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

Tim N Baldering; Christos Karathanasis; Marie-Lena I E Harwardt; Petra Freund; Matthias Meurer; Johanna V Rahm; Michael Knop; Marina S Dietz; Mike Heilemann

doi:10.1016/j.isci.2020.101895

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

iScience. 2020 Dec 7;24(1):101895. doi: 10.1016/j.isci.2020.101895. eCollection 2021 Jan 22.

Authors

Tim N Baldering¹, Christos Karathanasis¹, Marie-Lena I E Harwardt¹, Petra Freund¹, Matthias Meurer², Johanna V Rahm¹, Michael Knop^{2

3}, Marina S Dietz¹, Mike Heilemann¹

Affiliations

¹ Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 7, 60438 Frankfurt, Germany.
² Center for Molecular Biology of Heidelberg University (ZMBH), 69120 Heidelberg, Germany.
³ German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.

Abstract

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.

Keywords: Biochemistry; Biological Sciences Research Methodologies; Biophysics; Molecular Biology.