Figure S7.
Cortical accumulation of LIN-5 after furrow ingression is essential for the maintenance of midbody-localized proteins. (A and B) 3D projected confocal images of a one-cell embryo expressing GPR-1 tagged with YFP (YFP-GPR-1) during anaphase (A) and after furrow ingression (B). More than 13 recordings were made, and the representative images are shown here. Please note the robust localization of the YFP-GPR-1 signal at the juxtaposed membrane after furrow ingression. The scale bar is 10 µm. (C–F) Representative midplane confocal images of embryos coexpressing mNG-ECT-2 (in green) and ZEN-4-mScarlet (in red) after furrow ingression (C). Similarly, confocal microscopy images of embryos expressing CYK-4-mNG (D), GFP-NMY-2 (E), and mNG-ANI-1 (F) after furrow ingression. Insets of the corresponding images at the midbody region are also shown. More than eight embryos were imaged, and the representative embryos are shown in the figure panel. The scale bar is 10 and 5 μm for the representative embryo images and corresponding insets, respectively. (G and H) Representative time-lapse images of GFPTub.-expressing (inverted grayscale) embryos revealing bundling of astral microtubules (black arrow) in control (G) and in spd-1(ts) mutant embryos that are depleted for LIN-5 with failed abscission (H). The representative images are from −25 s w.r.t furrow involution, which is 0 s. Black dotted insets represent regions tracked for pseudokymograph analysis in I and J (see below). The scale bar is 10 µm. (I and J) Pseudokymograph analysis of astral microtubule bundling in control (I) and in spd-1(ts) mutant embryos that are treated with lin-5(RNAi) with failed abscission (J). Black arrows indicate astral microtubule bundling. Maximum projection of three z-sections is shown for lin-5(RNAi); spd-1(ts). Scale bar, 10 µm. (K and L) Representative pseudokymographs of the division plane (depicted on the top right) acquired by time-lapse confocal microscopy of control embryos expressing mNG-ANI-1 in spd-1(ts) embryos (K), or mNG-ANI-1 in spd-1(ts) embryos treated with lin-5(RNAi) (L). The pseudokymograph begins at −45 s w.r.t furrow involution, which is 0 s. The time interval in between each frame is 15 s. Please note that the mNG-ANI-1 is significantly enriched at the midbody after furrow ingression; in contrast, mNG-ANI-1 intensity spreads along the membrane in spd-1(ts) embryos treated with lin-5(RNAi) embryos. The scale bar is 5 µm. (M and N) Schematic of the method used to quantify mNG-ANI-1 fluorescence intensity at the midbody during 300 s after furrow involution. The red line drawn on the midbody represents the one-pixel-wide line scan of 10 μm (M). mNG-ANI-1 fluorescence intensity at 300 s in spd-1(ts) mutant embryos (black line), in spd-1(ts) mutant embryos that are treated with lin-5(RNAi) (pink line) (N). n is the number of embryos analyzed. Error bars shown by the shaded area are the SEM.

Cortical accumulation of LIN-5 after furrow ingression is essential for the maintenance of midbody-localized proteins. (A and B) 3D projected confocal images of a one-cell embryo expressing GPR-1 tagged with YFP (YFP-GPR-1) during anaphase (A) and after furrow ingression (B). More than 13 recordings were made, and the representative images are shown here. Please note the robust localization of the YFP-GPR-1 signal at the juxtaposed membrane after furrow ingression. The scale bar is 10 µm. (C–F) Representative midplane confocal images of embryos coexpressing mNG-ECT-2 (in green) and ZEN-4-mScarlet (in red) after furrow ingression (C). Similarly, confocal microscopy images of embryos expressing CYK-4-mNG (D), GFP-NMY-2 (E), and mNG-ANI-1 (F) after furrow ingression. Insets of the corresponding images at the midbody region are also shown. More than eight embryos were imaged, and the representative embryos are shown in the figure panel. The scale bar is 10 and 5 μm for the representative embryo images and corresponding insets, respectively. (G and H) Representative time-lapse images of GFPTub.-expressing (inverted grayscale) embryos revealing bundling of astral microtubules (black arrow) in control (G) and in spd-1(ts) mutant embryos that are depleted for LIN-5 with failed abscission (H). The representative images are from −25 s w.r.t furrow involution, which is 0 s. Black dotted insets represent regions tracked for pseudokymograph analysis in I and J (see below). The scale bar is 10 µm. (I and J) Pseudokymograph analysis of astral microtubule bundling in control (I) and in spd-1(ts) mutant embryos that are treated with lin-5(RNAi) with failed abscission (J). Black arrows indicate astral microtubule bundling. Maximum projection of three z-sections is shown for lin-5(RNAi); spd-1(ts). Scale bar, 10 µm. (K and L) Representative pseudokymographs of the division plane (depicted on the top right) acquired by time-lapse confocal microscopy of control embryos expressing mNG-ANI-1 in spd-1(ts) embryos (K), or mNG-ANI-1 in spd-1(ts) embryos treated with lin-5(RNAi) (L). The pseudokymograph begins at −45 s w.r.t furrow involution, which is 0 s. The time interval in between each frame is 15 s. Please note that the mNG-ANI-1 is significantly enriched at the midbody after furrow ingression; in contrast, mNG-ANI-1 intensity spreads along the membrane in spd-1(ts) embryos treated with lin-5(RNAi) embryos. The scale bar is 5 µm. (M and N) Schematic of the method used to quantify mNG-ANI-1 fluorescence intensity at the midbody during 300 s after furrow involution. The red line drawn on the midbody represents the one-pixel-wide line scan of 10 μm (M). mNG-ANI-1 fluorescence intensity at 300 s in spd-1(ts) mutant embryos (black line), in spd-1(ts) mutant embryos that are treated with lin-5(RNAi) (pink line) (N). n is the number of embryos analyzed. Error bars shown by the shaded area are the SEM.

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