagination of the spiracle primordium is clearly observed in the last segment of the embryo (A8). Images were taken every 2 minutes; the entire invagination process takes around 4 hours.