Table I. Peak and time-averaged NCC velocities along the AP axis Genotype/RNAi Wild-type N2 goa-1(−);gpa-16(−) fer-1(−) nmy-2(−) par-3(−) nmy-2(−) par-3(−) par-3(−);nmy-2(−) Fertilization site (number of zygotes) Normal (10) Normal (8) Reversed (3) Reversed (3) Reversed (3) Normal (6) Normal (8) Normal (4) v (peak)a μm/min 7.9 4.2 10.0 4.8 6.8 3.8 6.2 2.5 SD 1.8 0.9 2.1 0.9 1.4 0.9 1.3 0.8 n 30 24 9 9 9 18 24 12 v (mean)b μm/min 2.9 2.0 2.6 1.3 2.1 0.5 1.5 0.6 SD 3.2 1.8 3.5 1.7 2.5 2.0 2.8 1.2 n 160 156 93 147 119 199 165 48 The NCC velocities (v) are expressed as absolute rates of NCC displacement toward the anterior pole (micrometers/minute). av(peak) refers to the mean value of peak NCC velocities between pronuclear meeting and NEB among all zygotes for a given genotype or RNAi treatment (see Materials and methods). The NCC was moving toward the anterior pole for all velocity measurements included in peak values. The number of measurements used to calculate v(peak) is given (n; three measurements/zygote). bv(mean) refers to the mean (time averaged) value of all NCC velocity measurements between pronuclear meeting and NEB for a given genotype or RNAi treatment. The number of measurements used to calculate v(mean) is given (n). Both positive (toward the posterior) and negative (toward the anterior) velocity measurements were included in these calculations. both; n = 13 and n = 9, respectively). We conclude that the same actomyosin- and Gα-dependent forces that promote cen- tration also contribute to NCC rotation. Actomyosin opposes centration in let-99(−) mutant zygotes To further investigate the relationship between NMY-2, GOA-1/ GPA-16, and NCC movement, we altered actomyosin function in let-99(−) mutants, in which an abnormal GOA-1/GPA-16– dependent force prevents centration and causes excess NCC rock- ing (Tsou et al., 2003). Worms homozygous for a temperature- sensitive allele of let-99 that we isolated and named or513ts (see Materials and methods section C. elegans strains and main- tenance) produced zygotes (hereafter referred to as mutant zygotes) that exhibited an absence of centration and exces- sive NCC rocking at the restrictive temperature of 26°C (Fig. 2, A and B; and Video 2, available at http://www.jcb.org/cgi/ content/full/jcb.200703159/DC1). Strikingly, RNAi-mediated depletion of NMY-2 in the let-99(or513ts) mutant rescued centration (Fig. 2, A–C) and reduced NCC rocking by more than fivefold (Fig. 3 and Video 3). In most NMY-2–depleted let-99(or513ts) zygotes (n = 7), the pronuclei met posteriorly at a mean position of 63% EL, which is similar to wild-type (66% EL; n = 10) and let-99(or513ts) single mutants (67% EL; n = 8). NCC position at the time of NEB in the NMY-2– depleted let-99(or513ts) zygotes was significantly further an- terior than in let-99(or513ts) single mutants (51% vs. 63% EL; P < 0.0001) and was not significantly different from wild type (48% EL; P = 0.06). NMY-2 depletion also rescued centration and reduced NCC rocking in another let99(−) mutant, or204ts (un- published data). We conclude that NMY-2, like GOA-1/GPA-16, is required for the abnormal forces that prevent centration and promote NCC rocking in let-99(−) mutant zygotes. To further assess the role of myosin II activity in the let-99(−) phenotype, we inactivated the Rho-binding kinase called LET-502, which is required for wild-type levels of NMY-2 activity during cytokinesis and morphogenesis (Piekny et al., 2000; Piekny and Mains, 2002). In embryos from homozygous let-502(sb106ts);let-99(or513ts) double mutant worms (see Materials and methods section C. elegans strains and mainte- nance), centration was again rescued (Fig. 2 D), and NCC rock- ing was strongly reduced (Video 4, available at http://www.jcb.org/ cgi/content/full/jcb.200703159/DC1; and unpublished data). Thus, Rho-binding kinase, presumably acting through NMY-2, is re- quired to prevent centration and generate NCC rocking in the ab- sence of LET-99. We also examined let-99(or513ts) zygotes after the depletion of profilin (PFN-1), an F-actin assembly factor that is required for the accumulation of cortical F-actin in the zygote (Severson et al., 2002). In PFN-1–depleted let-99(−) mutant zygotes, the position of pronuclear meeting was highly variable, but, in each case, centration was rescued (Fig. 2 E). The rescue of centration was again accompanied by a marked reduction of NCC rocking compared with the let-99(or513ts) single mutant (Video 5 and unpublished data). We also used RNAi to simultaneously deplete two components of the Arp2/3 complex, another F-actin assembly factor required for cortical stability in the early embryo and for gastrulation but not for the myosin II–dependent processes of cell polarization and cytokinesis (Severson et al., 2002). Deple- tion of Arp2/3 in let-99(−) zygotes destabilized the cell cortex but did not restore centration (unpublished data), suggesting that the abnormal forces opposing centration in let-99(−) mutant zygotes are mediated specifically by profilin-dependent F-actin in association with NMY-2 but are not affected by a different perturbation of the F-actin cytoskeleton. We conclude that in the absence of LET-99 function, actomyosin-dependent contractile forces oppose centration and contribute to excessive NCC rocking. Because myosin II activity opposes centration in let-99(−) mutants, we reasoned that overactivating NMY-2 might drive the NCC further toward the posterior pole. To test this pre- diction, we used RNAi to deplete MEL-11, the C. elegans ortho- logue of MYPT (myosin phosphatase-targeting subunit), a protein phosphatase that inhibits NMY-2 activity (Piekny et al., 2000). Strikingly, MEL-11 depletion in let-99(or513ts) mutants caused the NCC to move toward the posterior in seven out of eight cases (Fig. 2 F and Video 6, available at http://www.jcb .org/cgi/content/full/jcb.200703159/DC1). To our surprise, the 1180 JCB • VOLUME 178 • NUMBER 7 • 2007