Figure 5.
Postmitotic localization of cortical LIN-5/GPR-1/2 is vital for midbody stability and accurate abscission. (A and B) 3D projected confocal images of one-cell embryo coexpressing LIN-5-mNG and ZEN-4-mScarlet during anaphase (A) and after furrow ingression (B). 25 images were acquired, and representative images are shown here; the scale bar is 10 µm. (C) Different z-sections (1 µm apart) of the selected region of LIN-5-mNG– and ZEN-4-mScarlet–coexpressing embryo, shown in B. Please note that LIN-5 localizes at the membrane surrounding the midbody (labeled with ZEN-4-mScarlet). A similar observation has been made for GPR-1/2 localization; see Fig. S7 B. The scale bar is 5 µm. (D) Schematic of a one-cell embryo highlighting the selected midbody region and the surrounding membrane. The midbody is composed of an antiparallel microtubule bundle (in asparagus)–, several midbody (in pink)–, and midbody membrane (in strawberry)–localized proteins, and its surrounding membrane is composed of LIN-5/GPR-1/2 complexes (in green). Also see Fig. S9 B. (E–H) Representative pseudokymographs of the division plane (depicted on the top right) acquired by time-lapse confocal microscopy of control embryos expressing CYK-4-mNG (E), CYK-4-mNG in spd-1(ts) background (F), CYK-4-mNG embryos treated with lin-5(RNAi) (G), or CYK-4-mNG in spd-1(ts) background that are depleted for LIN-5 (H). The pseudokymograph begins at −45 s w.r.t furrow involution, which is marked as 0 s. The furrow involution timing was determined using DIC microscopy (not shown). Please note that the CYK-4-mNG is absent at the spindle midzone in spd-1(ts) mutant embryos at furrow involution; however, it is regained later as the cell cycle progresses, as recently shown by Hirsch et al. (2022). In contrast, the spd-1(ts) mutant embryos, when treated with lin-5(RNAi), show significantly enriched localization of CYK-4-mNG at the spindle midzone during furrow involution, but CYK-4-mNG midzone localization is not maintained. The scale bar is 5 µm. (I–M) Schematic of the method used to quantify CYK-4-mNG fluorescence intensity at the spindle midzone during furrow involution (0 s) and 300 s after furrow involution (I). CYK-4-mNG fluorescence intensity at 0 and 300 s in control embryos (J), spd-1(ts) mutant embryos (K), lin-5(RNAi) embryos (L), and spd-1(ts) mutant embryos that are treated with lin-5(RNAi) (M). Please note that the midzone CYK-4-mNG fluorescence intensity is negligible in spd-1(ts) mutant embryos at 0 s but not at 300 s. In contrast, spd-1(ts) mutant embryos depleted for LIN-5 show robust CYK-4-mNG intensity at 0 s, but no enrichment is seen at 300 s. n is the number of embryos analyzed. The respective shaded color shows error bars in SEM. (N–Q) Representative pseudokymographs of the division plane (depleted on the top right) acquired by time-lapse confocal microscopy of GFPMem./Tub.;mCherryChr./Cent.-expressing untreated control embryos (N), in spd-1(ts) mutant background (O), treated with lin-5(RNAi) (P), or spd-1(ts) embryos that are also treated with lin-5(RNAi) (Q), as indicated. Only the GFPMem./Tub. signal is shown (in green) for easy comparative analysis of abscission failure. See the corresponding Videos 9, 10, 11, and 12. Control (n = 10); spd-1(ts) (n = 20); lin-5(RNAi) (n = 15); and lin-5(RNAi) in spd-1(ts) background (n = 17). Note that control, spd-1(ts), or lin-5(RNAi) does not show any abscission failure; however, spd-1(ts) embryos that are treated with lin-5(RNAi) show 41% (n = 7) abscission failure. n is the number of embryos analyzed. The pseudokymographs begin at 250 s after NEBD, as indicated. The scale bar is 5 µm. (R–U) Graphs represent furrow ingression kinetics in embryos coexpressing GFPMem./Tub.;mCherryChr./Cent. in control (R), spd-1(ts) mutant embryos (S), lin-5(RNAi) embryos (T), and spd-1(ts) mutant embryos that are treated with lin-5(RNAi) (U). Please note furrow ingression followed by regression in spd-1(ts) mutant embryos depleted for LIN-5. A similar observation was made for spd-1(ts) mutant embryos depleted for GPR-1/2 (see Fig. S8). n is the number of embryos analyzed. Time is w.r.t. NEBD.

Postmitotic localization of cortical LIN-5/GPR-1/2 is vital for midbody stability and accurate abscission. (A and B) 3D projected confocal images of one-cell embryo coexpressing LIN-5-mNG and ZEN-4-mScarlet during anaphase (A) and after furrow ingression (B). 25 images were acquired, and representative images are shown here; the scale bar is 10 µm. (C) Different z-sections (1 µm apart) of the selected region of LIN-5-mNG– and ZEN-4-mScarlet–coexpressing embryo, shown in B. Please note that LIN-5 localizes at the membrane surrounding the midbody (labeled with ZEN-4-mScarlet). A similar observation has been made for GPR-1/2 localization; see Fig. S7 B. The scale bar is 5 µm. (D) Schematic of a one-cell embryo highlighting the selected midbody region and the surrounding membrane. The midbody is composed of an antiparallel microtubule bundle (in asparagus)–, several midbody (in pink)–, and midbody membrane (in strawberry)–localized proteins, and its surrounding membrane is composed of LIN-5/GPR-1/2 complexes (in green). Also see Fig. S9 B. (E–H) Representative pseudokymographs of the division plane (depicted on the top right) acquired by time-lapse confocal microscopy of control embryos expressing CYK-4-mNG (E), CYK-4-mNG in spd-1(ts) background (F), CYK-4-mNG embryos treated with lin-5(RNAi) (G), or CYK-4-mNG in spd-1(ts) background that are depleted for LIN-5 (H). The pseudokymograph begins at −45 s w.r.t furrow involution, which is marked as 0 s. The furrow involution timing was determined using DIC microscopy (not shown). Please note that the CYK-4-mNG is absent at the spindle midzone in spd-1(ts) mutant embryos at furrow involution; however, it is regained later as the cell cycle progresses, as recently shown by Hirsch et al. (2022). In contrast, the spd-1(ts) mutant embryos, when treated with lin-5(RNAi), show significantly enriched localization of CYK-4-mNG at the spindle midzone during furrow involution, but CYK-4-mNG midzone localization is not maintained. The scale bar is 5 µm. (I–M) Schematic of the method used to quantify CYK-4-mNG fluorescence intensity at the spindle midzone during furrow involution (0 s) and 300 s after furrow involution (I). CYK-4-mNG fluorescence intensity at 0 and 300 s in control embryos (J), spd-1(ts) mutant embryos (K), lin-5(RNAi) embryos (L), and spd-1(ts) mutant embryos that are treated with lin-5(RNAi) (M). Please note that the midzone CYK-4-mNG fluorescence intensity is negligible in spd-1(ts) mutant embryos at 0 s but not at 300 s. In contrast, spd-1(ts) mutant embryos depleted for LIN-5 show robust CYK-4-mNG intensity at 0 s, but no enrichment is seen at 300 s. n is the number of embryos analyzed. The respective shaded color shows error bars in SEM. (N–Q) Representative pseudokymographs of the division plane (depleted on the top right) acquired by time-lapse confocal microscopy of GFPMem./Tub.;mCherryChr./Cent.-expressing untreated control embryos (N), in spd-1(ts) mutant background (O), treated with lin-5(RNAi) (P), or spd-1(ts) embryos that are also treated with lin-5(RNAi) (Q), as indicated. Only the GFPMem./Tub. signal is shown (in green) for easy comparative analysis of abscission failure. See the corresponding Videos 9, 10, 11, and 12. Control (n = 10); spd-1(ts) (n = 20); lin-5(RNAi) (n = 15); and lin-5(RNAi) in spd-1(ts) background (n = 17). Note that control, spd-1(ts), or lin-5(RNAi) does not show any abscission failure; however, spd-1(ts) embryos that are treated with lin-5(RNAi) show 41% (n = 7) abscission failure. n is the number of embryos analyzed. The pseudokymographs begin at 250 s after NEBD, as indicated. The scale bar is 5 µm. (R–U) Graphs represent furrow ingression kinetics in embryos coexpressing GFPMem./Tub.;mCherryChr./Cent. in control (R), spd-1(ts) mutant embryos (S), lin-5(RNAi) embryos (T), and spd-1(ts) mutant embryos that are treated with lin-5(RNAi) (U). Please note furrow ingression followed by regression in spd-1(ts) mutant embryos depleted for LIN-5. A similar observation was made for spd-1(ts) mutant embryos depleted for GPR-1/2 (see Fig. S8). n is the number of embryos analyzed. Time is w.r.t. NEBD.

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