![Functional midbody assembly is independent of central spindle assembly.(A) Representative images of GFP::TBB-2β-tubulin (green, top; inverted contrast, bottom) and mCherry:H2B (magenta, top) expressing embryos at 240 s after chromosome segregation [210 s in spd-1(oj5ts) embryos]. (B) Representative images of AIR-2::GFP-expressing (green, top; inverted contrast, bottom) and mCherry::H2B-expressing (magenta, top) embryos at 240 s after chromosome segregation [210 s in spd-1(oj5ts) embryos]. (C) Representative images of SPD-1::sfGFP-expressing (green, top; inverted contrast, bottom), TagRFP::ZEN-4–expressing, and mCherry::H2B-expressing (magenta, top; inverted contrast, bottom) embryos at 240 s after chromosome segregation (all genotypes). Schematic depicting Z-sectioning used for midbody imaging in A–C (right). (D) Line scan analysis of fluorescent protein–tagged midbody protein accumulation for cells with a visible midbody [except for zen-4(or153ts), for which all embryos were included; see Fig. S7 C] was done as for CYK-4::mNG (240 s; Fig. 3 D). n is listed on right side of graphs in the indicated color for each genotype [GFP::TBB-2β-tubulin: n = 21 control, n = 12 hcp-1Δ; hcp-2(RNAi), n = 15 hcp-1Δ, n = 13 hcp-2Δ, n = 17 control raised at 16°C, n = 13 spd-1(oj5ts), and n = 13 zen-4(or153ts); AIR-2::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 = 16 spd-1(oj5ts), and n = 14 zen-4(or153ts); SPD-1::sfGFP and TagRFP::ZEN-4: n = 17 control(RNAi), n = 14 hcp-1/2(RNAi), n = 9 hcp-1(RNAi), n = 10 hcp-2(RNAi), and n = 9 spd-1(RNAi)]. SPD-1::sfGFP quantification is shown for all embryos with visible midbodies by TagRFP::ZEN-4; error bars represent the SEM. (E) Representative central plane images of CYK-4::mNG-expressing (green), mCherry::H2B-expressing (magenta), and mCherry::PHPLC1δ1-expressing (magenta) embryos before (top) and after (bottom) midbody release from the plasma membrane (white arrows) after abscission. Black box behind spd-1(RNAi) images is for presentation purposes. (F) Quantification of abscission timing, determined by release of the CYK-4::mNG-labeled midbody from the plasma membrane [n = 19 control(RNAi), n = 19 hcp-1/2(RNAi), and n = 9 spd-1(RNAi)]. Time relative to anaphase onset (AO) of the second (AB) cell division. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test; error bars represent the SD; scale bars, 10 µm. n.s., P ≥ 0.05.](https://cdn.rupress.org/rup/content_public/journal/jcb/221/3/10.1083_jcb.202011085/4/jcb_202011085_fig4.png?Expires=1773605703&Signature=AIQVb0AZVwmVX9aEaTMiRqGbILiF0NXp-cDGytgSjN0v4pNUKm9e95FKQmJ7saLZwSH2RUZCz7tT7xS4ZSyJ-2ey4uHrvNg-U1OnJIgoMDV4T4TGVlCuemeHxO-6kxBv1OEsycAZRxC37-cZX6pxtBWlYwcv5vZmABBKzjPE-9GxZ4yw-1mGfxr0jvnkQR1ArJ-YOUzud-J8Gu4rGIswEFBxMBAWpBmXNjCZkxFo54n5I7HpboK5P4iMGRiEJNpbYKo-T3q~0Uw8fXmF9ZFLqt~UlPxg00R8oBMLI8ghWatr9IPlbkH9bd6eRR53Ou~F3sRr-4orfeP21pjhmPM5TQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Functional midbody assembly is independent of central spindle assembly. (A) Representative images of GFP::TBB-2β-tubulin (green, top; inverted contrast, bottom) and mCherry:H2B (magenta, top) expressing embryos at 240 s after chromosome segregation [210 s in spd-1(oj5ts) embryos]. (B) Representative images of AIR-2::GFP-expressing (green, top; inverted contrast, bottom) and mCherry::H2B-expressing (magenta, top) embryos at 240 s after chromosome segregation [210 s in spd-1(oj5ts) embryos]. (C) Representative images of SPD-1::sfGFP-expressing (green, top; inverted contrast, bottom), TagRFP::ZEN-4–expressing, and mCherry::H2B-expressing (magenta, top; inverted contrast, bottom) embryos at 240 s after chromosome segregation (all genotypes). Schematic depicting Z-sectioning used for midbody imaging in A–C (right). (D) Line scan analysis of fluorescent protein–tagged midbody protein accumulation for cells with a visible midbody [except for zen-4(or153ts), for which all embryos were included; see Fig. S7 C] was done as for CYK-4::mNG (240 s; Fig. 3 D). n is listed on right side of graphs in the indicated color for each genotype [GFP::TBB-2β-tubulin: n = 21 control, n = 12 hcp-1Δ; hcp-2(RNAi), n = 15 hcp-1Δ, n = 13 hcp-2Δ, n = 17 control raised at 16°C, n = 13 spd-1(oj5ts), and n = 13 zen-4(or153ts); AIR-2::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 = 16 spd-1(oj5ts), and n = 14 zen-4(or153ts); SPD-1::sfGFP and TagRFP::ZEN-4: n = 17 control(RNAi), n = 14 hcp-1/2(RNAi), n = 9 hcp-1(RNAi), n = 10 hcp-2(RNAi), and n = 9 spd-1(RNAi)]. SPD-1::sfGFP quantification is shown for all embryos with visible midbodies by TagRFP::ZEN-4; error bars represent the SEM. (E) Representative central plane images of CYK-4::mNG-expressing (green), mCherry::H2B-expressing (magenta), and mCherry::PHPLC1δ1-expressing (magenta) embryos before (top) and after (bottom) midbody release from the plasma membrane (white arrows) after abscission. Black box behind spd-1(RNAi) images is for presentation purposes. (F) Quantification of abscission timing, determined by release of the CYK-4::mNG-labeled midbody from the plasma membrane [n = 19 control(RNAi), n = 19 hcp-1/2(RNAi), and n = 9 spd-1(RNAi)]. Time relative to anaphase onset (AO) of the second (AB) cell division. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test; error bars represent the SD; scale bars, 10 µm. n.s., P ≥ 0.05.
