Figure 4.

Asymmetric distribution of the actin cortex and PM in the initiation of a directional polar bleb under temperature gradient. (A) Image sequences of fluorescently labeled F-actin (Lifeact-GFP) and PM (CellMask) in the same cell (created from the original images of Movie S17). ΔT = 15°C at T₀ = 25°C. Top images are in pseudocolor to highlight the dynamics of Lifeact-GFP. Scale bar, 10 μm. (B) Kymographs of Lifeact-GFP (green) and CellMask (magenta), and their merged images from the white rectangles in (A). (C) Time courses of fluorescence intensities measured at vertical black bars in (B). Arrows and red bars in (A)–(C) indicate the timing of initiation of bleb formation and the period of heating, respectively. The gray area in (C) also indicates the period of heating. The rapid fluorescence decrease at the onset of heating and increase at the end of heating are due to the effect of thermal quenching of fluorophores. (D) In-phase movement of the actin cortex and PM to the cooler side. These sequences were reconstructed from the polar transformed images of (A). Vertical red bars indicate the period of heating. Angle is defined as relative to the heat source (see Fig. S2: the position at 0° is the nearest to the heat source), and negative value is defined as the counterclockwise direction. See also Fig. S11. (E) Schematic model on the initiation of directional bleb formation by local heating. A possible Major case 1: (i and ii) local heating increases the contractile forces (orange arrows) more at the warmer side. (iii) The increased force at the warmer side detaches the cortex from the PM and the bleb formation begins. (iv) Although the cortex is ruptured at the warmer side, the bleb continues to extend. (v) The cortex rupture results in the movement of the cortex and PM toward the cooler side (green arrows). A possible Major case 2: cortex ruptures at the warmer side (ii’), initiating the bleb formation (iii’). A possible Minor case: in a small number of cases, the process stops at stage (iii), where cortex rupture is not observed (Fig. S13; Movie S18). Yellow circles indicate the positions of the heat source.