STED Microscopy of Actin Cytoskeleton

The experiments were performed using a Leica TCS SP5 STED microscope (Leica Microsystems, Mannheim, Germany), through a 100 × , 1.4 NA PL APO CS oil objective (Leica Microsystems), as described^{48 (link)}; or using a modified Quad Scan STED Microscope (Abberior) with a 100 × , 1.4 NA UPlanSApo oil objective (Olympus). Actin was labeled using ATTO647N phalloidin (Sigma Aldrich), using a previously published protocol^{49 (link)}. ATTO647N was excited using a pulsed diode laser at 635 nm (Leica Microsystems), and depleted using a 750 nm wavelength, provided by a Spectra-Physics Mai Tai tunable laser (pulsed at 80 MHz; Titanium Sapphire, Newport Spectra-Physics, Irvine, CA). For all stainings 3 independent experiments were analyzed and the total number of cells are presented in Supplementary Table S2.