Heterogeneity in VEGF Receptor-2 Mobility and Organization on the Endothelial Cell Surface Leads to Diverse Models of Activation by VEGF

Abstract

SUMMARY The dynamic nanoscale organization of cell surface receptors plays an important role in signaling. We determine this organization and its relation to activation of VEGF receptor-2 (VEGFR-2), a critical receptor tyrosine kinase in endothelial cells (ECs), by combining single-molecule imaging of endogenous VEGFR-2 in live ECs with multiscale computational analysis. We find that surface VEGFR-2 can be mobile or exhibit restricted mobility and be monomeric or non-monomeric, with a complex interplay between the two. This basal heterogeneity results in heterogeneity in the sequence of steps leading to VEGFR-2 activation by VEGF. Specifically, we find that VEGF can bind to monomeric and non-monomeric VEGFR-2 and that, when binding to monomeric VEGFR-2, its effect on dimerization depends on the mobility of VEGFR-2. Our study highlights the dynamic and heterogeneous nature of cell surface receptor organization and the need for multiscale, single-molecule-based analysis to determine its relationship to receptor activation and signaling.

Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane

“…These methods are based on the ability to localize single emitters beyond the diffraction limit with a precision down to the dimension of macromolecules ( Snyder et al., 2004 ; Thompson et al., 2002 ). Interactions between proteins in the PM can be reliably detected with very high spatial and temporal resolution by single-molecule co-localization or by co-tracking complexes ( Brameshuber and Schutz, 2012 ; da Rocha-Azevedo et al., 2020 ; Kasai et al., 2011 ; Low-Nam et al., 2011 ; Moller et al., 2020 ; Wilmes et al., 2015 ). However, reliable imaging and quantification of receptor interaction and dynamics in live cells by SMFM has remained challenging, as several major requirements have to be met: (1) selective and efficient labeling of target proteins in the PM with photostable fluorophores in multiple colors, (2) rapid time-lapse imaging of multiple channels with minimum photobleaching, and (3) comprehensive analysis including single molecule localization as well as spatial and spatiotemporal analysis with auto- and cross-correlation for all channels.…”

Four-Color Single-Molecule Imaging with Engineered Tags Resolves the Molecular Architecture of Signaling Complexes in the Plasma Membrane

“…beyond the diffraction limit with a precision down to the dimension of macromolecules (Snyder et al, 2004;Thompson et al, 2002). Interactions between proteins in the PM can be reliably detected with very high spatial and temporal resolution by single-molecule co-localization or by co-tracking complexes (Brameshuber and Schutz, 2012;da Rocha-Azevedo et al, 2020;Kasai et al, 2011;Low-Nam et al, 2011;Moller et al, 2020;Wilmes et al, 2015). However, reliable imaging and quantification of receptor interaction and dynamics in live cells by SMFM has remained challenging, as several major requirements have to be met: (1) selective and efficient labeling of target proteins in the PM with photostable fluorophores in multiple colors, (2) rapid time-lapse imaging of multiple channels with minimum photobleaching, and (3) comprehensive analysis including single molecule localization as well as spatial and spatiotemporal analysis with auto-and cross-correlation for all channels.…”

Analysis of conditional colocalization relationships and hierarchies in three-color microscopy images

“…We performed three-color IF imaging of VEGFR-2, TSAd, and paxillin or VEGFR-2, TSAd, and CHC using TIRFM, in the absence or presence of 2 nM VEGF (5 min stimulation before fixation, enough for VEGFR-2 activation [ da Rocha-Azevedo et al, 2020 ]) ( Figs. 5 A and 6 A ).…”