Frappa unit

Imaging and optical activation were performed using a spinning-disk confocal imaging system consisting of a Leica DMI6000B microscope with adaptive focus control, a Yokogawa CSU-X1 spinning-disk unit, an Andor iXon electron-multiplying charge-coupled device camera, a laser combiner with 445-, 488-, 515-, and 594-nm solid-state lasers, and an Andor FRAPPA unit for photoactivation of manually selected regions of the sample in real time, all controlled using Andor iQ2 software (Andor Technologies, Belfast, United Kingdom). For optical activation of iLID, the 445-nm laser was used at 5 μW and scanned across the selected region at a rate of 0.9 ms/μm². This was performed once every 3–5 s. Laser wavelengths of 515 and 594 nm were used for excitation of Venus and mCherry, respectively. Emission filters were Venus 528/20 and mCherry 628/20 (Semrock). All images were acquired using a 63× oil immersion objective. A single confocal plane was imaged at a rate of 1 frame/3 s or 1 frame/5 s. All imaging was performed inside a temperature-controlled chamber held at 37°C. The chamber was also maintained at 5% CO₂ during longer-duration experiments, that is, for samples kept on the microscope before and after treatment with Y-27632.

Confocal images were acquired on Nikon TI Eclipse with perfect focus system live at 37°C with 60× objective every 300 ms for 1 min. FRAP experiments were performed using a 488-nm laser at 100% power with an Andor FRAPPA unit with 200 µs dwell time and two repeats for each spot. The percent recovery after photobleaching for the mobile fraction was calculated by normalizing the bleached region to a control nonbleached region for each time point. The average of the baseline fluorescence was calculated and used for normalization. The minimum fluorescence signal for each bleached region was calculated to determine fluorescence remaining after bleaching. The minimum fluorescence intensity was subtracted from the normalized fluorescence intensity. Data were normalized a final time to the prebleached baseline values. These calculations constrain the prebleach fluorescence to 100% and the postbleach fluorescence to 0%, which isolates the percent mobile fraction recovery by taking into account the amount of the signal that was bleached and looking at its recovery over time. All images were analyzed and quantified using ImageJ and graphed in GraphPad Prism.

In vitro samples were mounted on pegylated glass slides (Alberti et al., 2018 (link)). Cells were grown in 35 mm glass bottom dishes (MatTek Coop.). Fluorescence bleaching experiments were carried out using Olympus IX81 Inverted Spinning Disc Microscope equipped with Andor iXON 897 EMCCD camera and a FRAPPA unit (Andor). Imaging was carried out using an UPlanSApo 100x (NA 1.4) oil immersion objective (Olympus). Fluorescence excitation was with 1% of a 50 mW 488 nm (Coherent Sapphire) and 5% of a 50 mW 561 nm laser (Cobolt Jive) for GFP and mCherry or Cy3 labeled proteins, respectively. Exposure time was typically 50 ms. A region of 10x10 pixels (∼0.82 μm x 0.82 μm) was selected within a droplet and bleached with 100% of a 100 mW 405 nm diode laser (Cairn OptoLED lite) and a 50 ms dwell time. The fluorescence intensity was recorded for 10 frames prior to the bleach and the recovery of fluorescence was recorded at 2 frames/s for 120 s or 240 frames.