TY  - JOUR
A1  - Baldering, Tim N.
A1  - Karathanasis, Christos
A1  - Harwardt, Marie-Lena
A1  - Freund, Petra
A1  - Meurer, Matthias
A1  - Rahm, Johanna V.
A1  - Knop, Michael
A1  - Dietz, Marina
A1  - Heilemann, Mike
T1  - CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics
T2  - iScience
N2  - 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.
Y1  - 2020
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/57416
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-574169
SN  - 2589-0042
VL  - 24.2021
IS  - 1, Art. 101895
PB  - Elsevier
CY  - Amsterdam [u.a.]
ER  -