Calyculin a

Cells were treated with the actin inhibitor cytochalasin-D (5 µM; Tocris), the ezrin inhibitor NSC668394 (50 µM; Calbiochem), Y27632 (25 µM; Hello Bio), calyculin A (10 nM; Cayman Chemical), Ouabain (30 nM; Tocris), and s-nitro-Blebbistatin 25 µM; Cayman Chemical). For cytochalasin-D and calyculin A treatment, an 18-slice Z-stack (140 nm step size) was acquired every minute for 5 min using a Plan Apochromatic 100× silicone oil immersion objective (Nikon; NA 1.35) as described above. After 5 min, a solution of cytochalasin-D dissolved in imaging media was injected into the well and the cells were imaged for another 15 min posttreatment. For NSC668394, Y27632, Ouabain, and S-nitro- Blebbistatin, cells were treated immediately before imaging and an 18-slice Z-stack (192.5 nm step size) was acquired every hour for 17 h using a Plan Apochromatic 40× air objective (Nikon; NA 0.95) and a 2× zoom lens (80× total magnification). Each drug treatment experiment was repeated in three separate wells, and multiple fields of view were collected per well.

Cells were imaged in culture media supplemented with 20 mM HEPES (SH3023701, HyClone) at 37°C, with or without 1 µM SiR-Actin (CY-SC001, Cytoskeleton) or 2 nM Calyculin A (101932-71-2, Cayman), on a Marianas Imaging System (Intelligent Imaging Innovations) consisting of an Axio Observer 7 inverted microscope (Zeiss) attached to a W1 Confocal Spinning Disk (Yokogawa) with Mesa field flattening (Intelligent Imaging Innovations), a Phasor photomanipulation unit (Intelligent Imaging Innovations), a motorized X,Y stage (ASI), and a Prime 95B sCMOS (Photometrics) camera. Illumination was provided by a TTL triggered multifiber laser launch (Intelligent Imaging Innovations) consisting of 405, 488, 561, and 637 lasers, using a 63 × 1.4 NA Plan-Apochromat objective (Zeiss). Temperature and humidity were maintained using a Bold Line full enclosure incubator (Oko Labs). The microscope was controlled using Slidebook 6 Software (Intelligent Imaging Innovations).

For Latrunculin B (Cayman Chemical, 10010631), Y-27632 2HCl (SelleckChem S1049), ROCKOUT, (Sigma, 555553), para-amino blebbistatin (Cayman Chemical, 22699) and calyculin A (Cayman Chemical, 19246) treatments, scales were removed and immediately placed in L-15 media. Imaging commenced at least 15 minutes before careful addition of chemicals while on the microscope stage. Final concentrations used in this study: Latrunculin B, 10 μM; Y-27632, 50 μM; ROCKOUT, 100 μM; para-amino blebbistatin, 100 μM; and calyculin A, 250 nm. Appropriate vehicle controls, either DMSO or ethanol, were used at equivalent %v/v.
For washout experiments, Latrunculin B or Y-27632 was added to 5 ml of L-15 and added to a dish of explanted scales. After 5 (LatB) or 20 (Y-27632) minutes, media was exchanged 4 times using 5 ml for each wash. The dish was immediately placed onto the microscope stage and imaging commenced.