CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics.
Biochemistry
Biological Sciences Research Methodologies
Biophysics
Molecular Biology
Journal
iScience
ISSN: 2589-0042
Titre abrégé: iScience
Pays: United States
ID NLM: 101724038
Informations de publication
Date de publication:
22 Jan 2021
22 Jan 2021
Historique:
received:
14
08
2020
revised:
12
11
2020
accepted:
02
12
2020
entrez:
28
12
2020
pubmed:
29
12
2020
medline:
29
12
2020
Statut:
epublish
Résumé
Single-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors
Identifiants
pubmed: 33364584
doi: 10.1016/j.isci.2020.101895
pii: S2589-0042(20)31092-0
pmc: PMC7753144
doi:
Types de publication
Journal Article
Langues
eng
Pagination
101895Informations de copyright
© 2020 The Author(s).
Déclaration de conflit d'intérêts
The authors declare no competing interests.
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