DEV017202 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Embryonic mop mutant phenotypes. All panels show early embryos with anterior to the left. (A,C,E) Wild type; (B,D,F) embryos derived from mop^T612 germline clones. (A,B) In situ hybridization with a hkb probe. Embryos lacking mop show normal hkb expression, indicating that Torso signaling is unaffected. (C-F) Embryos are stained for Slam, which marks the front of invaginating membranes (green), and with TOTO-3 to label nuclei (magenta). The first visible abnormality in embryos lacking mop is a loss of association of nuclei with the cortex (arrow in D). As cellularization proceeds, membrane invagination becomes irregular and more nuclei fall into the embryo (F).

• Supplemental Figure S2 -

  Fig. S2. mop expression. (A-F) In situ hybridization of embryos (A-D) or imaginal discs (E,F) with antisense (A,C,E) or sense (B,D,F) mop probes. mop RNA is maternally provided to early embryos (A) and expression is strongest in the nervous system and gut at later stages (C). mop is ubiquitously expressed in imaginal discs (E). (G,H) An eye disc with mop^T482 clones expressing a UAS-FlagMop transgene. Clones are positively marked by Flag staining (green in H) and stained with anti-Elav (G, magenta in H). Flag-tagged Mop rescues photoreceptor differentiation.

• Supplemental Figure S3 -

  Fig. S3. Interactions between mop, Cbl, Hrs and sty. (A-F) Eye discs stained with anti-Hrs (A,C,E, magenta in B,D,F). mop^T612 clones (A,B), Cbl^F165 clones (C,D) or mop^T612 Cbl^F165 double-mutant clones (E,F) are marked by the absence of GFP (green in B,D,F). Loss of Cbl does not restore normal Hrs localization to mop mutant clones. (G,H) An Hrs^D28/Df(2L)Exel6277 eye disc in which mop^T612 clones are marked by the absence of GFP (green in H), stained with anti-active Caspase 3 (G) and anti-Elav (magenta in H). Loss of Hrs does not rescue photoreceptor differentiation or cell survival in mop mutant clones. (I-L) Anti-Hrs staining of the dsRNA-treated D2F cells used in Fig. 6. Enlarged endosomes are seen on depletion of mop, Tsg101 or Vps28. (M) Western blot with antibodies to diphospho-MAPK and Tubulin of lysates from D2F cells treated with lacZ, mop, Cbl or sty dsRNA and transfected with Actin-GAL4, UAS-cblL as indicated, and incubated with Spi for 0 or 30 minutes. MAPK phosphorylation in Mop-depleted cells was partially rescued by overexpressing Cbl or by depleting Cbl or Sty.

• Supplemental Figure S4 -

  Fig. S4. Mop promotes lysosomal entry of Spi. (A-F) Examples of D2F cells treated with Alexa-labeled Spi and imaged 4 hours later, with Lysotracker shown in green (A,C,D,F) and Spi in magenta (B,C,E,F). (A-C) Cells treated with lacZ dsRNA and (D-F) with mop dsRNA. (G) Quantification of the percentage of vesicles containing Alexa-labeled Spi between 3 and 4 hours following Spi treatment that do not stain with Lysotracker or show weak or strong uptake of Lysotracker. Dark blue bars are from D2F cells treated with lacZ dsRNA and light blue bars are from D2F cells treated with mop dsRNA. n=452 vesicles for lacZ and 368 vesicles for mop, taken from two independent experiments.

• Supplemental Figure S5 -

  Fig. S5. Effects of mop on other signaling pathways. (A,B,D-H,J,K) Third instar wing discs; (C) a third instar eye disc; (I) an adult wing. mop mutant clones are marked by the absence of GFP (green in A-C, blue in F, green in H,K) or unmarked (I). mop^T612 (A,C-F,I-K) or mop^T482 (B,G,H) was used. Clones in A-C were generated in a Minute background. (A) Stained with anti-Cut (magenta). Normal Cut expression suggests that Notch signaling is unaffected. (B) Stained with anti-Ci (magenta); (C) stained with anti-β-galactosidase reflecting dpp-lacZ expression (magenta). Ci upregulation and dpp expression are normal in mop mutant clones, suggesting that Hh signaling is unaffected. (D-F) Stained with anti-Wg (D, red in F) and anti-Hrs (E, green in F). In mop mutant clones, Wg accumulates in large punctae that often colocalize with Hrs (arrows in F). (G,H) Stained with anti-Sens (G, magenta in H); (J,K) stained with anti-β-galactosidase reflecting Dll-lacZ expression (J, magenta in K). Sens expression is reduced, consistent with loss of the adult wing margin (I), but Dll expression is not significantly affected, suggesting that Wg signaling is only weakly reduced. Scale bars: 10 µm.