Supplementary Figure 1: Dynamic cortical clusters of aPARs.

(a) Schematic representation of a zygote undergoing embryonic polarization before symmetry breaking, during early (EE), middle (EM) and late (EL) establishment phases, and maintenance phase (MA), and nuclear envelope break down (NEBD) to cytokinesis. The EE to EM phases take place during mitotic prophase after symmetry breaking, when cortical actomyosin develops advected flows toward the anterior pole. The EL phase is the transition phase when cortical contraction ceases, leaving a deeper-furrow at the anteroposterior midline. The MA phase occurs during prometaphase. We used the onset of cytokinesis, which was visualized by the rotation of cortical NMY-2::Kate, PAR-3::GFP, PKC-3::GFP, and GFP::CDC-42, as a respective time point for all time-lapse movies. (b) Detection and classification of cortical clusters. Representative PAR-3::GFP images are shown of a raw image after background subtraction, a segmented binary image of cortical clusters, the local maxima within the segmented clusters defined by peak-detection algorithm, and classification of each peak based on goodness of fitting (R2) to the 2D Gaussian function. Peaks with an R2 value over and below 0.6 are shown in green and red, respectively. (c) Representative images of live zygotes expressing PAR-3::GFP and PKC-3::GFP taken by spinning-disc confocal microscopy (SDC) and total-internal-reflection-fluorescence microscopy (TIRF). All images are representative of 5 zygotes from 3 independent experiments. Scale bar, 5 μm. (d) Representative time-lapse images of a zygote expressing GFP::CDC-42 and mChery::PHPLC and a zygote expressing GFP::WSP-1CRIB. GFP::CDC-42, mCherry::PHPLC, and GFP::WSP-1CRIB appeared at the cortex as an anterior-enriched gradient and eventually formed cortical foci on the anterior cortex in the middle-to-late establishment phase (EML). The times stated are with respect to the onset of cytokinesis. Scale bar, 5 μm. (e) The graphs show relative fluorescence intensity of cortical GFP::CDC-42, mCherry::PHPLC, and GFP::WSP-1CRIB foci during the EE, EML, and MA phases. Mean ± s.d. from n = 5 zygotes from 3 independent experiments. Source data can be found in Supplementary Table 3. (f) The histograms show the distribution of intensity of PAR-3::GFP and PKC-3::GFP clusters during polarization in wild-type zygotes. Black and grey bars represent clusters with an R2 value from 2D Gaussian fitting over and below 0.6, respectively. Mean ± s.d. from n = 6 zygotes for EE phase and n = 7 zygotes each for other phases of polarization.