Single-Molecule Super-Resolution Microscopy Reveals Heteromeric Complexes of MET and EGFR upon Ligand Activation

Abstract

Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors.

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

“…We showed the endogenous expression by analyzing the cluster density (cluster/mm 2 ) of MET-mEos4b in HEK293T cells. Higher cluster numbers were reported for endogenous MET in HeLa cells in DNA-PAINT measurements (Harwardt et al, 2020). However, taking into account the different mRNA expression levels of MET in HeLa and HEK293T cells (the Human Protein Atlas; www.proteinatlas.org), we expect a lower receptor cluster density for HEK293T cells, which fits to our PALM data and supports an endogenous expression level.…”

“…Receptor mobility was found to respond to ligand binding for various receptor tyrosine kinases ( Schlessinger, 2002 ; Ibach et al., 2015 ; Harwardt et al., 2020 ). Therefore, we expected a reduced receptor mobility and an increased fraction of immobile receptors upon stimulation of MET with its physiological ligand HGF or the bacterial ligand InlB 321 .…”

Unbiased choice of global clustering parameters for single-molecule localization microscopy

“…DBSCAN is one of the most popular clustering algorithms for SMLM data [14, 15, 16, 17, 13, 32, 33, 34, 35]. Part of its appeal is the fact that its density-based clustering rule is intuitive and it can offer close-to-optimal results if parameters are chosen carefully [22].…”

“…This information can be the type of localization patterns to be detected or prior experience. One of the most widely used [13, 14, 15, 16, 17] algorithms is the ‘density-based spatial clustering algorithm’ (DBSCAN) [18, 19]. It is intuitive, simple, fast for 2D datasets [20] – but the parameter choice suffers from human bias.…”

Visualizing multi-protein patterns at the synapse of neuronal tissue with DNA-assisted single-molecule localization microscopy

“…Implementing an experimental protocol that exchanges imager strands in the buffer solution allows for imaging of more targets than if discrimination occurs on the basis of emission spectra (Jungmann et al 2014). Multiplexing and the excellent spatial resolution achieved with DNA-PAINT is now beginning to evolve as a tool in cell biology (Strauss and Jungmann 2020; Schröder et al 2020; Harwardt et al 2020).…”