Cortical centralspindlin and G have parallel roles in furrow initiation in early C. elegans embryos

JCS03447 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. ZEN-4::GFP localization near the cortex requires CYK-4 and AIR-2 but does not require furrowing, SPD-1, CAR-1 or Gα signaling. (A) In wild-type embryos, we can detect ZEN-4::GFP near the furrow during early furrow ingression (arrows) but it is not detectable at the equator before furrow ingression. (B) However, it is detectable at the equator (arrows) if we block furrow ingression by reducing the levels of actin with RNAi. This suggests that furrowing is not required to localize ZEN-4 to the furrow. (C-F) Depletion of cyk-4 (C) or air-2 (D) do disrupt ZEN-4::GFP localization whereas depletion of spd-1 (E) and car-1 (F) disrupts localization to the midzone but not near the furrow (arrows). (G-I) Depletion of par-2 (G), par-3 (H) or gpr-1/2 (I) does not disrupt ZEN-4::GFP localization to the midzone (asterisks) or furrow (arrows). gpr-1/2(RNAi) embryos were imaged at around 40 hours of feeding because ZEN-4::GFP hermaphrodites treated with this RNAi were sterile at 52 hours. Asterisks indicate regions where ZEN-4::GFP is associated with midzone microtubules; prominent labeled structures on the outside of embryos are extruded polar bodies. The frame in A is the earliest frame where ZEN-4::GFP is reliably detected at the furrow. For C-I we selected frames where the furrow appeared to reach the midzone or the furrow reached maximum ingression. We did not detect ZEN-4::GFP on the cortex at any time in cyk-4- or air-2-depleted embryos. Bar, 10 μm.

• Supplemental Figure S2 -

  Fig. S2. Average time of furrow initiation. Bars represent average time in seconds after the metaphase to anaphase transition for the appearance of a cleavage furrow. spd-1(oj5) embryos furrowed significantly earlier, whereas embryos depleted of air-2, par-2 and gpr-1/2 were delayed. Average of five embryos and error bars represent s.d.

• Supplemental Figure S3 -

  Fig. S3. Spindle elongation. Average length of the spindle (in microns) measured from the center of each pole over time in five embryos for each treatment. Time 0 is the metaphase to anaphase transition.

• Supplemental Figure S4 -

  Fig. S4. Distribution of microtubule ends at the cortex of individual embryos. The number of microtubule ends in a 5 micron-wide window at the time of furrow ingression in each wild-type embryo. 0 is the position where the furrow begins ingression, anterior is left (negative values) and posterior is right (positive values). The location of the furrow does not correlate with the location of the global minimum of microtubule ends.

• Supplemental Figure S5 -

  Fig. S5. Distribution of microtubules ends at the cortex at different timepoints prior to furrow ingression. The number of microtubule ends in a 5 micron-wide window 30 seconds, 20 seconds and 10 seconds before or at the time of furrow ingression in wild-type embryos. Average of 10 embryos. 0 is the position where the furrow begins ingression, anterior is left (negative values) and posterior is right (positive values). The localization of the furrow does not correlate with a minimum of microtubules at any timepoint before furrow ingression.

• Supplemental Figure S6 -

  Fig. S6. Different window sizes to analyze distribution of microtubule ends at the cortex. The number of microtubule ends in a 1, 3, 5 or 7 micron-wide window at the cortex at the time of furrow ingression in wild-type embryos. Average of 10 embryos. 0 is the position where the furrow begins ingression, anterior is left (negative values) and posterior is right (positive values). As the window becomes larger the data becomes smoother. Using smaller windows, the localization of furrow ingression correlates with a local maximum of microtubules, although there is no consistent increase or decrease in the number of microtubules at the equator in individual embryos (data not shown).

• Movie 1 -

  Movie 1. zen-4(or153) embryos furrow but do not complete cytokinesis. The movie starts after pronuclear migration before entry into the first round of mitosis. Posterior is to the right. Embryos are approximately 50 microns long. Frames are 15 seconds apart and played back at seven frames per second.

• Movie 2 -

  Movie 2. zen-4(or153) par-2(RNAi) embryos do not initiate furrow ingression. The movie starts after pronuclear migration before entry into the first round of mitosis. Posterior is to the right. Embryos are approximately 50 microns long. Frames are 15 seconds apart and played back at seven frames per second.

• Movie 3 -

  Movie 3. spd-1(oj5) embryos furrow and complete cytokinesis. The movie starts after pronuclear migration before entry into the first round of mitosis. Posterior is to the right. Embryos are approximately 50 microns long. Frames are 15 seconds apart and played back at seven frames per second.

• Movie 4 -

  Movie 4. spd-1(oj5) par-2(RNAi) embryos furrow and complete cytokinesis. The movie starts after pronuclear migration before entry into the first round of mitosis. Posterior is to the right. Embryos are approximately 50 microns long. Frames are 15 seconds apart and played back at seven frames per second.