DEV026963 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Actin furrow extension assay. Surface views of actin rings in a representative cycle-12 metaphase wild-type or Apc2^g10 embryo at four depths (0, −0.8, −1.6 and −2.2 µm). These embryos were evaluated for complete actin rings (green) and incomplete actin rings (red), to assay actin furrow extension.

• Supplemental Figure S2 -

  Fig. S2. Microtubules in Apc2^g10 embryos appear normal. (A-J) Still images from movies of microtubules during metaphase to late telophase in wild-type (A-E) and Apc2^g10 (F-J) mutant embryos expressing ZeusGFP, a microtubule marker. There are no apparent spindle morphology defects in the Apc2^g10 mutants. (K-L′) Early telophase microtubules in wild-type (K) and Apc2^g10 mutants (L) have similar densities and organization. Images K,K′ and L,L′ are from the same embryos as in Fig. 3D,D′ and E,E′, respectively, but taken 30 seconds later. (M,N) Interphase microtubules in wild-type (M) and Apc2^g10 mutants (N) appear to have similar densities and organization. Scale bars: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Astral microtubule organization is similar in wild-type and Apc2^g10 anaphase embryos. (A-B′) Single optical slices of cycle-9 anaphase asters in wild-type (A,A′) and Apc2^g10 (B,B′) embryos. Deconvolved (Autodeblur Gold CF software version X2.1.1, Media Cybernetics) three-dimensional volume-rendered (Volume Viewer plug-in for ImageJ) versions of these embryos are shown in Fig. 3G,G′,H,H′. (C) We compared the overall density of microtubules between wild-type and Apc2-null astral arrays using 3D Sholl analysis (Sholl, 1953). Using a custom MatLab program, we chose astral centers based on the intensity of fluorescence and generated a sphere from that point around the astral array with a radius of 6.5 µm. We analyzed the number of microtubules penetrating that spherical shell at lines of latitude (planes) every 0.8 µm in the central part of the sphere (center ± 3.2 µm) and did not analyze the spherical caps (shaded area). (D) This analysis revealed no significant differences between wild-type and mutant asters, either in cycle 9 or cycle 10 (P>0.05, two-tailed t-test assuming unequal variance). (E-H) Select non-deconvolved images from the embryos analyzed. (I) Quantification of the distribution of astral microtubules in the z-axis in wild-type and Apc2^g10 cycle 9 and 10 embryos. Both genotypes have a Gaussian distribution of microtubules, with the highest percentage of microtubules at the equator (0 µm) and with decreasing numbers both into the interior of the embryo and towards the cortex at 0.8 µm intervals. The percentage of microtubules at each plane was calculated from the average number of total microtubules in all planes. In the wild type, the average total number of microtubules was 173 from 52 astral centers, and in the mutant the average total number of microtubules was 198 from 49 astral centers. (J) Overall maximum astral microtubule length is similar in wild-type and Apc2-null cycle 9 and 10 embryos. The average length of the ten longest microtubules in each of nine astral centers for each genotype was determined. No significant difference was detected (P>0.05, two-tailed t-test assuming unequal variance). Scale bars: 10 µm.

  Reference

  Sholl, D. A. (1953). Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Anat. 87, 387-406.

• Supplemental Figure S4 -

  Fig. S4. APC2 and DIA localize with actin pseudocleavage furrows throughout the cell cycle. (A1-F4) Cross-sections of wild-type cycle-12 embryos throughout the cell cycle, labeled for actin (rows 1 and 4, red), APC2 (rows 2 and 4, blue) and DIA (rows 3 and 4, green). In actin furrows, APC2 (row 2, B-D) and DIA (row 3, B-D) localize with actin (row 1, B-D). DIA is enriched at the furrow tip and sometimes extends beyond APC2 and actin in the furrow tip (row 4, arrows). Scale bars: 10 µm.