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.

 

Figure 3: SIngle-particle tracking of the MET receptor tyrosine kinase with uPAINT. (a) Principle of uPAINT. Single MET receptors are labeled with fluorophore-labeled ligands, imaged with TIRF microscopy and followed over time. (b) Single-particle trajectory map of MET in a HeLa cell. Inset: bright-field image. (c) Diffusion coefficient distribution of resting and activated MET in HeLa cells. MET was either tracked with a fluorophore-labeled non-activating antibody fragment (Fab) or with site-specifically labeled internalin B activating MET.

 

Publications

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

Dietz MS, Fricke F, Krüger CL, Niemann HH, Heilemann M (2014) Receptor–Ligand Interactions: Binding Affinities Studied by Single-Molecule and Super-Resolution Microscopy on Intact Cells. ChemPhysChem 15, 671.

Volz Y, Koschut D, Matzke-Ogi A., Dietz MS, Karathanasis C, Richert L, Wagner MG, Mély Y, Heilemann M, Niemann HH, Orian-Rousseau V (2015) Direct binding of hepatocyte growth factor to CD44v6. Bioscience Reports 35, e00236.

Harwardt MLIE, Young P, Bleymüller WM, Meyer T, Karathanasis C, Niemann HH, Heilemann M & Dietz MS (2017) Membrane dynamics of resting and InlB-bound MET receptor tyrosine kinase studied by single-molecule tracking. FEBS Open Bio 7, 1422.

Harwardt MLIE, Dietz MS, Heilemann M & Wohland T (2018) SPT and imaging FCS provide complementary information on the dynamics of plasma membrane molecules. Biophysical Journal, 114, 2432.