Wiskostatin

Alexa 647 (A647; 20–30 μM in bathing solution, Invitrogen) and Alexa 488 dyes (A488; 20–30 μM in bathing solution, Invitrogen) were excited by a solid-state 638 nm (30 mW output) and 488 nm lasers (20 mW output) with an inverted confocal microscope (TCS SP8, Leica, Germany; original magnification, × 63/1.40 oil objective). Unless mentioned otherwise, the 638 nm laser was set at 20–25% of the maximum power and the 488 nm laser was set at 0.5–2% of the maximum power. A647 fluorescence was collected with a photomultiplier at 639–767 nm, whereas A488 was collected with a hybrid GaAsP spectral detector at 489–596 nm. For time-lapse A647/A488 imaging, images were collected with 40 ms inter-frame interval at 45–70 nm per pixel in an imaging area of ∼160–320 μm². In some experiments, cells were pre-treated with 3 μM latrunculin A (Enzo Life Sciences), 4 μM Cyto D (Enzo Life Sciences), wiskostatin (10 μM, Tocris Bioscience) and SMIFH2 (25 μM, Tocris Bioscience) for 20 min in the bath solution, or whole-cell dialysis of phalloidin–FITC (1.3 μM, Invitrogen) for 2–3 min before imaging.