Fig. S7

Actin Comet Formation in Ooplasm-Yolk Granules Segregation, Related to Figure 7

(A) Normalized ooplasm accumulation along the lateral (Lat) axis of the oocyte. N = 2 experiments, n = 6 oocytes. Error bars, SEM 0 and 300 μm correspond to the center and margin of the oocyte, respectively. (B) Shape of the blastodisc-YGs interface (BYI) of the oocyte at 30 mpf (red) and 35 mpf (blue). N = 3 experiments, n = 3 oocytes. Error bars, SEM (C) Kymograph of ooplasm and YGs marked by Rac1-NGreen taken along a midline in Figure 7B. White dashed line outlines BYI during the first three cell cycles. Scale bars, 25 μm (y axis) and 10 min (x axis). (D) Fluorescence image of oocytes expressing Utr-GFP to mark F-actin (red) and exposed to NileRed to mark YGs (green). Same oocyte as in Figure 7D. Yellow ROI indicates the area used for zoomed-in images in (D’). Scale bar, 100 μm. (D’) Zoomed-in view of YGs and bulk actin at the ROI indicated in (D). Arrowheads indicate the formation of actin comets on the ooplasm facing side of YGs. Scale bar, 25 μm. (E) Fluorescence image of oocytes expressing Utr-GFP exposed to DMSO (control), CK666 (Arp2/3 inhibitor, 300μM) or SMIFH2 (Formin inhibitor, 300μM). Yellow and white boxes indicate the ROIs used for measuring actin on YGs surfaces in (E’). Scale bar, 25 μm. (E’) Differential actin intensities during actin bulk actin polymerization wave propagation (white boxes) normalized to actin prior to wave formation (yellow boxes) on YGs surface for control (DMSO, blue, N = 3 experiments, n = 5 oocytes), CK666-treated (green, N = 3 experiments, n = 6 oocytes) and SMIFH2-treated (red, N = 3 experiments, n = 6 oocytes) oocytes. Error bars, SEM ^∗∗∗∗p<0.0001, ns: not significant, Mann-Whitney test. (F) Fluorescence images of oocytes expressing Utr-GFP to mark F-actin with additional YGs transplanted from a donor oocyte into its blastodisc prior to bulk actin polymerization wave formation at 24, 30 and 41 mpf. Scale bar, 100 μm. (F’) Kymograph of actin intensity marked by Utr-GFP expression along the yellow dashed line in (F) as a function of time. Hot-to-cold color coding corresponds to high-to-low actin intensity. Scale bars, 50 μm (y axis) and 2 min (x axis). (G) Schematic illustrating the vegetalward movement of the bulk actin polymerization wave (green), flows of bulk actin toward the animal pole (red), and actin comet formation at BYI, pushing YGs toward the vegetal pole. Green dashed line outlines the leading front of the bulk actin polymerization wave. (H) Fluorescence images of dchsmutant oocytes expressing Utr-GFP to mark F-actin, with several of the “escaping” YGs being segmented. Scale, 100 μm.