Figure 9. Forces on the NCC during centration. (A) We hypothesize that GOA-1/GPA-16 and NMY-2 contribute to a force-generating mechanism that can promote NCC movement in opposite directions, with LET-99 favoring the anterior direction. (B) Based on the results of this and previous studies, we specu- late that two independent forces drive NCC movement: a G∝- and NMY-2–dependent force (depicted in A) that is oriented by LET-99 (red arrows) and an- other force independent of these factors (blue arrows). NCC velocity depends on the sum of the two forces. This model accounts for the effects of varying NMY-2 activity in a let-99(−) mutant background. (C) Cartoon of two proposed mechanisms driving centration, with arrows indicating force vectors; yellow arrows represent forces on centrosomes. Mechanism 1: ﬁxed MT-based motors (blue arrows) pull cortical sites toward MT minus ends. Mechanism 2: NMY-2 acts as a force generator (red arrows) by pulling MT attachment sites away from centrosomes in the cortical plane. In wild type, both mechanisms act in parallel. In Gα-depleted zygotes, only mechanism 1 acts. In let-99(−) zygotes, mechanism 1 acts normally, whereas the force vectors of mechanism 2 are spatially reversed. (Fig. 7), this result suggests that the directionally biased active transport of NMY-2 aggregates along the transverse axis de- pends on both GOA-1/GPA-16 and LET-99. Discussion We have shown that the Gα proteins GOA-1/GPA-16 and the myosin II NMY-2 act similarly to promote NCC centration and rotation in wild type and to oppose centration and provoke ex- cessive NCC rocking in let-99(−) mutants. In addition to NMY-2, the F-actin assembly factor PFN-1 and the Rho-binding kinase LET-502, which activates NMY-2 by phosphorylating its regula- tory light chain, are also required to prevent centration in the ab- sence of LET-99. Moreover, depletion of the inhibitory myosin II light chain phosphatase MEL-11 in a let-99(−) mutant promotes posterior movement of the NCC. Based on these findings, we propose that the Gα proteins, acting either through or in parallel to myosin regulatory light chain phosphorylation, activate an actomyosin-dependent force that promotes NCC movement and that the direction of this force is regulated by LET-99 (Fig. 9 A). Our findings also suggest that the actomyosin-dependent force acts in parallel with a separate, unidentified centration-promoting force that is independent of actomyosin, Gα, and LET-99. Finally, we have shown that elements of the actomyosin cortex undergo intrinsically polarized movement that could directly generate a pulling force on centrosomes, and we propose that Gα and LET-99 act by regulating this movement. Our findings challenge the view that actomyosin is not required for centrosome movement in the early C. elegans em- bryo (see Introduction). Analysis of direct roles of actomyosin in centrosome positioning in the C. elegans embryo has previ- ously been complicated by an earlier requirement for the polar- ization of cortical PAR (partitioning) proteins, which, in turn, are thought to act upstream of Gα and LET-99 to control centro- some movement (Colombo et al., 2003; Tsou et al., 2003). However, F-actin appears to play PAR-independent roles in the Gα-dependent processes of anaphase spindle pole flattening and NCC rotation (Severson and Bowerman, 2003). Our finding that NMY-2 promotes centration in par-3(it71) zygotes likewise indicates a direct role for actomyosin and further suggests that centration is independent of cortical polarity, as neither NMY-2 nor LET-99 is asymmetrically distributed in par-3(it71) zygotes at this stage (Tsou et al., 2002; Munro et al., 2004). In contrast, NCC rotation appears to depend on PAR-3 as well as on Gα, LET-99, and NMY-2 (Tsou et al., 2002). Two force-producing pathways promote centration in the C. elegans zygote Centration was slowed but never completely blocked by the depletion of GOA-1/GPA-16 or NMY-2 (see Materials and NUCLEAR MOVEMENT IN C. ELEGANS ZYGOTES • GOULDING ET AL. 1187