CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics

  • 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 in vivo in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.

Download full text files

Export metadata

Additional Services

Share in Twitter Search Google Scholar
Metadaten
Author:Tim N. Baldering, Christos KarathanasisORCiDGND, Marie-Lena Harwardt, Petra Freund, Matthias MeurerORCiDGND, Johanna V. RahmORCiD, Michael Knop, Marina DietzORCiDGND, Mike HeilemannORCiDGND
URN:urn:nbn:de:hebis:30:3-574169
DOI:https://doi.org/10.1016/j.isci.2020.101895
ISSN:2589-0042
Parent Title (German):iScience
Publisher:Elsevier
Place of publication:Amsterdam [u.a.]
Document Type:Article
Language:English
Year of Completion:2020
Year of first Publication:2020
Publishing Institution:Universit├Ątsbibliothek Johann Christian Senckenberg
Release Date:2020/12/27
Volume:24.2021
Issue:1, Art. 101895
Page Number:21
HeBIS-PPN:477686141
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Sammlungen:Universit├Ątspublikationen
Licence (German):License LogoCreative Commons - Namensnennung 4.0