with LET-99 regulating a redundant pair of Gα subunits called GOA-1 and GPA-16 (Tsou et al., 2003). These two Gα pro- teins promote forces that pull on spindle pole centrosomes during anaphase in the C. elegans embryo (Miller and Rand, 2000; Gotta and Ahringer, 2001; Colombo et al., 2003), as in other systems (for review see Hampoelz and Knoblich, 2004), but their importance for centrosome movement at other stages is unclear. GOA-1/GPA-16 depletion in let-99(−) mutant zy- gotes eliminates NCC rocking, indicating that during wild- type centration, LET-99 restricts excessive force stimulation by Gα signaling (Tsou et al., 2003). Strikingly, GOA-1/GPA-16 depletion also rescues the centration defect in let-99(−) mu- tants, indicating that in the absence of LET-99, inappropriate Gα signaling promotes a net force on the NCC directed away from the cell center toward the posterior pole. However, GOA-1 and GPA-16 have not been shown to influence centration in wild-type zygotes. In this study, we address two related questions about the control of NCC centration and rotation: the molecular basis of the driving force and the spatial regulation of this force. We show that Gα function is required for wild-type rates of centra- tion and rotation and that the nonmuscle myosin II NMY-2 is required to a similar degree for both processes. Like Gα, acto- myosin opposes centration in let-99(−) mutants, suggesting that Gα and actomyosin act together to generate a force on the NCC whose direction is determined by LET-99. Finally, we show that cortical NMY-2–GFP aggregates anterior to the NCC move with an anterior bias in wild-type zygotes; this bias is strongly reduced in Gα-depleted zygotes and is reversed in let-99(−) mutant zygotes, supporting our conclusion that polar- ized actomyosin contraction, which is promoted by Gα and spa- tially directed by LET-99, generates part of the force driving centration and rotation. Results The wild-type rate of centration depends on G function and myosin II Centration, the movement of the NCC to the cell center, oc- curs during mitotic prophase in wild-type one-cell C. elegans zygotes (Video 1, available at http://www.jcb.org/cgi/content/ full/jcb.200703159/DC1). To determine whether the redundantly acting Gα proteins GOA-1 and GPA-16 contribute to centration, we used RNAi to deplete both simultaneously and measured the rate of NCC movement along the AP axis during centration (see Materials and methods section DIC video microscopy and analysis…). Although not absolutely required for centration, our measurements showed that Gα activity is required for the wild-type rate of centration (Fig. 1 B and Table I). In wild-type zygotes, the speed of the NCC (vAP, plotted as incremental velocity in Fig. 1 B, with peak and mean velocity values in Table I) increased during the first half of centration to a peak value of 7.9 μm/min and a mean value of 6 μm/min followed by a gradual decrease to zero. In contrast to wild-type zygotes, vAP in Gα-depleted zygotes remained relatively constant during centration (Fig. 1 B), and the peak values of vAP were re- duced relative to wild type by a factor of approximately two (4.2 μm/min [t test; P < 0.00001] compared with 7.9 μm/min in wild type; Table I). The time-averaged value of vAP was also reduced to 2.0 μm/min compared with 2.9 μm/min in wild type (P < 0.0001). These results suggest that GOA-1 and GPA-16 contribute to an anterior-directed net force on the NCC during centration. The lack of an absolute requirement could reflect a failure to fully deplete these proteins using RNAi or the pres- ence of multiple additive force-generating mechanisms. Further depletion of GOA-1/GPA-16 results in sterility (see Materials and methods section C. elegans strains and maintenance), com- plicating our ability to distinguish between these two explana- tions (see Discussion). The molecular basis of force production downstream of Gα signaling during mitotic spindle positioning is unknown. We tested the role of NMY-2, a nonmuscle myosin II required for zygote polarity and cytokinesis (Guo and Kemphues, 1996; Cuenca et al., 2003). A requirement for NMY-2 in centration has not been tested previously because the pronuclei meet near the cell center in most nmy-2(RNAi) zygotes (Shelton et al., 1999). To assess the effect of NMY-2 depletion on the cen- tration velocity profile, we examined nmy-2(RNAi) zygotes in which fertilization occurred at a site near the oocyte meiotic spindle rather than the typical fertilization site opposite the meiotic spindle (see Materials and methods section C. elegans strains and maintenance). In such cases of reversed fertilization, both pronuclei form near one pole and then move together to the cell center (Albertson, 1984; Goldstein and Hird, 1996). In comparison with NMY-2(+) zygotes with reversed fertilization, NMY-2–depleted zygotes with reversed fertilization showed substantial reductions in both peak and time-averaged values of vAP (4.8 μm/min and 1.3 μm/min, respectively [P < 0.0001 for both], compared with 10.0 μm/min and 2.6 μm/min in NMY-2(+); Table I). As observed in GOA-1/GPA-16–depleted zygotes, vAP remained at a constantly low level throughout cen- tration in NMY-2–depleted zygotes (Fig. 1 C), and the highest values of vAP were similar to those measured in GOA-1/GPA-16– depleted zygotes (P = 0.08; Table I). Still lower values of vAP were measured in NMY-2–depleted zygotes after normal fer- tilization, in which the NCC moved only a short distance after pronuclei met near the cell center (Table I). The reduced NCC velocity in nmy-2(RNAi) zygotes could result indirectly from previously documented defects in cell polarity that occur as a result of NMY-2 depletion (Guo and Kemphues, 1996; Shelton et al., 1999). To address this possi- bility, we examined centration in par-3(it71) zygotes, which exhibit extensive polarity defects (Kemphues et al., 1988; Cheng et al., 1995; Etemad-Moghadam and Kemphues et al., 1995; Munro et al., 2004). In par-3(it71) mutant zygotes with re- versed fertilization (Table I and Fig. 1 C), time-averaged (2.5 μm/min) and peak NCC velocity measurements (6.8 μm/min) were intermediate between wild-type and NMY-2–depleted zygotes (P < 0.0001 for both comparisons; Table I). We also examined NCC movement in normally fertilized par-3(it71) zygotes, in which pronuclei met near the center of the zygote, at 58% egg length (EL; compared with 66% EL in wild type). The NCC moved toward the anterior at speeds similar to those observed after reversed fertilization (Table I), invariably moving 1178 JCB • VOLUME 178 • NUMBER 7 • 2007