Ultra 888 emccd

Cell migration was stimulated by making an infinite scratch wound. The cells were allowed to recover for a period of 2 hours before imaging. Images were acquired using a 10X phase objective on a Lecia DMI 4000 equipped with a Hamamatsu ImagEM EMCCD camera. Images were captured every 5 minutes for 10 hours. Migration rates were measured as the area covered by the edge of the wound in the field of view per unit time using Fiji (NIH). For TIRF wound healing experiments, cells were imaged on a Nikon Ti with a 100X, 1.49 NA objective using the 488nm laser, and an Andor iXon Ultra 888 EMCCD.

In vitro motility assays were performed as described [89] . Briefly, 60 μl of NM2A-GFP-Flag HMM molecules (0.2 mg/ml) in motility buffer (MB: 20 mM MOPS, pH 7.3, 0.1 mM EGTA, 2 mM MgCl 2 ) with 0.5 M NaCl were trapped in a flow cell. NM2A-GFP-Flag HMM molecules attached to the coverslip were further incubated with 50 μl MB with 0.5 M NaCl and 1 mg/ml BSA, washed in MB with 50 mM NaCl (LS buffer) and incubated for 4 min with 35 μl of 1 mM ATP, 0.2 mM CaCl 2 , 1mM ATP, 1 μM CaM, 1 nM MLCK and 10 μM of unlabeled actin in LS buffer. Coverslips were washed in LS buffer and incubated with 30 μl of labelled actin filaments (20 nM Rhodamine phalloidin actin in 50 mM DTT) for 35 s. The excess solution was removed and the flow cell was loaded for 1 min with 40 μl of MB with 0.7% methylcellulose, 1mM ATP, 50 mM KCl, 50 mM DTT, 2.5 mg/ml glucose, 2.5 μg/ml glucose oxidase, and 45 μg/ml catalase. The slides were imaged at 25ºC, at 5 s intervals for 2 min, on an inverted Nikon Eclipse Ti-E microscope with an H-TIRF module attachment, a CFI60 Apochromat TIRF 100x Oil Immersion Objective Lens (N.A. 1.49, W.D. 0.12 mm, F.O.V 22 mm) and an EMCCD camera (Andor iXon Ultra 888 EMCCD, 1024 × 1024 array, pixel size: 13 μm). Velocity of the actin filaments on top of the different NM2A-GFP-Flag HMM molecules was quantified using the FAST algorithm described in [90] .