Frap wizard

For FRAP measurements, HL-1 cardiomyocytes were seeded in ibidi 15 μ-Slide 8 Well Glass Bottom chambers (ibidi, 80827). Twenty-four hours after seeding, cells were transfected with 1 μg DSG2-GFP construct as described before (31 (link)), using TurboFect (Thermo Fisher Scientific, R0531). Images were captured with a Leica SP5 confocal microscope (Leica) with the LAS-AF software starting 60 minutes after treatment with the vehicle or erlotinib. Using the Leica FRAP Wizard, 5 pictures were taken before bleaching; then, an area of interest was bleached with 20 pulses at 100% laser intensity for 0.523 s. After that, 60 pictures were taken every second, 20 pictures were taken every 3 s, and 10 pictures were taken every 10 s. Halftime of recovery, as well as immobile fractions, were calculated using the Leica FRAP Wizard.

Live cell confocal microscopy was performed using a TSC SP5 confocal laser-scanning microscope (Leica) and a 63x Leica Objective (HC-PL-APO-CORR-CS2, NA = 1,20), with cells grown to midlog (OD₆₀₀ = 0.4–0.6) labeled with FM4-64 5 min before imaging. Cells were mounted on concavalin A coated cover slides. FRAP was carried in perivacuolar regions of interest containing Vps4-eGFP and FM4-64; bleaching was achieved by 10 ms exposure of an Argon laser beam (digital power 40%, intensity 40%) emitting at 488 nm (point-bleaching setting, Leica-FRAP Wizard). Vps4-eGFP was imaged with 488 nm (10% intensity, HyD 100% gain), FM4-64 was imaged with 561 nm (3% intensity, HyD detector 100% gain). The time-series were acquired with an imaging speed of 1400 Hz. For analysis, fluorescence intensity was corrected for background and bleaching, and normalized to the fluorescence intensity from the first frame. The rate of recovery after photobleaching and the fitted time constants were determined using a single-exponential fitting custom-made MATLAB script.

FRAP was performed using the FRAP‐Wizard of the Leica LAS‐AF microscope software which allowed to control and tune the scanning conditions: prebleach, photobleach, and postbleach phases. Rhodamine‐labeled ELP‐polymer conjugates were excited and bleached with the 561 nm laser line. ROIs were defined on the dextran/PEG interface with a diameter of 10 µm. FRAP acquisition was started with ten images scan at low laser power. Then, the dye was bleached locally inside the ROIs at 100% laser power using a scan of 10 frames. Finally, fluorescence recovery was monitored by the acquisition of a series of 150 images at the same low laser power as the prebleach phase.