Much has been leaned about the spatiotemporal organization of ESCRTs in cells through quantitative high-resolution imaging techniques. Yet, the mechanism for ESCRT induced membrane remodeling remained unknown. This may stem from the inability of current imaging techniques to document the entire process with sufficient resolution in live cells. On top of that, tagging ESCRT proteins with fluorescent proteins may influence the physiological function of the ESCRT membrane fission machine, which is composed of relatively small proteins that polymerize to mediate their function. Genetic code expansion and bioorthogonal labeling offer, for the first time, a non-invasive way to directly attach fluorescent dyes to proteins in live cells. Employing the superb photophysical properties and small size of fluorescent dyes to high resolution imaging of ESCRTs is expected to improve the spatiotemporal resolution of the techniques and at the same time to better preserve physiological context. Recently, we have demonstrated the applicability of this approach for studying cellular polymers in live cells, using tubulin as a benchmark. Fluorescent dye labeling the ESCRT proteins CHMP4B and IST1 indicate that this approach is also applicable for imaging ESCRTs in live cells. We now expand the applicability of this labeling approach and adapt it to live super-resolution imaging which will ultimately be applied to study ESCRTs in a manner that better recapitulate their natural state and in a yet unprecedented spatiotemporal resolution.
