DEV008078 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. In situ hybridizations. (A,A′) Fluorescent osk mRNA in (A) wild-type and (A′) mutant (rab6^D23D germ line clone) oocyte. osk mRNA mislocalized to the center and the oocyte nucleus. (B) Mosaic egg chamber stained for Osk protein (red). Drab6 mutant follicle cell clones were labeled by the absence of GFP (green), and are outlined by dotted lines. Osk protein accumulation to the posterior is not affected. (C,C′) Projections of several optical sections of stage 10 egg chambers stained for Stau (red) and with Phalloidin (green). Stau localized to the posterior in wild type (C). In Drab6 mutants, Stau accumulated in the center of the oocyte and close to the nucleus (C′). (D,D′) Cuticle preparations of wild type (D) and Drab6 mutant (D′) lacking abdominal structures. (E,E′) Egg chambers expressing Kin-β-gal to label MT plus-ends. Wild type (E) and Drab6 mutant (E′). (F,F′) Fluorescent grk mRNA in situ hybridization. grk localized to the anterodorsal corner of wild-type (F) and Drab6 mutant (F′) oocytes. (G,G′) Fluorescent bcd mRNA in situ hybridization. bcd localization in a characteristic ring shape to the anterior cortex in wild type (G) and Drab6 mutants (G′). (H,H′) Grk signaling to the posterior follicle cells is unaffected in Drab6 mutants (H′) as indicated by the presence of a posterior aeropyle. Anterior micropyles (arrows); posterior aeropyles (arrowheads); asterisk, oocyte nucleus. Scale bars: 20 μm.

• Supplemental Figure S2 -

  Fig. S2. Drab6 affects plasma membrane integrity. (A,A′) Stage 9 egg chambers stained with LE. (A) Control. (A′) Drab6 mutant. Absence of Drab6 leads to massive accumulation of LE-positive patches in the nurse cells (arrow) and to LE labeling of ring-like structures in the oocyte (see inset, arrowhead). (B,B′) EM to monitor integrity of plasma membranes between neighbouring nurse cells in a control egg chamber (B) and in a Drab6 mutant (B′). Continuous membranes were observed in the control (arrow), but ruptures were frequently present in the mutant (arrow). (C) Drab6 mutant egg chamber stained to visualize F-actin and DNA. Inset, control stained to visualize F-actin. The strong phenotype affecting nurse cell membrane integrity also caused disorganization of the actin cytoskeleton. DAPI staining revealed that these cells were not degenerated. (D,D′) Drab6 mutant oocytes, rescued by GFP-Drab6 expression. (D) During stage 7, Drab6 was scarcely detectable on membranes (arrow). (D′) Recorded with the same confocal settings, during stage 10, Drab6 massively accumulated juxtaposed to nurse cell membranes (arrow). Scale barS: 20 μm; 500 nm in EM micrographs.

• Supplemental Figure S3 -

  Fig. S3. GFP-Drab6 and RFP-Drab6 expression do not affect the localization of Golgi-associated proteins. (A) Distribution of Golgi-associated proteins in stage 9 egg chambers. (I-III) Egg chambers stained with Lva. (IV,V) Egg chambers expressing GalT-GFP. (I) W¹¹¹⁸ (wild-type) egg chamber; (II) Drab6^D23D/Cyo; ubi-RFP-Drab6/Tm2 egg chamber; (III) Drab6^D23D/Cyo; _matαtub-GFP-Drab6/Tm2 egg chamber; (IV) W¹¹¹⁸; GalT-GFP egg chamber; (V) Drab6^D23D/GalT-GFP; ubi-RFP-Drab6/Tm2 egg chamber. The distribution of GFP-GalT was not analyzed in _matαtub-GFP-Drab6 egg chambers; both Drab6 and GalT were tagged with GFP. In Drab6-overexpressing oocytes, the distribution of the Golgi-associated proteins Lva and GalT-GFP is similar to that observed in wild-type oocytes. Scale bars: 20 μm. (B) Level of expression of RFP-Drab6 and GFP-Drab6. Western blots of ovarian extracts from flies expressing different levels of Drab6: W¹¹¹⁸ (wild-type) ovaries, Drab6^D23D/Drab6^D23D; ubi-RFP-Drab6/Tm2 ovaries, Drab6^D23D/Cyo; ubi-RFP-Drab6/Tm2 ovaries, Drab6^D23D/Cyo; _matαtub-GFP-Drab6/Tm2 ovaries. (I) Western blot probes with anti-Drab6 antibody. The bands at 22 kD correspond to endogenous Drab6 and the bands at 50 kD correspond to the different forms of GFP- and RFP-tagged Drab6. In the absence of Drab6 (Drab6^D23D/Drab6^D23D; ubi-RFP-Drab6/Tm2 ovaries) the band at 22 kD is not detectable, confirming the specificity of the antibody for Drab6. (II) Western blot probes with anti-α-Tubulin for loading control. In Drab6^D23D/Cyo; ubi-RFP-Drab6/Tm2 and Drab6^D23D/Cyo; _matαtub-GFP-Drab6/Tm2 ovaries, the amount of Drab6 is approximately twice that in the wild type (W¹¹¹⁸). Signal quantification was performed with MultiGauge software (Fujifilm). We used α-Tubulin as a loading reference. Drab6 antibody (Eurogentec) was obtained from rabbit after immunization with two peptides (sequences available upon request); Rab6 antibody does not function in whole-mount.