Fig. S5

Fig. S5

Rheological Analysis of the Ooplasm, F-Actin, and Yolk Granules, Related to Figures 4 and 5

(A) Normalized and averaged intensity of the bulk actin within the ooplasm along the animal-vegetal (AV) axis of the oocyte prior to actin wave formation in unconfined (WT, data taken from Figure 4A′) and spatially confined (N = 3 experiments, n = 8 oocytes) oocytes. Error bars, SEM. Note that the gradient of bulk actin along the AV axis of the oocyte is preserved even when the oocyte is flattened (spatially confined), suggesting that the observed actin gradient is not due to signal quenching by YGs in the oocyte center. (B) Kymograph of actin intensity along the animal-vegetal (AV) axis of the oocyte as a function of time. Yellow arrowheads trace F-actin bundles flowing toward the animal pole (AP). Scale bars, 25 μm (y axis) and 2 min (x axis). (C) Fluorescence images of F-actin (magenta, left panel), Myosin-2 (cyan, middle panel) and merged F-actin & Myosin-2 (right panel), illustrating their colocalization. Scale bars, 50 μm. (D) Kymograph of F-actin intensity along the line perpendicular to the laser ablation site and prior to actin polymerization wave. Hot-to-cold color coding corresponds to high-to-low actin intensity. White arrowheads trace the recoil of the bulk actin after ablation. Scale bars, 5 μm (y axis) and 5 s (x axis). (E) Kymograph of F-actin intensity changes perpendicular to the laser ablation site after ablation during the bulk actin polymerization wave. Hot-to-cold color coding corresponds to high-to-low actin intensity. White arrowheads trace the recoil of the bulk actin network after ablation. Scale bars as in D. (E’) Initial recoil velocities measured for laser cuts performed prior (red) and during (blue) bulk actin polymerization wave formation, obtained by fitting a linear curve to the first four post-cut data points in Figure 5F and measuring the slope. Error bars, SEM ^∗∗p=0.0037, Mann-Whitney test. (F) Fluorescence images of oocytes injected with 70 KDa Dextran-Alexa Fluor 647 to label the ooplasm for Fluorescence Recovery After Photobleaching (FRAP) experiments. Golden box outlines the bleached area (41.5 ^∗ 8.3 μm2). Scale bar, 25 μm. (F’) Normalized and averaged Dextran intensity of the bleached area over time. N = 4 experiment, n = 36 oocytes. Calculated diffusion coefficient and corresponding ooplasmic viscosity (from Stokes-Einstein relationship; for details see STAR Methods): 1.9 μm2/s and 20 mPa.s (G) Bright-field images of oocytes before (left column) and at the end (right column) of pipette aspiration to measure ooplasm viscosity within the blastodisc dominated by F-actin. Scale bar, 100 μm. Black arrowhead indicates how far the ooplasm has flown in the pipette. (G’) Box and whiskers plot of measured blastodisc viscosities from pipette aspiration assay. “+” sign indicates the mean. N = 3 experiments, n = 23 oocytes. (G’’) Box and whiskers plot of measured blastodisc viscosities from pipette aspiration assay in DMSO (Cyan, N = 3 experiments, n = 15 oocytes) and Cytochalasin B (Cyto B, magenta, N = 4 experiments, n = 16 oocytes) treated oocytes. This result indicates that the bulk actin accounts largely for the measured blastodisc viscosity in control oocytes. “+” signs indicate the mean. ^∗∗∗∗p < 0.001, Mann-Whitney test. (H) Box and whiskers plot of YGs mesh size obtained by measuring the shortest axis of fluid pockets between YGs. “+” sign indicates the mean. N = 3 experiments, n = 3 oocytes. (I) Bright-field images of oocytes before (left column) and at the end (right column) of pipette aspiration to measure YGs viscosity. Scale bar, 100 μm. Black arrowhead indicates how far yolk has flown in the pipette. (I’) Averaged yolk tongue displacement (deformation) during the aspiration and relaxation time points. Black arrowhead indicates the end of aspiration. N = 3 experiments, n = 21 oocytes. Error bars, SEM (I’’) Box and whiskers plot of measured yolk viscosities from pipette aspiration assay. “+” sign indicates the mean. N = 3 experiments, n = 26 oocytes. (J) Normalized ooplasm flow velocities away from the peak of the flows and along the animal-vegetal (AV) axis of the oocyte (averaged for the duration of the first cell cycle). Solid line, exponential fit with the length scale of ∼160μm. Error bars, SD. N = 1 experiment, n = 3 oocytes.