Figure 7.
Polar relaxation by dynein-mediated removal of cortical myosin II induces furrow formation in parallel with equatorial stimulation. Synthetic effect of the simultaneous inhibition of dynein activity (gpr-1/2(RNAi)) and centralspindlin-mediated signaling (cyk-4(GAP)) was examined by observing embryos with these mutations in the following combinations: cyk-4(+) control(RNAi) (n = 12), cyk-4(+) gpr-1/2(RNAi) (n = 12), cyk-4(GAP) control(RNAi) (n = 13), and cyk-4(GAP) grp-1/2(RNAi) (n = 13). In contrast to cyk-4(RNAi), a point mutation in the GAP domain of CYK-4 (cyk-4(GAP)) specifically inactivates the equatorial stimulation via the centralspindlin-ECT2 pathway without affecting the spindle geometry. Since the cyk-4(GAP) allele is temperature-sensitive, embryos were imaged at 23°C. (A) Density and flow of myosin II in the cell cortex for the indicated strains and conditions were obtained in a similar manner to Fig. 5, E and F. (B) Temporal change of the density of myosin II in the posterior cortex within 10 µm from the posterior tip was plotted (mean across embryos ± SEM). (C) Cumulative activities of the centrosome-directed cytoplasmic transport of myosin II particles were plotted (mean ± SEM). Inhibition of dynein activity almost completely abolished the myosin transport irrespective of the genotype of cyk-4 (gpr-1/2(RNAi) or cyk-4(GAP) gpr-1/2(RNAi)). (D) Timing of the initial sign of furrow formation. The numbers on the top indicate the number of embryos that formed the cleavage furrow out of the total number of observed embryos. The defect in the centralspindlin-dependent equatorial stimulation did not severely affect the patterns of the myosin II distribution and the cortical flow (A, ii, cyk-4(GAP) vs. A, i, wild-type). In contrast, inactivation of the cortical dynein activity, which nearly completely eliminated the cytoplasmic transport of myosin II (shown in C), drastically changed the cortical distribution and flow of myosin II (A, iii, gpr-1/2(RNAi) vs. A, i, wild-type, and B gpr-1/2(RNAi)), although it still allowed a weaker bidirectional flow (A, iii) and furrow formation in the presence of wild-type centralspindlin (D, gpr-1/2). Inhibition of both the centralspindlin-dependent pathway and the dynein-dependent pathway almost completely abolished the bidirectional flow (A, iv, cyk-4(GAP) gpr-1/2(RNAi), and B, cyk-4 gpr-1/2) and severely affected the furrow formation (D, cyk-4 gpr-1/2; see also Fig. S5).

Polar relaxation by dynein-mediated removal of cortical myosin II induces furrow formation in parallel with equatorial stimulation. Synthetic effect of the simultaneous inhibition of dynein activity (gpr-1/2(RNAi)) and centralspindlin-mediated signaling (cyk-4(GAP)) was examined by observing embryos with these mutations in the following combinations: cyk-4(+) control(RNAi) (n = 12), cyk-4(+) gpr-1/2(RNAi) (n = 12), cyk-4(GAP) control(RNAi) (n = 13), and cyk-4(GAP) grp-1/2(RNAi) (n = 13). In contrast to cyk-4(RNAi), a point mutation in the GAP domain of CYK-4 (cyk-4(GAP)) specifically inactivates the equatorial stimulation via the centralspindlin-ECT2 pathway without affecting the spindle geometry. Since the cyk-4(GAP) allele is temperature-sensitive, embryos were imaged at 23°C. (A) Density and flow of myosin II in the cell cortex for the indicated strains and conditions were obtained in a similar manner to Fig. 5, E and F. (B) Temporal change of the density of myosin II in the posterior cortex within 10 µm from the posterior tip was plotted (mean across embryos ± SEM). (C) Cumulative activities of the centrosome-directed cytoplasmic transport of myosin II particles were plotted (mean ± SEM). Inhibition of dynein activity almost completely abolished the myosin transport irrespective of the genotype of cyk-4 (gpr-1/2(RNAi) or cyk-4(GAP) gpr-1/2(RNAi)). (D) Timing of the initial sign of furrow formation. The numbers on the top indicate the number of embryos that formed the cleavage furrow out of the total number of observed embryos. The defect in the centralspindlin-dependent equatorial stimulation did not severely affect the patterns of the myosin II distribution and the cortical flow (A, ii, cyk-4(GAP) vs. A, i, wild-type). In contrast, inactivation of the cortical dynein activity, which nearly completely eliminated the cytoplasmic transport of myosin II (shown in C), drastically changed the cortical distribution and flow of myosin II (A, iii, gpr-1/2(RNAi) vs. A, i, wild-type, and B gpr-1/2(RNAi)), although it still allowed a weaker bidirectional flow (A, iii) and furrow formation in the presence of wild-type centralspindlin (D, gpr-1/2). Inhibition of both the centralspindlin-dependent pathway and the dynein-dependent pathway almost completely abolished the bidirectional flow (A, iv, cyk-4(GAP) gpr-1/2(RNAi), and B, cyk-4 gpr-1/2) and severely affected the furrow formation (D, cyk-4 gpr-1/2; see also Fig. S5).

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