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
Affiliations
Tim N. Baldering
Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 7, 60438 Frankfurt, Germany
Christos Karathanasis
Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 7, 60438 Frankfurt, Germany
Marie-Lena I.E. Harwardt
Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 7, 60438 Frankfurt, Germany
Petra Freund
Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 7, 60438 Frankfurt, Germany
Matthias Meurer
Center for Molecular Biology of Heidelberg University (ZMBH), 69120 Heidelberg, Germany
Johanna V. Rahm
Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 7, 60438 Frankfurt, Germany
Michael Knop
Center for Molecular Biology of Heidelberg University (ZMBH), 69120 Heidelberg, Germany; German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
Marina S. Dietz
Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 7, 60438 Frankfurt, Germany
Mike Heilemann
Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 7, 60438 Frankfurt, Germany; Corresponding author
Summary: 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.
