(A and B) Spinning disk confocal images of bodipy-conjugated ActA (top left) and rhodamine-labeled actin (top right) in water-in-oil emulsions incubated at 20°C (A) or at 30°C (B). A polar actin cortex with a single cap is observed at 20°C, in contrast to the uniform spherical cortex observed at 30°C. The fluorescence intensity profiles as a function of the angle along the contour are shown for different water-in-oil emulsions at 20°C (A; bottom panels) or at 30°C (B). Data for individual emulsions normalized to the mean intensity in each contour (grey lines) is shown together with the population average (thick line). The actin distributions (bottom right) are peaked at the center of the cap for all the emulsions at 20°C, in contrast to the uniform distributions at 30°C. The ActA distributions (bottom left) are essentially flat at both temperatures. The residual ActA observed outside the polar caps at 30°C suggests that ActA is in excess in our system (Figure 2—figure supplement 5). (C) Spinning disk confocal images from a time-lapse video (Video 2) showing the development of a polar actin cap in an emulsion incubated at 20°C. The time after droplet formation is indicated. Cortex assembly starts uniformly along the interface, and becomes polar within minutes. (D) A kymograph of the video in (C), showing the fluorescence intensity along the contour of the emulsion (vertical axis) as a function of time (horizontal axis). The cortical actin flow which slows down as the cap contracts is evident in the kymograph. (E) A histogram of the initial cortical flow speeds measured for a population (N = 32) of emulsions followed by time-lapse microscopy during the symmetry breaking process as in (C). Inset- a schematic illustration of the cortical actin flow during the symmetry breaking process. (F) Bulk assay for actin network contractility. The relative width of the actin containing strip in comparison to the width of the incubation chamber after 30 min is plotted as a function of temperature. At temperatures <25°C the network contracts into a thin strip (left inset), while at higher temperatures (>25°C) no bulk contractility is observed (right inset). (G) Samples were incubated for 30 min at 30°C to generate homogenous cortices, and then moved to 20°C. Cortex polarity developed over time, with symmetry breaking often initiating in more than one position (Figure 2—figure supplement 4A). The relative proportion of symmetric (dark) and polar cortices (light) with one (bare) or multiple (stripes) actin caps, at different time windows following the temperature shift are shown, in comparison to control samples incubated at 30°C or at 20°C.