DEV01956 Supplementary Material

Files in this Data Supplement:

• Movie 1 - Movie 1. Normal slow cytoplasmic streaming in a wild-type stage 9 oocyte. Slow, multidirectional endosome movements are visible at the anterior end (upper region). Little movement is visible at the posterior end (lower region). This 8 second time-lapse movie represents 30 minutes of real time. Scale bar: 25 mm.

• Movie 2 - Movie 2. Absence of streaming in a stage 9 Khc²³ oocyte. Endosomes saltate slightly within the ooplasm, and there is slight motion because of specimen drift as the egg chamber flattens. No coordinated currents are evident. This 8 second time-lapse movie represents 30 minutes of real time. Scale bar: 25 mm.

• Movie 3 - Movie 3. Absence of streaming in a stage 9 Khc²⁷ oocyte. Some endosomes oscillate in the anterior region of the oocyte, but directed endosome movement is not seen. This 8 second time-lapse movie represents 30 minutes of real time. Scale bar: 25 mm.

• Movie 4 - Movie 4. Fast streaming in a stage 11 wild-type oocyte. The majority of fluorescent endosomes move in robust, well-ordered currents. This 8 second time-lapse movie represents 30 minutes of real time. Scale bar=25 mm.

• Movie 5 - Movie 5. A stage 10b Khc¹⁷ oocyte with a fast streaming area. Ordered motion of endosomes is seen only in the posterior (bottom-left) region of the oocyte. Other endosomes oscillate but show little directed motion. The bright material flowing beside the oocyte is fluid between the egg chamber and surrounding halocarbon oil. This 8 second time-lapse movie represents 30 minutes of real time. Scale bar: 25 mm.

• Movie 6 - Movie 6. Lack of streaming in a stage 11 Khc²⁷ oocyte. Fluorescent endosomes oscillate a bit, and may drift due to influx of nurse cell cytoplasm during dumping, but no directed streaming currents are seen. The oocyte grows substantially because of massive influx of nurse cell cytoplasm at the anterior, yet retains stratified ooplasm, with endosomes concentrated in posterior regions, indicating that nurse cell dumping does not provide sufficient force for mixing the ooplasm. This 8 second time-lapse movie represents 30 minutes of real time. Scale bar: 25 mm.

• Movie 7 - Movie 7. Injection of dynein intermediate chain antibody induces premature fast streaming. Before injection, the wild-type stage 9 oocyte exhibits slow streaming. After injection, streaming becomes more rapid and well-ordered, although it is not as robust as normal fast streaming. This 15 second time-lapse movie represents 50 minutes of real time. Scale bar: 25 mm.

• Movie 8 - Movie 8. Injection of dynein heavy chain antibody induces premature fast streaming. Before injection, the wild-type stage 10a oocyte has slow streaming near the anterior end. After injection, streaming currents become more rapid and robust. This 11 second time-lapse movie represents 40 minutes of real time. Scale bar: 25 mm.

• Movie 9 - Movie 9. Control IgG injection has no effect on fast streaming in a late stage oocyte. A wild-type stage 10b oocyte shows fast streaming throughout the cell. Upon IgG injection the oocyte changes shape and imaging shifts to a focal plane close to the cortex, where less endosome movement is visible. Focus is then restored to a deeper level within the oocyte where rapid streaming currents are apparent. This 8.9 second time-lapse movie represents 33 minutes of real time. Scale bar: 50 mm.

• Movie 10 - Movie 10. Injection of anti-KHC hinders fast streaming in a wild-type stage 11 oocyte. This 8.8 second time-lapse movie represents 33 minutes of real time. Scale bar: 50 mm.

• Movie 11 - Movie 11. Injection of anti-Khc antibody blocks premature fast streaming in a spir mutant oocyte. This 6.7 second time-lapse movie represents 25 minutes of real time. Scale bar: 50 mm.

• Movie 12 - Movie 12. GFP::a-tubulin microtubules in a stage 9 oocyte. Microtubules, most abundant near the oocyte anterior, appear randomly organized and move. The oocyte nucleus is visible at the right side of the anterior. This 8 second time-lapse movie represents 30 minutes of real time. Scale bar: 25 mm.

• Movie 13 - Movie 13. GFP::a-tubulin microtubules form dynamic parallel arrays during fast streaming in a stage 10b oocyte. Posterior is towards the bottom right. Yolk endosomes appear as grey and white spheres. Note that parallel microtubule arrays align with rapid streaming currents. This 8 second time-lapse movie represents 30 minutes of real time. Scale bar: 25 mm.

• Supplemental Figure 1 - Fig. S1. grk mRNA localizes to the anterodorsal corner in the absence of streaming. Fluorescent in situ hybridization in stage 9 shows strong dorsal-anterior localization of grk mRNA in (A) wild-type, (B) Khc¹⁷ and (C) Khc²³ oocytes. (D) Khc²⁷ oocytes; no concentrated grk message is visible. Scale bar: 25 mm.

• Supplemental Figure 2 - Fig. S2. Multi-stage views of osk mRNA in hypomorphic and null Khc mutants. Fluorescent in situ hybridization of late stage 8, stage 10A and stage 10B egg chambers shows the progression of osk mRNA localization over time in (A-C) wild-type, (D-F) Khc¹⁷, (G,H) Khc²³ and (J-L) Khc²⁷ oocytes. In wild-type oocytes, osk formed a transient central spot in stage 8 (A) that disappeared as osk localized to the posterior pole (B,C). (D-I) In the hypomorphs Khc¹⁷ and Khc²³, the central osk localization appeared later and persisted longer, throughout posterior osk localization. (J-L) In Khc²⁷ oocytes, no central or posterior concentrations of osk were detected.