JCS033027 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A-D) Yl and PH(PLCδ)-GFP localization in control (A) and rab5² GLC (B-D) stage 10 egg chambers. (B, green in A, D) PH(PLCδ)-GFP (C, red in A, D) Yl. Note that in rab5² GLC large PH(PLCδ)-GFP-positive structures in the oocyte cytoplasm are present and associated with Yl. (E, H) Electron micrographs showing the early endocytic intermediate aggregate indicated by the green arrowhead in (Fig. 2B). (F) High magnification of the region inside the red box in (E). (G, H) Actin-rich follicle cell microvilli indicated by the asterisk in Fig. 2E. Note the presence of fiber bundles inside the aggregate cytoplasm (F, triple arrowheads) resembling actin fiber bundles seen in follicle cell microvilli (G, H, triple arrowheads). The annotation of endocytic intermediates is described in the legend to Fig. 2. Bars (A, B) 500 nm, (C, D) 100 nm. (I) F-actin staining on rab5² GLC reveal the presence of abnormal intracytoplasmic actin patches independently of the presence of PH(PLCδ)-GFP transgene. (J-K′′) F-actin and PH(PLCδ)-GFP localization in control (J-J′′) and Rab5 RNAi (K-K′′) S2 cells. (J′, K′, green in J, K) PH(PLCδ)-GFP (K′′, J′′, red in J, K) F-actin. Note that the PH(PLCδ)-GFP-positive structures in the cytoplasm in Rab5 RNAi cells are associated with F-actin. Bar, 10 µm. (L) Quantification of F-actin and PH(PLCδ)-GFP localization defect in control and Rab5 RNAi-treated cells. n, number of cell examined. (M) Quantification of F-actin localization defect in control and Rab5 RNAi-treated cells. n, number of cell examined. (N) Rab5 and α-tubulin levels in control and rab5 RNAi-treated cells. In rab5 RNAi-treated cells Rab5 is not detectable while α-tubulin levels in the extract are comparable to those in control.

• Supplemental Figure S2 -

  Fig. S2. Synaptojanin loss of function in the germ line does not affect Yolk endocytosis and actin distribution. Yolk internalization and F-actin distribution in control (A-C) and synj¹ GLC (D-F). (Blue in A, D) DNA, (green in A, D) GFP, (B, E) DIC image, (C, F) F-actin. The absence of nuclear GFP in the germline cells marks the presence of a GLC in D. In synj¹ GLC Yolk granules content of the oocyte visible on the DIC images is similar to control (compare B and E). F-actin distribution is also similar to control (compare C and F).

• Supplemental Figure S3 -

  Fig. S3. Anti-Skittles antibody allows Skittles detection. Western blot from wt, w;;UAS-GFP-Skittles and sktl^Δ5/ sktl^2.3 ovarian extract incubated with anti-Skittles antibody. Skittles protein is detected at its predicted size of 88 kDa in wt, w;;UAS-GFP-Skittles extracts and absent from sktl^Δ5/sktl^2.3 extract. GFP-Skittles fusion is detected at its predicted size of 113 kDa in w;;UAS-GFP-Skittles extract. The 75-kDa band corresponds to aspecific immunoreactivity, equivalent in all extracts.

• Supplemental Figure S4 -

  Fig. S4. Rab5 controls the restriction of PtdIns(3,4,5)P₃ from the membrane of early endocytic intermediates. PH(GRP1)-GFP localization in wt (A) and rab5²-GLC (B) stage 10 egg chambers. Note the presence of large PH(GRP1)-GFP-positive structures in the oocyte cytoplasm when Rab5 is absent (arrows in B) which are never seen in control. Bar, 50 µm.