JCB: ARTICLE Control of nuclear centration in the C. elegans zygote by receptor-independent Gα signaling and myosin II Morgan B. Goulding,1 Julie C. Canman,1 Eric N. Senning,1,2 Andrew H. Marcus,1,2 and Bruce Bowerman1 1Institute of Molecular Biology and 2Department of Chemistry, University of Oregon, Eugene, OR 97403 Y G O L O I B L L E C F O L A N R U O J E H T M elegans zygote involves microtubule-dependent itotic spindle positioning in the Caenorhabditis pulling forces applied to centrosomes. In this study, we investigate the role of actomyosin in centration, the movement of the nucleus–centrosome complex (NCC) to the cell center. We ﬁnd that the rate of wild-type centra- tion depends equally on the nonmuscle myosin II NMY-2 and the Gα proteins GOA-1/GPA-16. In centration- defective let-99(−) mutant zygotes, GOA-1/GPA-16 and NMY-2 act abnormally to oppose centration. This sug- gests that LET-99 determines the direction of a force on the Introduction Nuclear movement in animal cells can be driven by pulling forces applied to astral microtubules (MTs) emanating from centrosomes bound to the nuclear envelope (Reinsch and Gonczy, 1998). In the one-cell Caenorhabditis elegans zygote, asymmetric cleavage is preceded by a series of stereotyped nuclear movements (Fig. 1 A; for review see Cowan and Hyman, 2004). Female and male pronuclei invariably meet in the poste- rior cytoplasm and move as a unit with the paired centrosomes to the cell center during mitotic prophase; this centration of the nucleus–centrosome complex (NCC) is accompanied by a 90° rotation that aligns the axis of the centrosome pair with the zygote’s longer anteroposterior (AP) axis (Albertson, 1984). Shortly after centration and rotation are completed, nuclear envelopes break down, and the mitotic spindle forms. Laser ablation experiments have revealed that during cen- tration, centrosomes are pulled strongly toward the anterior and weakly toward the posterior (Labbe et al., 2004). These forces are likely transmitted by astral MTs, which extend from each Correspondence to Morgan B. Goulding: goulding@uoregon.edu Abbreviations used in this paper: AP, anteroposterior; DIC, differential interfer- ence contrast; EL, egg length; F-actin; ﬁlamentous actin; MSD, mean-squared displacement; MT, microtubule; NCC, nucleus–centrosome complex; NEB, nuclear envelope breakdown. The online version of this article contains supplemental material. © The Rockefeller University Press $30.00 The Journal of Cell Biology, Vol. 178, No. 7, September 24, 2007 1177–1191 http://www.jcb.org/cgi/doi/10.1083/jcb.200703159 NCC that is promoted by Gα signaling and actomyosin. During wild-type centration, NMY-2–GFP aggregates anterior to the NCC tend to move further anterior, sug- gesting that actomyosin contraction could pull the NCC. In GOA-1/GPA-16–depleted zygotes, NMY-2 aggregate displacement is reduced and largely randomized, whereas in a let-99(−) mutant, NMY-2 aggregates tend to make large posterior displacements. These results suggest that Gα signaling and LET-99 control centration by regulating polarized actomyosin contraction. centrosome to the cell cortex and are required for centration and rotation (for review see Cowan and Hyman, 2004). The molecu- lar motors that pull MTs are unknown, as is the molecular basis of their attachment to the cell cortex. Cortically enriched fila- mentous actin (F-actin) could provide such anchorage; however, disruption of F-actin by cytochalasin D does not prevent NCC centration (Hill and Strome, 1988) or rotation (Hyman and White, 1987), leading to suggestions that cortical F-actin is un- likely to be involved (Grill et al., 2001). The latter conclusion is surprising, as centrosome movement in other systems is clearly F-actin dependent (Euteneuer and Schliwa, 1985). A related problem is how the forces that drive centration and rotation are spatially regulated. Although spatial regulation is not in principle necessary for centration (Grill and Hyman, 2005), the C. elegans zygote is a polarized cell, and an asymmet- rically distributed DEP domain–containing protein encoded by the let-99 gene is required for centration (Rose and Kemphues, 1998; Tsou et al., 2002). The LET-99 protein is enriched in a posterior cortical band coinciding with the initial position of the NCC before centration. In let-99(−) mutant zygotes, cen- tration fails, and the NCC undergoes a series of abnormally rapid and extensive oscillatory rocking movements. DEP do- mains have been implicated in the regulation of heterotrimeric G proteins (Koelle and Horvitz, 1996; Chen and Hamm, 2006), JCB 1177