Fig. 3

Comparison of endosomal compartments in wild-type and suf/spastizin mutant oocytes.

Small icon next to figures indicates the level of the optical section (black line) in the oocyte (blue circle). (A) Surface view of immuno-labeled stage III oocytes showing Rab5, Rab7 and Rab11 (green). The nuclei of the surrounding follicle cells are labeled with DAPI (blue). Note that Rab11-positive staining accumulates in patches below the layer of follicle cell nuclei. Scale bar: 50 μm. (B) Optical section of stage III oocytes showing no change in the number of Rab5, a slight increase of Rab7 and a strong increase of Rab11 positive vesicles. The oocyte cytoplasm was counterstained with the lysosomal marker Lysotracker (red) labeling yolk globules. Scale bar: 50 μm. (C) Summary of the results of panel B quantified in Figure S3. The bars display the ratio of Rab-positive foci in mutant oocytes to wild-type. Equal numbers correspond to one fold (baseline). (D) Functional analysis of the endosomal trafficking routes by cargo assay. LDL (red) as a marker for the degradative route accumulates within 10 min in fragmented lysosomes. Transferrin (Tfn; red) as a recycling cargo shows no difference between wt and mutant oocytes after 35 (middle) or 55 min (lower panel) pulse/chase. Note that Transferrin accumulates in a Rab11b-negative compartment. Scale bar: 50 μm. (E) In wt oocytes, Rab5 (green; upper panel) colocalizes with Suf/Spastizin (red), but Rab7 does not overlap (middle panel). Suf/Spastizin (red) and Rab11b (green) colocalize predominantly, but independent localization of Rab11 and Suf is also observed (lower panel). Scale bar: 50 μm.