methods section C. elegans strains and maintenance). Consider- ing this result with prior evidence that centration can occur in- dependently of F-actin (Hill and Strome, 1988), we propose that wild-type centration results from the additive effects of two par- allel force-generating mechanisms, one of which is promoted by Gα/actomyosin and spatially oriented by LET-99. In this scenario, the near-zero net axial movement of the NCC ob- served in let-99(−) mutants can be explained as the result of a balanced conflict between two opposing forces that normally cooperate to drive centration (Fig. 9 B). If one or both of the opposing forces in let-99(−) varies with centrosome position and velocity, the conflict between them might drive excessive NCC rocking (Grill et al., 2005; Pecreaux et al., 2006; Kozlowski et al., 2007), and this rocking could be reduced by tipping the balance of force in either direction. We were indeed able to suppress NCC rocking by either reducing or increasing the posterior-directed actomyosin- dependent force in a let-99(−) mutant. Interestingly, simulation of wild-type anaphase centrosome oscillations (which occur with roughly the same period as the prophase oscillations in let-99(−) zygotes) shows that rocking can be reduced to zero by either increasing or decreasing a cortical pulling force (Pecreaux et al., 2006). Wild-type NMY-2 aggregate dynamics suggest that cortical actomyosin acts as a force generator Based on the behavior of wild-type NMY-2–GFP aggregates, we can distinguish between different models for the role of actomyosin in centration. Several current models of centrosome movement in the C. elegans embryo posit that force generators bind and effectively shorten astral MTs while anchored to fixed cortical sites (Fig. 9 C, mechanism 1; for review see Cowan and Hyman, 2004; Grill and Hyman, 2005; Kozlowski et al., 2007). Our finding that NMY-2–GFP aggregates anterior to the NCC move with an aster-independent anterior bias suggests instead that a pulling force may be generated by the intrinsic motility of MT attachment sites within the cortical plane (Fig. 9 C, mechanism 2). Such actomyosin-based motility of cortical sites indeed appears to move asters in yeast (Hwang et al., 2003) and mammalian cells (Rosenblatt et al., 2004). However, we measured peak centro- some velocities during centration that far exceed the highest rates of NMY-2– GFP aggregate movement. We speculate that both types of pulling mechanisms (attachment site motility and tethered MT shortening) may operate in tandem so that the re- sulting forces are coupled. Alternatively, rapid MT binding and release by NMY-2 aggregates could ratchet them toward MT minus ends, and many such small pulling movements could gen- erate a large summed force on the NCC. Remarkably, an anterior bias in aggregate displacement was not found in the most anterior cortical regions but only in regions a short distance anterior to the NCC. Any pulling force exerted on the NCC by these biased movements would there- fore be predicted to diminish toward the end of centration. This prediction is consistent with the sinusoidal NCC displacement profile observed in wild type (Kimura and Onami, 2005), cor- responding to the bell-shaped velocity profile shown in Fig. 1. As the velocity increase during early centration depends on NMY-2, it is plausible that the local regulation of polarized cor- tical contraction acts to generate a position-dependent pulling force on centrosomes. Roles of GOA-1/GPA-16 and LET-99 in cortical NMY-2 aggregate motility Given the similar requirements of NMY-2 and GOA-1/GPA-16 for promoting centration in wild-type zygotes and opposing it in let-99(−) mutants, we hypothesized that Gα signaling influ- ences actomyosin contractility to promote centration in wild- type zygotes. Indeed, the loss of anteriorly biased NMY-2–GFP aggregate movement in cortical regions anterior to the NCC is consistent with a role of Gα signaling in directing biased actomyosin contraction. Gα is not simply required for NMY-2 aggregate movement, however, as directed movement was evi- dent in octants near and posterior to the NCC in Gα-depleted zygotes. An alternative mechanism of Gα action is suggested by the bidirectional bias of large AP displacements toward the NCC in goa-1(−);gpa-16(−) zygotes. We speculate that Gα signaling might induce the relaxation of actomyosin in the vicinity of asters, allowing long-range contractile flow toward distal cortical regions. Supporting this model, GOA-1/GPA-16 and their positive regulator GPR-1/2 are also required for the spindle poles to transmit a cytokinesis-promoting signal during anaphase, and GPR-1/2 is enriched at this stage in the polar cor- tex, where relaxation is thought to occur (Bringmann et al., 2007). Other Gα proteins are known to affect actomyosin as- sembly and contraction through regulation of the small G pro- teins Rho and Rac (Sugimoto et al., 2003; Rogers et al., 2004), raising the possibility that GOA-1/GPA-16 may influence aster- associated actomyosin through the local regulation of one of these pathways. Because LET-99 has been shown to negatively regulate Gα-dependent forces that promote NCC rocking (Tsou et al., 2003), we expected LET-99 inactivation to also perturb NMY-2– GFP aggregate dynamics, but in a manner distinct from the effect of GOA-1/GPA-16 depletion. Indeed, NMY-2–GFP ag- gregates in let-99(−) mutant zygotes exhibited a characteristic pattern of abnormal movements throughout a large region of the cortex. In contrast to the anterior direction of large displace- ments near the forward-moving centrosome in wild type, the largest displacements near and anterior to the NCC in let-99(−) zygotes were directed toward the posterior. Thus, LET-99 ap- pears to reverse the net directional force acting on a fast-moving subpopulation of cortical NMY-2–GFP aggregates near the NCC. If these aggregates bind to astral MTs, their behavior in the let-99(−) mutant could explain the reversal of the actomyosin- dependent force that promotes centration in wild-type zygotes. Interestingly, posteriorly biased large displacements were simi- larly observed in let-99(−) zygotes near the NCC, where active transport predominated, and anteriorly adjacent to this region, where constraining forces predominated. This relationship sug- gests that NMY-2–GFP aggregates in these two regions are mechanically linked and that polarized actomyosin contraction in the posterior cortex of let-99(−) zygotes exerts a pulling force on the anterior cortex. 1188 JCB • VOLUME 178 • NUMBER 7 • 2007