Super-Resolution Imaging of Autophagy by a Preferred Pair of Self-Labeling Protein Tags and Fluorescent Ligands.
Anal Chem
; 94(43): 15057-15066, 2022 11 01.
Article
en En
| MEDLINE
| ID: mdl-36262049
ABSTRACT
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 microenvironment. Herein, a representative autophagy marker LC3 protein is selected to develop two hybrid self-labeling systems with tetramethylrhodamine (TMR) fluorophores through SNAP/Halo-tag technologies. 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 â¼50 nm, which disclosed reduced sizes of autophagosomes (â¼650 nm, ferritinophagy) than those in nonselective (â¼840 nm, mammalian target of rapamycin (mTOR)) and selective autophagy (â¼900 nm, mitophagy).
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Autofagia
/
Colorantes Fluorescentes
Idioma:
En
Revista:
Anal Chem
Año:
2022
Tipo del documento:
Article
País de afiliación:
China