MET receptor tyrosine kinase activation studied at the single-molecule level

The receptor tyrosine kinase MET is a central player in vertebrate development, wound healing, and tissue regeneration. MET mediates these responses by activating different signaling pathways including signal transduction via the mitogen-activated protein kinase (MAPK) and the phosphoinositide 3-kinase (PI3K) pathways. Next to its important physiological function, MET is involved in numerous diseases. In cancer, MET was reported to be aberrantly expressed leading to constitutively active receptors inducing tumor growth and metastasis. MET is also exploited by pathogenic bacteria during infection, e.g. Listeria monocytogenes, the causative agent of listeriosis. L. monocytogenes induces its uptake via proteins of the internalin family and MET is one of their targets.

We use single-molecule techniques to study the initial processes occurring directly on the membrane upon activation of MET by its bacterial ligand internalin B (InlB). Single-molecule photobleaching microscopy reveals MET receptor dimerization upon activation with InlB as well as pre-formed dimers in the absence of activating ligands (Figure 1). We established a method to measure ligand affinities directly on cells, and determined the binding constant of InlB321 to MET (Figure 2). With single-molecule tracking in live cells, we studied MET mobility and its changes following ligand binding as well as its link to different endocytosis pathways (Figure 3).

 


Figure 1: Single-molecule intensity analysis of InlB321-bound MET reveals receptor dimerization. (a) TIRF image of InlB321-ATTO647N bound to HeLa cells. (b) Analysis of single fluorescence spots reveals the presence of dimeric MET receptors. (c) Fluorescence intensity distribution of InlB321-activated HeLa cells reveals two populations which can be assigned to monomeric and dimeric receptors. (d) Comparison of resting and InlB321-activated cells reveals that the dimer fraction increases in activated cells.

 

Figure 2: Binding study on InlB321-induced HeLa cells via single-molecule imaging. Least-square fitting of the InlB binding curve with a Langmuir-binding model reveals a dissociations constant of 3.1 nM. On the right representative localization images at two different ligand concentrations are shown. The single-molecule images correspond to the respective data points with light and dark blue circles in the binding curve.

 

Single-particle tracking of MET receptors with uPAINT in living HeLa cells
Figure 3: Single-particle tracking of MET receptors with uPAINT in living HeLa cells. a) Single MET receptors are studied with TIRF microscopy at the cell membrane of HeLa cells. Either a non-activating monoclonal Fab fragment or InlB321 labeled with ATTO 647N was added to the cells at 250 pM concentration. b) Map of single-particle traces. Recorded movies were analyzed by localizing single molecules and reconstructing single-particle traces from these localizations (scale bar 5 µm). Inset: Transmitted light image of the same cell. c) Relative frequency distributions of resting and InlB-activated MET diffusion coefficients D. The Fab-ATTO 647N-bound receptors exhibit fast diffusion with a small fraction of immobile receptors. In contrast, InlB321-ATTO 647N shows a pronounced shoulder at lower diffusion coefficients representing slowed MET motion probably due to an increased immobilization prior to endocytosis.

 

Publications

Harwardt, M.-L. I. E., Young, P., Bleymüller, W. M., Meyer, T., Karathanasis, C., Niemann, H. H., Heilemann, M. & Dietz, M. S. (2017) Membrane dynamics of resting and InlB-bound MET receptor tyrosine kinase studied by single-molecule tracking. FEBS Open Bio. doi: 10.1002/2211-5463.12285.

Volz, Y., Koschut, D., Matzke-Ogi, A., Dietz, M. S., Karathanasis, C., Richert, L., Wagner, M. G., Mély, Y., Heilemann, M., Niemann, H. H. & Orian-Rousseau, V. (2015) Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6. Bioscience Reports. 35 (4), e00236.

Dietz, M. S., Fricke, F., Krüger, C. L., Niemann, H. H. & Heilemann, M. (2014) Receptor–ligand interactions: Binding affinities studied by single-molecule and super-resolution microscopy on intact cells. ChemPhysChem. 15 (4), 671.

Dietz, M. S., Haße, D., Ferraris, D. M., Göhler, A., Niemann, H. H. & Heilemann, M. (2013) Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells. BMC Biophysics 6, 6.