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...
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| Hauptverfasser: | , , , , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
2021
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| In: |
iScience
Year: 2020, Jahrgang: 24, Heft: 1 |
| ISSN: | 2589-0042 |
| DOI: | 10.1016/j.isci.2020.101895 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1016/j.isci.2020.101895 Verlag, lizenzpflichtig, Volltext: https://www.sciencedirect.com/science/article/pii/S2589004220310920 
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| Verfasserangaben: | 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 |
| Zusammenfassung: | 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. |
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| Beschreibung: | Available online 7 December 2020 Supplemental information Seite 11-20 Gesehen am 07.04.2021 |
| Beschreibung: | Online Resource |
| ISSN: | 2589-0042 |
| DOI: | 10.1016/j.isci.2020.101895 |