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...
Saved in:
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article (Journal) |
| Language: | English |
| Published: |
2021
|
| In: |
iScience
Year: 2020, Volume: 24, Issue: 1 |
| ISSN: | 2589-0042 |
| DOI: | 10.1016/j.isci.2020.101895 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1016/j.isci.2020.101895 Verlag, lizenzpflichtig, Volltext: https://www.sciencedirect.com/science/article/pii/S2589004220310920 
|
| Author Notes: | Tim N. Baldering, Christos Karathanasis, Marie-Lena I.E. Harwardt, Petra Freund, Matthias Meurer, Johanna V. Rahm, Michael Knop, Marina S. Dietz, and Mike Heilemann |
| 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. |
|---|---|
| Item Description: | Available online 7 December 2020 Supplemental information Seite 11-20 Gesehen am 07.04.2021 |
| Physical Description: | Online Resource |
| ISSN: | 2589-0042 |
| DOI: | 10.1016/j.isci.2020.101895 |