Figure 7.

Photoconversion or photoactivation assay. (A) Schematic overview of a photoconversion or photoswitching assay to specifically visualise the proteins that are stably associated with a complex. The protein of interest tagged with a photoconvertible or photoswitchable fluorescent protein is expressed in the cell. Before photoconversion (t_pre), the entire complex (e.g., an FA) is one colour (here, green). A small region tightly enclosing the protein complex is briefly exposed to a low intensity of 405 nm laser light (t₀), converting the fluorescent proteins in this area to a different colour (here, red). After sufficient time has passed for the dynamic fraction to exchange, another image is taken (t_post). As the volume exposed to the 405 nm laser is small, most fluorescent proteins remain unconverted (green); therefore, exchanging proteins from the converted complex will almost certainly be replaced by unconverted fluorescent proteins. In this way, at t_post, any remaining converted (red) fluorescence signal represents the stably bound fraction, while the unconverted signal represents the dynamically exchanging fraction. (B) Data from a photoconversion experiment where paxillin-mMaple3 is expressed in U2OS cells. Left image shows the ratio of converted signal at t_post over the converted signal at t₀ on a pixel-by-pixel basis. Blue/purple indicates a low ratio representing the dynamically bound fraction, white/yellow a high ratio and the stably bound fraction. The right image is threshold showing the stably bound fraction in red and the dynamically bound fraction in green. Scalebar: 1 µm. Adapted from Legerstee et al. 2019 [58].