Figure 3. Effects of NMY-2 inactivation and hyperactivation on centration and NCC rocking in let-99(−) mutant zygotes. (A) NCC rocking in let-99(or513ts) mutant zygotes is reduced by the depletion of either NMY-2 or the inhibitory myosin phosphatase MEL-11. The angle of the centrosomal axis relative to the AP axis is plotted over time during a 1-min interval before NEB for individual wild-type and let-99(or513ts) zygotes and for let-99(or513ts) mutant zygotes depleted of NMY-2 or MEL-11 (in which NCC rocking was partially suppressed). The variation in the angle over time in let-99(or513ts) zygotes depicts the rocking of the centrosomal axis. This angle varied among individual NMY-2–depleted and MEL-11–depleted let-99(−) zygotes but did not change substantially over time in any one zygote (not depicted). (B) NCC position along the AP axis at NEB plotted against total angular displacement of the centrosomal axis during a 1-min interval preceding NEB in wild-type (n = 9), let-99(or513ts) (n = 5), and let-99(or513ts) mutant zygotes depleted of NMY-2 (n = 6) or MEL-11 (n = 6); error bars show one SD. Total angular displacement refers to the sum of absolute angular displacements in either direction during the 1-min interval. be reflected by the movements of NMY-2 aggregates. For example, pulling force might be entirely generated by an MT-based mo- tor such as dynein, with actomyosin simply providing cortical anchorage for the motor. In this case, every component of the cortical attachment site, including NMY-2, should tend to move toward the centrosomes if at all. Alternatively (or additionally), NMY-2 itself could itself act as a force generator by pulling the actomyosin cortex and attached MT plus ends away from centro- somes. This model predicts that NMY-2 aggregates in at least some region anterior to the NCC will tend to move away from the NCC during centration. To test these ideas, we used automated particle tracking (Margineantu et al., 2000) to measure displace- ments of brightly labeled cortical NMY-2 aggregates in the corti- cal plane of wild-type zygotes (n = 10) during the early stage of centration (Fig. 5, A–D; and Video 8, available at http://www.jcb .org/cgi/content/full/jcb.200703159/DC1; see Materials and meth- ods section Particle tracking for details of tracking procedure). To detect local behaviors of NMY-2 aggregates, we di- vided the region of the cortical plane from 25 to 75% EL into eight subregions (octants), which were defined with respect to the cell poles and the direction of NCC rotation. Each octant comprised a lateral half of one AP segment with length equal to 12.5% EL (6–7 μm), and the zygotes were oriented such that NCC rotation was clockwise. This convention positioned centrosomes initially near the anterior edge of the posterior-most pair of octants, with the presumptive anterior spindle pole centro- some subsequently swinging through the full breadth of octant g (Fig. 5 E). We first asked whether NMY-2 aggregate movement in any region of the cortex is directionally biased. For every cortical octant, we obtained the frequency distribution of all displacement sizes at a given time scale projected onto the AP axis (Fig. 6) or the transverse axis (Fig. 7). At short time scales (0.25–2 s), these distributions resembled smooth Gaussian curves with peaks very close to zero, indicating that random thermal forces influence NMY-2 aggregate movement (unpublished data). At longer time scales (4–16 s), the appearance of directed nonran- dom movements along one or both axes became increasingly prominent in some octants (Fig. S2, available at http://www.jcb .org/cgi/content/full/jcb.200703159/DC1). At the time scale of 12 s, wild-type NMY-2 aggregate dis- placements throughout much of the cortex were biased toward the anterior (Fig. 6). This bias was evident in the major peaks of displacements in the central octants (b, c, f, and g) near and anterior to the NCC. Large displacements (>1 μm/12 s) were biased toward the anterior on the side of the presumptive ante- rior spindle pole centrosome (octants e, f, and h) and toward the posterior on the opposite side (octants a–c), suggesting a posi- tive correlation between large displacements of NMY-2 aggre- gates and the angular displacement of the nearest centrosome. Anterior movements of NMY-2 aggregates occur independently of mitotic asters The correlated movements of NMY-2 aggregates and the NCC suggested a mechanical link. To test the possibility that NMY- 2 aggregate movement is driven by NCC movement, we blocked centration and rotation by inactivating ZYG-9, a protein re- quired for astral MT stability and, consequently, for centrosome movement. In zyg-9(or634ts) mutant zygotes (n = 8), centra- tion and rotation failed as reported previously (Bellanger et al., 2007), but NMY-2 aggregates in most octants still showed a pronounced anterior bias in peak displacement (Fig. 6). In octants c and g, large displacements in both directions occurred more frequently in the zyg-9(−) mutant than in wild type; in octants b and f, large displacements occurred more frequently toward the posterior. We conclude that NMY-2 aggregate motility 1182 JCB • VOLUME 178 • NUMBER 7 • 2007