A fluorogenic array for temporally unlimited single-molecule tracking.
Nat Chem Biol
; 15(4): 401-409, 2019 04.
Article
em En
| MEDLINE
| ID: mdl-30858596
ABSTRACT
We describe three optical tags, ArrayG, ArrayD and ArrayG/N, for intracellular tracking of single molecules over milliseconds to hours. ArrayG is a fluorogenic tag composed of a green fluorescent protein-nanobody array and monomeric wild-type green fluorescent protein binders that are initially dim but brighten ~26-fold on binding with the array. By balancing the rates of binder production, photobleaching and stochastic binder exchange, we achieve temporally unlimited tracking of single molecules. High-speed tracking of ArrayG-tagged kinesins and integrins for thousands of frames reveals novel dynamical features. Tracking of single histones at 0.5 Hz for >1 hour with the import competent ArrayG/N tag shows that chromosomal loci behave as Rouse polymers with visco-elastic memory and exhibit a non-Gaussian displacement distribution. ArrayD, based on a dihydrofolate reductase nanobody array and dihydrofolate reductase-fluorophore binder, enables dual-color imaging. The arrays combine brightness, fluorogenicity, fluorescence replenishment and extended fluorophore choice, opening new avenues for tracking single molecules in living cells.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Corantes Fluorescentes
/
Imagem Individual de Molécula
/
Microscopia de Fluorescência
Limite:
Humans
Idioma:
En
Revista:
Nat Chem Biol
Assunto da revista:
BIOLOGIA
/
QUIMICA
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Estados Unidos