Super-Resolution Imaging of Autophagy by a Preferred Pair of Self-Labeling Protein Tags and Fluorescent Ligands

Autophagy is a core recycling process for homeostasis, with its dysfunction associated with tumorigenesis and various diseases. Yet, its subtle intracellular details are covered due to the limited resolution of conventional microscopies. The major challenge for modern super-resolution microscopy deployment is the lack of a practical labeling system, which could provide robust fluorescence with fidelity in the context of the dynamic autophagy microenviron-ment. Herein, a representative autophagy marker LC3 protein is selected to develop two hybrid self-labeling systems with tetrame-thylrhodamine (TMR) fluorophores through SNAP/Halo-tag tech-nologies. A systematic investigation indicated that the match of the LC3-Halo and TMR ligand remarkably outperforms that of LC3-SNAP, as the former Halo system exhibited more robust single-molecule brightness (440 vs 247), total photon numbers (45600 vs 13500), and dwell time of the initial bright state (0.82 vs 0.40 s) than the latter. With the aid of this desirable Halo system, for the first time, live-cell ferritinophagy is monitored with a spatial resolution of -,50 nm, which disclosed reduced sizes of autophagosomes (-,650 nm, ferritinophagy) than those in nonselective (-,840 nm, mammalian target of rapamycin (mTOR)) and selective autophagy (-,900 nm, mitophagy).

Automated summary

Autophagy is a crucial cellular recycling process associated with tumorigenesis and various diseases. Conventional microscopy has limited resolution, leading to a lack of detailed understanding of autophagy. Researchers have developed a practical labeling system that provides robust fluorescence for studying the autophagy microenvironment. With the help of this system, they have successfully monitored live-cell ferritinophagy for the first time.