DEV011437 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Sisyphus antibody specificity and developmental expression. (A) Syph antibody binds in vitro translated (IVT) Syph tail (lane 1) and IVT-full length (FL) Syph (lane 2). (B) Syph antibody recognizes both endogenous and ectopic Syph protein (green) in stage 14-15 embryos. Syph was ectopically expressed in otherwise wild-type embryos as a UAS-Syph-RFP fusion (red) with an Engrailed-Gal4 driver (stripes). Scale bar: 20 µm. (C) Syph protein expression visualized in confocal micrographs of successively older wild-type embryos. Syph is expressed in both the nucleus and the cytoplasm throughout the embryo, except during cell division when it is observed on the condensed chromosomes/spindle. Whole-embryo images are shown in the left column, with higher magnification views of the central or posterior (pole-cell-containing) regions in the middle and right columns, respectively. Embryo age is indicated by nuclear cycle (NC) for precellular stages or by embryonic stage for postcellular embryos. Scale bars: 20 µm. (D) Distribution of Syph during three different stages of mitosis, with Syph (green), tubulin (red) and DAPI (blue). Syph localizes to condensed chromosomes on the mitotic spindle of S2R+ cells. In addition, puncta of Syph can be observed on microtubules. Gross mitotic defects (i.e. binucleate cells) were not observed in Syph RNAi cells. Scale bars: 2 µm.

• Supplemental Figure S2 -

  Fig. S2. Sisyphus protein-protein interactions. (A) Syph interacts physically with its putative cargo proteins through its C-terminal tail domain. With the exception of Tango, all ³⁵S-labelled in vitro translated (IVT) putative Syph cargos tested bind to GST-Syph C-terminal tail (see also Table 1). Syph cargo clones for IVT were obtained from the Drosophila Gene Collections (DGC-1 or -2). (B) Coomassie-stained gels of GST fusion proteins used in the study to examine Sisyphus protein interactions. (C) CadICD pieces show specific binding to known interacting proteins. IVT-³⁵S-p120-catenin (middle panel) binds the juxtamembrane domain (JMD) present in cad-A (lane 4), whereas IVT-³⁵S-β-catenin (lower panel) binds the catenin-binding domain (CBD) present in cad-C, as expected. (D) DE-cadherin’s C-terminal Syph-binding region (cad-D) is conserved with human cadherins. Alignment of the C-termini of the human (HS) cadherin protein indicated with the last 40 amino acids of DE-cadherin.

• Supplemental Figure S3 -

  Fig. S3. Sisyphus RNAi controls. (A) Western blot analysis of lysates from S2R+ cells or stage 14 nuclear GFP-expressing embryos (see Fig. 5A,B) that were treated with Syph dsRNAs, GFP dsRNA or buffer alone. Per lane, 15 µg protein was loaded and the lysates were probed with α-Groucho (recognizing an unrelated protein) as loading controls. (B-C′) Confocal micrographs showing dorsal view of a stage 15 embryos expressing GFP under the control of the En-Gal4 driver (striped pattern) that were injected with buffer alone (B) or with Syph dsRNA (C,C′). C was taken with the same confocal settings as B. C′ is an overexposure of C to show that injection of dsRNA to GFP does not result in stripe mismatching. Scale bar: 20 µm. (D-E′) Confocal micrographs showing dorsal view of stage 15 embryos expressing a Syph-GFP fusion protein under the control of the En-Gal4 driver that were injected with buffer alone (D) or with Syph dsRNA (E-E′). E was taken with the same confocal settings as D. E′ is an overexposure of E to show that injection of dsRNA to Syph efficiently knocks down the ectopically expressed Syph-GFP fusion protein. Scale bar: 20 µm. (F-H) Confocal micrographs showing dorsal views of stage 15 wild-type embryos that were injected with buffer alone (F) or with Syph dsRNA (G-H) then stained with Engrailed (green) and DAPI (blue) to show that reducing Syph also results in epithelial mismatching (arrowheads in H compared with F) and failure to fully close at dorsal closure in the absence of reporter genes (arrows in G). Scale bar: 20 µm.

• Movie 1 -

  Movie 1. Movie of Syph localization to the leading edge and filopodia shown in Fig. 2F-F′′′.

• Movie 2 -

  Movie 2. Movie of Syph trafficking within filopodia shown in Fig. 2G-J.

• Movie 3 -

  Movie 3. Movies of GFP-cadherin trafficking within filopodia of control and syph-deficient embryos shown in Fig. 4K-M.

• Movie 4 -

  Movie 4. Movies of EB1-EYFP trafficking within filopodia of control and syph-deficient embryos shown in Fig. 6A-C.