DEV01828 Supplementary Material

Files in this Data Supplement:

• Supplemental Table (Adobe PDF)
• Supplemental Figure 1 -

  Fig. S1. C. elegans anillin-related proteins. (A) Schematics comparing the three C. elegans anillin homologs with the human (Hs) and Drosophila (Dm) anillins. The C-terminal region, including the PH domain (dark grey) and the ~280 amino acids N-terminal to it (pink), which we refer to as the Anillin Homology domain, exhibits the greatest conservation. ANI-1 also contains regions with sequence homology to the actin-binding (green) and myosin-binding (blue) domains defined in human and Drosophila anillins. (B) Sequence alignments of the actin- (green bar) and myosin-binding (blue bar) domains of the human and Drosophila proteins with the predicted corresponding domains of ANI-1. Residues conserved among ANI-1 and one (orange) or both (light yellow) of the human and Drosophila proteins are highlighted. (C) Sequence alignments of the AH (pink bar) and PH (grey bar) domains. Residues conserved among the human, Drosophila and one (orange) or more (light yellow) of the C. elegans proteins are highlighted. Residues shown to be functionally important in the Drosophila protein (Field et al., 2005) are highlighted in light blue and marked with an asterisk.

• Supplemental Figure 2 -

  Fig. S2. ANI-1 depletion does not affect PAR-6 localization. Timelapse sequences of paired confocal and DIC images were acquired for control and ani-1(RNAi) embryos expressing PAR-6:GFP. In both control and ani-1(RNAi) embryos, PAR-6 localizes to the entire cortex as meiosis II ends and becomes progressively enriched in the anterior half during pronuclear migration where it remains. Scale bar: 10 mm.

• Supplemental Figure 3 -

  Fig. S3. ANI-1 depletion initiated during embryogenesis results in a variety of developmental defects. rde-1 mutant hermaphrodites were mated with wild-type males and then their gonads were either injected with dsRNA directed against ani-1 (bottom) or not treated (top). The development of embryos collected between 6 and 11 hours after injection, which were loaded with the maximal amount of injected dsRNA, was monitored. ANI-1-depleted animals exhibited malformed cuticles, protruding vulvae and extruded gonads (hermaphrodites), and defects in tail structure (males). ANI-1 depleted worms were not sterile. Scale bars: 25 mm.

• Supplemental Figure 4 -

  Fig. S4. ANI-2 is not required for early embryonic contractile events. Selected still images from timelapse DIC sequences of ani-2(RNAi) embryos (see also Movie 3). Embryos are oriented with their anterior on the left and their posterior (defined by the site of sperm entry) on the right. Scale bar: 10 mm.

• Supplemental Figure 5 -

  Fig. S5. ANI-2 localizes to the lining of the rachis of the syncytial gonad. Control, ani-2(RNAi) and unc-61(e226) mutant C. elegans syncytial gonads were fixed and stained for DNA, myosin II (NMY-2), ANI-2 and UNC-59 septin. Images were collected every 0.4 mm through the volume of the gonad, and sections through the bottom half of the gonad were projected to a single plane as for Fig. 5. ANI-2 staining is present on the surface of the rachis (arrows) in unc-61(e228) gonads, and the septin UNC-59 is present on the remnant structures in ANI-2-depleted gonads. Scale bar: 10 mm.

• Movie 1 -

  Movie 1. A wild-type control embryo was imaged by DIC microscopy for 25 minutes 50 seconds from pronuclear migration until the two-cell stage. Images were acquired every 5 seconds; timelapse playback is at 60× real time.

• Movie 2 -

  Movie 2. An ANI-1 depleted embryo was imaged by DIC microscopy for 25 minutes 50 seconds from pronuclear migration until the two-cell stage. Images were acquired every 5 seconds; timelapse playback is at 60× real time. Ruffling and pseudocleavage fail, but cytokinesis appears normal. An abnormal, blebbing polar body is visible at the anterior (left) of the embryo.

• Movie 3 -

  Movie 3. An ANI-2-depleted embryo was imaged by DIC microscopy from pronuclear migration until the two-cell stage. Images were acquired every 5 seconds; timelapse playback is at 60× real time. Polar body extrusion, ruffling, pseudocleavage and cytokinesis occur normally.

• Movie 4 -

  Movie 4. A control embryo expressing a GFP fusion with myosin II heavy chain (NMY-2) was imaged by spinning disc confocal microscopy for 8 minutes, 20 seconds preceding pronuclear meeting. For each timepoint, three z-sections were collected at 1 mm intervals and projected. Patches of NMY-2:GFP move to the embryo anterior (left). Timelapse playback is at 60× real time.

• Movie 5 -

  Movie 5. An ANI-1-depleted embryo expressing NMY-2:GFP was imaged as for Movie 4. Cortical NMY-2:GFP does not form large patches but small fluorescence discontinuities move to the anterior (left).