For dcFRAP experiments, transfected HEK293 cells grown in four-compartment 35-mm glass-bottom dishes were washed three times with BiotinElation buffer (150 mM NaCl, 2.5 mM KCl, 10 mM Hepes, 12 mM glucose, 0.5 mM CaCl2, and 0.5 mM MgCl2) (66). Cell-impermeable chemical cross-linking using EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific) was used to immobilize cell surface proteins. If intracellular proteins are interacting with immobilized cell surface proteins, their respective mobility will be reduced. This setup was used to visualize the assembly of immobilized FZD5 with heterotrimeric G proteins according to previously published protocols (35). Live-cell imaging experiments were carried out using a Zeiss 710 laser scanning microscope. Images were acquired using a 40×, 1.2 numerical aperture C-Apochromat objective, and the 488-nm and the 561-nm laser lines were used to excite GFP/Venus and mCherry fluorophores, respectively. Photobleaching was performed by 100% laser illumination of a 1.80-μm by 1.80-μm area placed over the cell PM. Fluorescence was measured before and after bleaching using low-intensity illumination for a total time period of 100 s. Average pixel intensity was measured using the ZEN2013 software, corrected for background fluctuations and bleaching artifacts, and normalized to prebleached intensity (67). The recovered mobile fraction (Fm) was calculated as follows: Fm = (IPI0) / (III0), where II is the initial intensity measured before bleaching, I0 is the immediate fluorescence intensity after bleaching, and IP is intensity value after bleaching. The mobile fraction was defined by the average of the fluorescence recovery assessed between time 85 to 101 s. We excluded experiments in which V5-FZD5-mCherry was not sufficiently immobilized after the cross-linking procedure (cutoff at 40% cross-linking–induced reduction in mobile fraction).

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