![Midbody assembly can occur in the absence of a central spindle via astral MT bundling.(A) Quantification of peak levels of GFP::TBB-2β-tubulin at the midbody above intracellular background; related to Fig. 4 A and the normalized line scans in Fig. 4 D. (B) Quantification of peak levels of AIR-2Aurora-B::GFP at the midbody above intracellular background; related to Fig. 4 B and the normalized line scans in Fig. 4 D. (A and B) Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test; error bars represent the SD. (C) Bar graphs showing the percentage of embryos with a visible midbody (as assessed qualitatively) and a measurable peak in fluorescence intensity at the division site. Data correspond to embryos from Fig. 4, A–D. n is listed to the right side of bars in the indicated color for each genotype [GFP::TBB-2β-tubulin: n = 21 control, n = 13 hcp-1Δ; hcp-2(RNAi), n = 16 hcp-1Δ, n = 13 hcp-2Δ, n = 17 control raised at 16°C, n = 21 spd-1(oj5ts), and n = 13 zen-4(or153ts); AIR-2Aurora-B::GFP: n = 13 control, n = 13 hcp-1(RNAi); hcp-2Δ, n = 15 hcp-1Δ, n = 14 hcp-2Δ, n = 13 control raised at 16°C, n = 22 spd-1(oj5ts), and n = 14 zen-4(or153ts); SPD-1PRC1::sfGFP and TagRFP::ZEN-4MKLP1: n = 17 control(RNAi), n = 14 hcp-1/2(RNAi), n = 9 hcp-1(RNAi), n = 10 hcp-2(RNAi), and n = 14 spd-1(RNAi)]. (D) Representative time-lapse images of GFP::TBB-2β-tubulin-expressing (green, top; inverted grayscale, middle) and mCherry::H2B-expressing (magenta) embryos undergoing midbody assembly; see also Video 8. White arrows indicate bundled astral MTs at the furrow. Time is relative to midbody assembly. Bottom: Time-lapse pseudokymographs showing GFP::TBB-2β-tubulin during midbody assembly. Image series begins 150 s before midbody assembly. A maximum projection of three Z-sections is shown. Dashed orange box (middle) indicates area selected for pseudokymograph. White scale bar and vertical black scale bar, 10 µm. Horizontal black scale, 15 s. n.s., P ≥ 0.05; **, P < 0.01; and ****, P < 0.0001.](https://cdn.rupress.org/rup/content_public/journal/jcb/221/3/10.1083_jcb.202011085/4/jcb_202011085_figs7.png?Expires=1773623877&Signature=DLwxeBNskhlpIINNTDPpRuJjRMNpzC~TPpvMGAJkMDLUaTbcDufKE1isOvoMx1w36LNB3V2vIC16rPYN1dTw~ApWtrn~nkEa7XTHc97yll~5Jmpnf7ZR-JROn0IdS3wJSMKMCpgS~T1wL9rFweO6Afw5EMc1u2SqLADNz7JuN02Cab-ehBxQXY2DWc8v9qp3fJgOPQJjkqj4o9yCPRP1iJ7lWUt2oGeUI4xLJyWO8d5NQ2nVkfD4Bkz9KYQDNyqOLFuxefQfkA81i3GzS3jgHZZEgJO~NOCUPb~zClbPa6FIeN13HjHO7-jyR6aZLvyTqk-j6meYUfRtwfZLL2INjw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Midbody assembly can occur in the absence of a central spindle via astral MT bundling. (A) Quantification of peak levels of GFP::TBB-2β-tubulin at the midbody above intracellular background; related to Fig. 4 A and the normalized line scans in Fig. 4 D. (B) Quantification of peak levels of AIR-2Aurora-B::GFP at the midbody above intracellular background; related to Fig. 4 B and the normalized line scans in Fig. 4 D. (A and B) Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test; error bars represent the SD. (C) Bar graphs showing the percentage of embryos with a visible midbody (as assessed qualitatively) and a measurable peak in fluorescence intensity at the division site. Data correspond to embryos from Fig. 4, A–D. n is listed to the right side of bars in the indicated color for each genotype [GFP::TBB-2β-tubulin: n = 21 control, n = 13 hcp-1Δ; hcp-2(RNAi), n = 16 hcp-1Δ, n = 13 hcp-2Δ, n = 17 control raised at 16°C, n = 21 spd-1(oj5ts), and n = 13 zen-4(or153ts); AIR-2Aurora-B::GFP: n = 13 control, n = 13 hcp-1(RNAi); hcp-2Δ, n = 15 hcp-1Δ, n = 14 hcp-2Δ, n = 13 control raised at 16°C, n = 22 spd-1(oj5ts), and n = 14 zen-4(or153ts); SPD-1PRC1::sfGFP and TagRFP::ZEN-4MKLP1: n = 17 control(RNAi), n = 14 hcp-1/2(RNAi), n = 9 hcp-1(RNAi), n = 10 hcp-2(RNAi), and n = 14 spd-1(RNAi)]. (D) Representative time-lapse images of GFP::TBB-2β-tubulin-expressing (green, top; inverted grayscale, middle) and mCherry::H2B-expressing (magenta) embryos undergoing midbody assembly; see also Video 8. White arrows indicate bundled astral MTs at the furrow. Time is relative to midbody assembly. Bottom: Time-lapse pseudokymographs showing GFP::TBB-2β-tubulin during midbody assembly. Image series begins 150 s before midbody assembly. A maximum projection of three Z-sections is shown. Dashed orange box (middle) indicates area selected for pseudokymograph. White scale bar and vertical black scale bar, 10 µm. Horizontal black scale, 15 s. n.s., P ≥ 0.05; **, P < 0.01; and ****, P < 0.0001.
