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

Abstract

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.

Fluorescent reporters, calibration standards and endogenous protein labeling for quantitative single-molecule localization microscopy in cells

“…and 𝑞-values of CD86-mEos2 and CTLA-4-mEos2 were also determined in HEK293 and MEF cells (Krüger et al 2017;Karathanasis et al 2020) and thus demonstrate the robustness of this approach. Additionally, the 𝑝and 𝑞-values of CD86-mEos4b and CTLA-4-mEos4b (𝑝 = 0.27, 𝑞 = 0.35) are very similar to that of CD86-mEos3.2 and CTLA-4-mEos3.2.…”

“…Round cover glasses (diameter 25 mm, VWR International, Radnor, USA) or square cover glasses (35 × 64 mm, # 1.5, Thermo Fisher Scientific) were incubated in isopropanol (VWR Chemicals) for 20 min and plasmacleaned with oxygen or nitrogen for 15 min (Diener Electronic GmbH, Ebhausen, Germany). After that, cover glasses were coated with 100 µg/mL poly-L-lysine (Sigma-Aldrich) or 0.8 mg/mL PLL-PEG-RGD (for details see (Harwardt et al 2020)) in PBS for 1.5-2 hours and washed with distilled water. Cover glasses were dried with nitrogen, and used immediately or stored under argon atmosphere at -20 °C.…”

“…In Karathanasis et al (2020), the oligomeric state of the tumor necrosis factor (TNF) receptor was analyzed (Karathanasis et al 2020) using the original model (Hummer et al 2016). This study reported an arrangement of TNF receptors into trimers and nonamers upon stimulation with the natural ligand TNF𝛼.…”

“…The deviations between model 1 and 2 are small if oligomers of lower orders are analyzed and a high detection efficiency is used. Therefore, the obtained oligomer fractions in previous studies should be sparsely affected (Krüger et al 2017;Baldering et al 2019b;Karathanasis et al 2020). Nevertheless, the use of the revised model is recommended in future studies to obtain precise oligomer fractions.…”

“…This section shows the simplicity and high efficiency of PCR tagging in combination with 4 Results & Discussion 75 PALM (Baldering et al 2020). Two receptor tyrosine kinases, MET and EGFR, were labeled with the fluorescent protein mEos4b using this novel labeling technology and the specific integration was verified by PCR and western blots.…”