excitation and emission light was ﬁltered using two ﬁlter wheels (MetalTek) controlled by a driver (Lambda 10–2; Sutter Instrument Co.). In the case of DIC imaging, the emission was ﬁltered according to polarization; for GFP ﬂuorescence imaging, the emission was ﬁltered using a 505-nm-long pass ﬁlter (Z485LP; Chroma Technology Corp.). Focusing of the objective was controlled by a XYZ automated stage (PZ-2000; Applied Scientiﬁc Instrumentation) with a piezo z-axis top plate. All image acquisition was controlled by MetaMorph imaging software version 6.2r6 (Molecular Devices). Zygotes were collected and mounted as for DIC microscopy except that the slide was warmed to 26°C using a Bionomic cell (BC-300W; 20/20 Technologies). To quantitate cortical NMY-2–GFP intensity, we imaged the entire thickness of the zygote cortex in a z-series of eight images 0.2 μm apart and used ImageJ to make a two-dimensional projection by summing the signal at each point. We selected a rectangular area corresponding to the median 50% of the zygote (25% egg width on each side of a line corre- sponding to the AP axis) and plotted the distribution proﬁles of ﬂuores- cence intensity at pronuclear meeting and NEB by adding the signal at each point along the normalized length of the AP axis. To eliminate varia- tion arising from curvature of the cortex at the cell poles, we analyzed only a segment from 0.2 to 0.8 EL. For each zygote, relative distribution of NMY-2–GFP along the AP axis was calculated as the fraction of total corti- cal signal at each point along the axis. Data presented in Fig. S1 A are mean values for each genotype/RNAi condition. A t test (two-tailed, as- suming unequal variance) was used to compare values between genotypes at each axial position. To simultaneously visualize cortical NMY-2–GFP and centrosomes (Fig. S1 B), we collected a cortical ﬂuorescence z-series and a DIC image of the NCC every 10 s. Fluorescence z-series corre- sponding to each time point were projected as a sum, and stacks were normalized for EL. Each stack was cropped to a median 50% of egg width, and then the reslice command was used to generate a three-dimensional kymograph. This was ﬂattened by projecting the average (mean) of all slices, thus reducing the AP axis to a row of pixels and generating a kymograph that showed change in the AP intensity proﬁle over time. All kymographs were autoadjusted for brightness and contrast, aligned with reference to the time of NEB, and averaged. Corresponding DIC stacks were likewise normalized for EL, and the centrosomes were marked at each 10-s time point; these marked stacks were used to generate kymo- graphs using the same method as used for ﬂuorescence. Particle tracking To quantify the movements of actomyosin aggregates, we measured NMY-2–GFP displacements occurring at time scales ranging from 0.25 to 16 s. Fig. 5 (C and D) shows the trajectories of all cortical NMY-2–GFP aggregates in a wild-type zygote imaged over 60 s using 1- and 12-s time scales. The trajectory of each aggregate is described by a single track; sampling the aggregates’ displacements at time intervals of 1 s (time scale = 1 s) will yield a set of displacements (in this case, up to 59; shown in Fig. 5 C). Sampling at a longer time scale (12 s) yields a smaller number of displacements of more widely distributed magnitude (Fig. 5 D). Importantly, sampling at this longer time scale reveals directional trends in aggregate movement that may not be apparent at shorter time scales. Note that from the same set of trajectories, a different set of displacements over a 12-s interval would be obtained by shifting the initial sampling time point. Therefore, when measuring displacements over a time scale longer than the acquisiton frame rate, the most complete description is obtained by sampling all possible overlapping time frames in each image sequence. Thus, we extracted all displacements at a given time scale and analyzed frequency distributions of displacement size and direction. Zygotes were observed using DIC optics until the time of pronuclear meeting; the objective was then focused on the upper cortex, and images of a single focal plane were collected at intervals of 0.25–1 s beginning 30 s after pronuclear meeting. After recording for 30–60 s, we determined the direction of NCC rotation and conﬁrmed that the cell successfully di- vided and that its daughters reentered interphase. Although we could not simultaneously record movements of NMY-2–GFP aggregates and the NCC, we found that during the longer 60-s period of data collection, the NCC in wild-type zygotes moves anteriorly a mean distance of 12 ± 2% of EL and rotates through a mean angle of 28 ± 14° (n = 25). In Gα- depleted zygotes, the NCC moves 5.5 ± 2% of EL toward the anterior and rotates through 14 ± 13° (n = 16). In let-99(or513ts) zygotes, the NCC moves 1 ± 2% of EL toward the anterior and changes its direction of rota- tion four to ﬁve times (n = 16). Image sequences were opened using ImageJ, background was sub- tracted, and the stack was rotated to align the zygote horizontally with anterior to the left before saving as a series of 8-bit TIFF images. Further image processing and analysis were performed using IDL (Research Systems, Inc.). NMY-2–GFP aggregates were tracked as described previously (Marcus et al., 1996; Margineantu et al., 2000; Knowles et al., 2002) The ﬁrst image of the sequence was imported and digitally ﬁltered using a Gaussian ﬁlter of 11-pixel (0.7 μm) diameter. A set of features was deﬁned (Fig. 5, A and B) corresponding to the brightest regions distinguishable through the Gaussian ﬁlter; most of these were too faint to be discerned by eye and were re- moved by selecting for brightness. The remaining set of features was over- laid on the original image to conﬁrm identity with observed NMY-2–GFP aggregates. The features identiﬁed were compiled into a time-dependent trajectory that linked features corresponding to the same aggregates in successive frames. Trajectories were plotted on the x and y coordinates to visualize aggregate displacements (Fig. 5, C and D). Histograms of aggre- gate displacements as a function of time scale were constructed from the aggregate trajectories. Distributions of aggregate displacements were ana- lyzed at the time scale of 12 s. This choice represented a compromise be- tween the objectives of detecting biologically driven behaviors (increasingly prominent with increasing time scale) and maintaining a representative dataset: at longer time scales, considerably fewer data points were avail- able from a subset of recordings that lasted 30 s. MSDs were calculated as a function of the time scale τ from the aggregate trajectories according to the method outlined by Margineantu et al. (2000). The two orthogonal projections of the MSD—parallel and perpendicular to the AP axis—are given by the formulas ⎡⎣x(t )−x(t+τ)⎤⎦2 and ⎡⎣y(t )−y(t+τ)⎤⎦2 , respectively. The angle brackets indicate that the x- and y-component MSDs are averaged over all aggregates within a designated spatial region (i.e., an octant) and over all possible starting times (t) of the image frame sequence. Online supplemental material Fig. S1 quantitatively shows the spatial distribution of cortical NMY-2– GFP in wild-type, goa-1(RNAi);gpa-16(RNAi), and let-99(or513ts) at the times of pronuclear meeting and NEB (A). It also shows for each geno- type a time course of averaged NMY-2–GFP intensity proﬁle over the AP axis during 240 s before NEB together with a time course of centrosome positions in the same zygotes (B). Figs. S2–4 show histograms of NMY-2– GFP aggregate displacements at time scales of 4, 8, 12, and 16 s for wild type (Fig. S2), goa-1(−);gpa-16(−) (Fig. S3), and let-99(−) (Fig. S4). Videos show wild-type centration (Video 1), defective centration in let-99(−) and its suppression or enhancement after actomyosin manipulation (Videos 2–6), wild-type distribution of cortical NMY-2–GFP from pronuclear migration through cytokinesis (Video 7), and high speed image sequences used for particle tracking analysis of cortical NMY-2–GFP during centration in wild-type, goa-1(−);gpa-16(−), and let-99(−) zygotes (Videos 8–10). Online supplemental material is available at http://www.jcb.org/cgi/ content/full/jcb.200703159/DC1. We thank Ed Munro and Pierre Gönczy for supplying NMY-2–GFP transgenic strains in advance of publication and a goa-1;gpa-16 feeding RNAi bacterial strain, respectively, and for engaging us in discussions. Theresa Stiernagle at the Caenorhabditis Genetics Center, which is funded by the NIH, supplied some of the worm strains. We thank Chris Doe, Ken Prehoda, Sean O’Rourke, Sarah Siegrist, and three anonymous referees for helpful comments on the manuscript. This work was supported by an NIH National Research Service Award fellowship to M.B. Goulding, a Jane Cofﬁn Childs postdoctoral fellowship to J.C. Canman, an NIH grant (R01GM49869) to B. Boweman, and an NIH grant (R01GM67891) to A.H. Marcus. Submitted: 26 March 2007 Accepted: 27 August 2007 References Albertson, D.G. 1984. Formation of the first cleavage spindle in nematode embryos. Dev. Biol. 101:61–72. Bellanger, J.-M., J.C. Carter, J.B. Phillips, C. Canard, B. Bowerman, and P. Gonzcy. 2007. ZYG-9, TAC-1 and ZYG-8 together ensure correct micro- tubule function throughout the cell cycle of C. elegans embryos. J. Cell Sci. 120:2963–2973. Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics. 77:71–94. Bringmann, H., C.R. Cowan, J. Kong, and A.A. Hyman. 2007. LET-99, GOA-1/ GPA-16, and GPR-1/2 are required for aster-positioned cytokinesis. Curr. Biol. 17:185–191. 1190 JCB • VOLUME 178 • NUMBER 7 • 2007