Figure 2. Centration in let-99(−) mutant zygotes is opposed by actomyosin. (A–F) Still frames (top two rows) show pronuclear meeting and NEB in wild- type and mutant zygotes. Below, kymographs show pronuclear meeting and centration or centration defects in the same zygotes. At the bottom, data from multiple zygotes of each genotype are summarized. Kymographs represent the projection over time of a line of pixels through the midline of the cell (shown in wild-type still frames) and are aligned by the time of NEB. Centration is shown for wild-type (A), is defective in let-99(or513ts) mutant zygotes (B), is res- cued in the let-99(or513ts) mutant background by depletion of either NMY-2 (C), the Rho-binding kinase LET-502 (D), or the proﬁlin PFN-1 (E), and is reversed by depletion of the inhibitory myosin phosphatase MEL-11 (F). In diagrams at the bottom, line segments show net axial displacement of the NCC from pronuclear meeting (ﬁlled ovals) to NEB (arrows) in individual zygotes. Bar, 10 μm. depletion of MEL-11 also reduced NCC rocking by more than threefold (Fig. 3), suggesting that the amount of rocking is not a simple function of NMY-2 activity. To summarize, we conclude that although NMY-2 nor- mally acts to promote centration, loss of LET-99 reverses the mechanical output of NMY-2 activity to oppose centration. To account for the suppression of NCC rocking by either the deple- tion or overactivation of NMY-2, we speculate that the mis- directed actomyosin-dependent force in let-99(−) mutants is counteracted by an independently generated centration-promoting force and that a balance of roughly equal but oppositely acting forces promotes excess rocking (see Discussion). Polarized distribution of cortical NMY-2 aggregates during wild-type centration Seeking to understand how NMY-2 contributes to NCC move- ment, we used spinning disc confocal microscopy to image cor- tical NMY-2–GFP in transgenic zygotes during centration. In agreement with previous studies (Munro et al., 2004; Motegi and Sugimoto, 2006), we found the onset of centration to co- incide with a sudden reorganization of cortical NMY-2. At the time of pronuclear meeting in wild type, an anteriorly enriched cortical network of relatively large actomyosin bundles is re- placed by a more finely reticular F-actin network in which NMY-2 is focused in small, isolated aggregates. Both F-actin and NMY-2 localize most densely to a cap covering the anterior half of the zygote, and a separate, less dense cap of actomyosin also is observed over the posterior pole. The lowest density of NMY-2 aggregates is consistently found in an intermediate zone (50–75% EL) that encircles the position of the NCC at the be- ginning of centration. Interestingly, the highest NMY-2 density is always found at the boundary between this intermediate zone and the anterior cap; until a late stage of centration, this myosin- dense ring is several micrometers anterior to the advancing NCC (Fig. 4, Fig. S1, and Video 7, available at http://www.jcb .org/cgi/content/full/jcb.200703159/DC1). Although aggregates appeared to be highly mobile, the overall NMY-2 distribution pattern persisted past the end of centration. Thus, cortical acto- myosin is well positioned to play a role in generating forces that pull centrosomes strongly toward the anterior and weakly toward the posterior (Labbe et al., 2004). Although the relative enrichment of NMY-2 in the anterior cap at NEB was slightly reduced in Ga-depleted and let-99(−) mutant zygotes (Fig. S1), the significance of this is unclear. Our finding that centration occurs at a near wild-type rate in par-3(it71) zygotes, in which NMY-2 aggregates are distributed uniformly throughout the cortex (Munro et al., 2004), suggests that a polarized distri- bution is unnecessary for the action of NMY-2 in centration. Therefore, rather than focus on the overall distribution of NMY-2, we examined the behavior of individual NMY-2 aggregates for clues to actomyosin’s mechanical role. Wild-type NMY-2 aggregates undergo anteriorly biased movement If cortical actomyosin is directly involved in generating a pulling force on centrosomes, the mechanism of force generation should NUCLEAR MOVEMENT IN C. ELEGANS ZYGOTES • GOULDING ET AL. 1181