Flow chambers were attached to PEGylated coverslips as previously described (15). For ensemble experiments, chambers were prepared with 100 nM double-labeled sensor and incubated at room temperature for 30 min. For the single-molecule assay, 100 nM unlabeled sensor with 100 pM labeled sensor was mixed in PBS and added to the flow cells for 30 min. The chambers were then washed with 200 μl of PBS and Pluronic F-127 (0.2% w/v) for ~1 min to prevent nonspecific cell attachment. The chambers were washed again with PBS to remove excess Pluronic. Cells were then added and incubated for at least 1 hour at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) high-glucose medium. FRET measurements were made within 3 hours of plating the cells and acquired with an objective heater (Bioptechs) set to 37°C. Images were prepared in Fiji (48) and analyzed using custom MATLAB scripts.

For immunofluorescence, cells were fixed with 4% paraformaldehyde for 15 min at 37°C and washed with PBS. Cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min, washed with PBS, and then blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature. Antibodies for myosin IIa (Sigma-Aldrich, no. M8064; 1/100 dilution) and phosphorylated myosin light chain (Cell Signaling Technologies, no. 3675S; 1/200 dilution) were incubated with 5% BSA for 45 min at room temperature. Secondary antibodies (anti-rabbit 647, Cell Signaling Technologies, no. 4414S; and anti-mouse 555, Cell Signaling Technologies, no. 4409S; 1/200 dilution) were incubated with 5% BSA for 45 min at room temperature.

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