Abstract
SummarySingle-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.
Highlights
Super-resolution microscopy enables the investigation of protein organization and dynamics at the nanoscale and opens the door for a molecular view on protein function in cells (Schermelleh et al, 2019)
We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative Single-molecule localization microscopy (SMLM) and single-particle tracking
We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality
Summary
Super-resolution microscopy enables the investigation of protein organization and dynamics at the nanoscale and opens the door for a molecular view on protein function in cells (Schermelleh et al, 2019). Singlemolecule localization microscopy (SMLM) is a super-resolution technique that generates images from stochastic activation and detection of single fluorescent emitters (Sauer and Heilemann, 2017). Photoactivated localization microscopy (PALM) is a variant of SMLM that uses photoactivatable or photoconvertible fluorescent proteins that are activated stochastically by irradiation with violet light (Betzig et al, 2006). Fluorescent proteins allow stoichiometric labeling by genetic coupling to a target protein, which avoids unspecific labeling as it can occur with fluorophorelabeled antibodies. Fluorescent proteins allow targeting intracellular proteins and protein domains without the need of cell membrane permeabilization which may induce damage to cellular structures (Whelan and Bell, 2015)
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