It is intriguing that the transverse movement of NMY-2 aggregates was greatly reduced in both zyg-9(−) and let-99(−) mutants. As this movement requires the normal activity of astral MTs (absent in zyg-9(−)), we expected it instead to be increased by the hyperactive aster movement in let-99(−). We speculate that signaling from astral MTs may induce the directed move- ment of actomyosin through a mechanism that operates on a relatively long time scale compared with that of NCC rocking. Such a positive feedback effect on actomyosin movement could play an important role in generating smooth and directed cen- trosome movement during centration. To further elucidate how actomyosin interacts with astral MTs to drive centrosome move- ment, it will be interesting to examine the correlated trajectories of astral MTs and associated NMY-2 aggregates in doubly la- beled zygotes. Materials and methods C. elegans strains and maintenance N2 Bristol was used as the wild-type strain and was maintained according to Brenner (1974). Alleles listed by chromosome number were used as follows: fer-1(hc1ts), let-502(sb106ts) I; zyg-9(or634ts) II; dpy-19(e1259), glp-1(q339), let-99(or204ts), let-99(or513ts), lon-1(e185), par-3(it71) III; unc-17(e245) IV; him-5(e1467) V; and lin-2(e1309) X. par-3(it71) mutant embryos were obtained by picking long adults from the strain KK571 (lon-1(e185) par-3(it71)/qC1[dpy-19 (e1259) glp-1(q339)] III). To visualize cortical NMY-2–GFP, we used the strain JJ1473 (unc-119(ed3) III; zuIs45[nmy-2–NMY-2–GFP+unc-119(+)] V). NMY-2–GFP localization throughout early cleavage in live embryos is indistinguishable from the immunolocalization of endogenous NMY-2 (Munro et al., 2004). let-99(or204ts) and let-99(or513ts) were isolated in screens for temperature-sensitive embryonic-lethal mutations affecting early cell divisions (Encalada et al., 2000). Both were outcrossed to N2 males through six generations before phenotypic analysis. In both mutants, the defective cen- tration phenotype was strongly expressive immediately after shifting worms from 15°C to room temperature, suggesting that the mutant proteins rapidly inactivate. or204ts and or513ts mutant worms were maintained at 15°C and shifted to 26°C for at least 1 h before beginning experiments. Broods of embryos produced by hermaphrodites homozygous for either or204ts or or513ts were nearly 100% viable at 15°C and >99% lethal at 26°C. Both alleles were mapped to linkage group IV by mating with strains carry- ing visible mutations. or204ts is strictly recessive: 914/945 (97%) em- bryos from heterozygotes grown at 26°C hatched. or513ts may be weakly dominant: 818/898 (91%) embryos from heterozygotes grown at 26°C hatched. or204ts failed to complement a known recessive and noncondi- tional let-99 allele, or81 (Tsou et al., 2002), and further failed to comple- ment or513ts. To identify the molecular lesions in or204ts and or513ts, genomic DNA encompassing the K08E7.3 (let-99) locus was ampliﬁed from each mutant by PCR using primer sequences not found in the paralo- gous locus F55H2.4 (lrg-1). Sequences from pooled PCR reactions were obtained using a genetic analysis sequencer (CEQ 800; Beckman Coulter) at the University of Oregon Sequencing Facility and were compared with published wild-ype sequences as well as the background strain used for mutagenesis, CB1309 (lin-2(e1309) X) Sequence analysis revealed that or204ts carries two missense mutations at the let-99 locus: a T→A transi- tion that converts leucine 409 to histidine and a G→A transversion that converts arginine 515 to histidine. or513ts also carries two missense muta- tions in let-99: a G→A transversion that converts aspartate 371 to aspara- gine and a T→C transversion that converts methionine 405 to threonine. None of these four mutations was present in CB1309. The phenotypes of or204ts or or513ts are indistinguishable from other let-99(−) mutants described previously (Rose and Kemphues, 1998). A double mutant strain of the genotype let-502(sb106ts) I;let-99 (or513ts) IV was constructed by mating let-502(sb106ts) males with unc-17 (e245) let-99(or513ts) hermaphrodites. The let-502 mutation confers a visible phenotype with fewer oocytes than normal present in the hermaph- rodite uterus; from F2 self-progeny that were Unc with few oocytes, we recovered a strain homozygous for both sb106ts and or513ts. Double mutant worms were maintained at the restrictive temperature of 26°C for at least 6 h before collecting embryos. We conﬁrmed the presence of the or513ts mutation by outcrossing the double mutant strain with N2 males to recover an unc-17 or513ts strain that was wild type at the let-502 locus. To obtain wild-type embryos with reversed fertilization, fer-1(hc1ts) hermaphrodites were shifted to the restrictive temperature of 26°C for several hours and were mated with wild-type males (Goldstein and Hird, 1996). All RNAi was performed by feeding worms on lawns of RNase III– deﬁcient Escherichia coli–expressing double-stranded RNA from inducible promoters. Most bacterial strains were obtained from the library of Kamath et al. (2003) and distributed by Geneservice Ltd. The goa-1;gpa-16 dou- ble RNAi feeding strain was provided by P. Gonczy (Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland; Colombo et al., 2003). Bacterial lawns were grown overnight at 37°C on feeding RNAi plates containing 1 mM IPTG + 75 μg/ml ampicillin (Kamath et al., 2003) from 200 μl of a twofold concentrated stationary-phase liquid culture (uninduced) and were stored for up to 2 wk at 4°C. Worms were trans- ferred to feeding RNAi plates as suspensions of unfed L1 hatchlings and grown at 15°C until adulthood. With the exception of mel-11, all RNAi treatments caused a proportion of animals to become sterile and produce no eggs. In cases in which treatment from L1 produced sterility, worms were plated at later stages of development. All RNAi treatments were pre- dicted to affect phenotypic traits other than NCC movement; the presence of these phenotypic markers in zygotes or their siblings was used to assess the efﬁcacy of gene inactivation. For example, to ensure that NMY-2 was strongly depleted, we collected data only from zygotes showing severe de- fects in the NMY-2–dependent processes of cytokinesis and interphase cor- tical contractility. In the case of goa-1;gpa-16 double RNAi, zygotes were used whose older siblings showed large multinucleate cells, which is a re- sult of cytokinesis defects during late cleavage that indicates the effective depletion of both GOA-1 and GPA-16. DIC video microscopy and analysis of NCC movement Zygotes were dissected out in M9 salts, placed on 3% agarose pads, and overlaid with a coverslip, under which was placed enough M9 to de- lay evaporation from the agar pad. For Nomarski differential interference contrast (DIC) microscopy, 63× 1.4 oil immersion objectives (Carl Zeiss MicroImaging, Inc.) were used with microscopes (Axioskop; Carl Zeiss MicroImaging, Inc.). DIC images were collected at room temperature (23 ± 1°C) at frame rates of 0.1–2.0 Hz using a digital video camera (307,200 pixels2; CCDX; Dage-MTI) connected to a frame-grabber card controlled by a computer (G4; Macintosh). The frame-grabber software was Image 1.62 (Scion) run on an OS9 operating system (Macintosh). These image sequences were collected as TIFF stacks and analyzed using ImageJ (National Institutes of Health [NIH]) and Excel (Microsoft). Image stacks were rotated to align the zygote’s AP axis horizontally with anterior to the left. The NCC position, relative to the zygote’s anterior pole at the time of NEB, was tracked at 10-s intervals beginning just before pronuclear meeting and terminating at NEB. The center of the NCC (midpoint between the diametrically opposed centrosomes) was approximated by eye. NCC velocity was calculated for each 10-s interval. The mean velocity and SD for each genotype/RNAi treatment were computed for the period between pronuclear meeting and NEB. To estimate peak velocities, incremental ve- locity measurements for each zygote of a given genotype/RNAi treatment were sorted in descending order; the three highest values obtained for each zygote were selected, and the mean of these values was computed for all zygotes. Net NCC rotation in wild-type, nmy-2(RNAi), and goa-1 (RNAi);gpa-16(RNAi) zygotes was measured during the 60 s preceding NEB. NCC angular position was measured as the angle of a line connect- ing the two centrosomes relative to the AP axis, and the change in this angle over the 60-s period was calculated. NCC oscillations in let-99(−) single and double mutants were also measured during the 60 s preceding NEB, when the NCC in let-99(−) single mutants is abnormally rotating in place. At each reversal of NCC rotation, angular position was measured as the angle of a line connecting the two centrosomes relative to the AP axis. To compare any two sets of measurements, we used the t test (two- tailed, assuming unequal variance). Fluorescence microscopy and analysis of NMY-2–GFP localization All ﬂuorescence imaging was performed by spinning disc confocal micros- copy. NMY-2–GFP was excited using a 300-mW argon laser (Dynamic Laser; Solamere Technology Group) coupled via optical ﬁber to a spinning disc confocal attachment (Yokogawa; PerkinElmer). The spinning disc head was mounted onto a side port of an inverted ﬂuorescence microscope (TE2000U; Nikon), which was used with either a 60× or 100× plan Apo DIC 1.4 objective (Nikon). Transmitted light was shuttered using a cus- tom adapted shutter (Uniblitz; Applied Scientiﬁc Instrumentation), whereas NUCLEAR MOVEMENT IN C. ELEGANS ZYGOTES • GOULDING ET AL. 1189