DEV02527 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. RHO-1 and CDC-42 act in separate pathways to control polarity establishment. Still images of GFP-PAR-2 and GFP-PAR-6 during polarity establishment in cdc-42(RNAi);rho-1(RNAi) embryos. Times (seconds) are relative to nuclear envelope breakdown.

• Supplemental Figure 2 -

  Fig. S2. RHO-1 is involved in regulating the PAR-2 domain size. (A-E) Time-lapse images of GFP-PAR-2 polarity establishment in (A) control, (B,C) rho-1(RNAi) and (D,E) ect-2(RNAi) embryos. Times (seconds) are relative to nuclear envelope breakdown. Rho-1(RNAi) and ect-2(RNAi) embryos display similar phenotypes and fall into two classes based on their GFP-PAR-2 localization. (B,D) GFP-PAR-2 spreads along the cortex, resulting in a large GFP-PAR-2 domain. (C,E) GFP-PAR-2 appears late or never at the cortex.

• Supplemental Figure 3 -

  Fig. S3. RHO-1 is required for the correct timing of PAR-2 domain establishment. (A) Appearance of GFP-PAR-2 at the cortex relative to nuclear envelope breakdown (NEBD). In control embryos, GFP-PAR-2 appears at the cortex at 14.15±1.92 minutes before NEBD. GFP-PAR-2 localization to the cortex was delayed in both rho-1(RNAi) (4.19±1.71 minutes before NEBD) and ect-2(RNAi) (4.72±3.64 minutes before NEBD) embryos. (B) The GFP-PAR-2 domain size was manually tracked after the GFP-PAR-2 domain reached its maximal extent, whereas the GFP-PAR-6 domain was tracked after pseudocleavage regression. The domain size was calculated as a fraction of the embryo circumference. In control embryos, the GFP-PAR-2 domain extent amounts to 53±2% (n=8), the GFP-PAR-6 domain to 46±2% (n=9). In rho-1(RNAi) embryos, the GFP-PAR-2 domain extent amounts to 70±15% (n=10), whereas GFP-PAR-6 is distributed uniformly along the cortex (100%, n=5).

• Supplemental Figure 4 -

  Fig. S4. PAR-2 and PAR-6 localization in ect-(RNAi) embryos. Control and ect-2(RNAi) embryos expressing GFP-PAR-2 were stained for GFP and PAR-6. Overlay of PAR-2 (red) and PAR-6 (green) images shows that in ect-2(RNAi) embryos both proteins overlap.

  Movies 1-11. All supplementary movies were recorded on a spinning disk confocal microscope. Posterior is to the right.

• Movie 1 -

  Movie 1. GFP-PAR-2 dynamics of a wild-type embryo.

• Movie 2 -

  Movie 2. Cortical YFP-CDC-42 dynamics of a wild-type embryo.

• Movie 3 -

  Movie 3. Cortical YFP-CDC-42 dynamics in a par-3(it71) embryo.

• Movie 4 -

  Movie 4. Cortical NMY-2-GFP dynamics of a wild-type embryo.

• Movie 5 -

  Movie 5. Cortical NMY-2-GFP dynamics in an ect-2(RNAi) embryo.

• Movie 6 -

  Movie 6. Cortical NMY-2-GFP dynamics in a cdc-42(RNAi) embryo.

• Movie 7 -

  Movie 7. Cortical YFP-CDC-42 dynamics in a ect-2(RNAi) embryo.

• Movie 8 -

  Movie 8. GFP-PAR-6 dynamics of a wild-type embryo.

• Movie 9 -

  Movie 9. GFP-PAR-6 dynamics in a rho-1(RNAi) embryo.

• Movie 10 -

  Movie 10. GFP-PAR-2 dynamics in an ect-2(RNAi) embryo. (Note, no GFP-PAR-2 localization at the cortex.)

• Movie 11 -

  Movie 11. GFP-PAR-2 dynamics in a rho-1(RNAi) embryo.