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.

Colocalization analysis of multicolor microscopy images is a cornerstone approach in cell biology. It provides information on the localization of molecules within subcellular compartments and allows the interrogation of known molecular interactions in their cellular context. However, almost all colocalization analyses are designed for two-color images, limiting the type of information that they reveal. Here, we describe an approach, termed “conditional colocalization analysis,” for analyzing the colocalization relationships between three molecular entities in three-color microscopy images. Going beyond the question of whether colocalization is present or not, it addresses the question of whether the colocalization between two entities is influenced, positively or negatively, by their colocalization with a third entity. We benchmark the approach and showcase its application to investigate receptor-downstream adaptor colocalization relationships in the context of functionally relevant plasma membrane locations. The software for conditional colocalization analysis is available at https://github.com/kjaqaman/conditionalColoc.

Aggravation of ER stress by AECHL-1 uncovers a novel pathway for selective elimination of cancer cells.

Significance StatementVascular Endothelial Growth Factor Receptor 2 (VEGFR-2) is a critical receptor on the surface of endothelial cells (which line the blood vessels). It promotes the formation of new blood vessels in response to its ligand VEGF. Currently we know little about the organization and dynamics of this receptor on the cell surface, with multiple proposed models regarding its activation mechanism by VEGF. To fill this knowledge gap, we conducted a quantitative single-molecule imaging study of endogenous VEGFR-2 in primary endothelial cells, revealing the dynamic nanoscale organization of VEGFR-2 in its native environment. Our study also addresses the interplay between this organization, ligand binding and receptor activation, shedding light on the cell biophysical properties of this developmentally important receptor.

Triple negative breast cancer (TNBC) features among the most aggressive manifestations of cancer due to its enhanced metastatic potential and immunity to therapeutics which target hormone receptors. Under such scenarios, anti-cancer compounds with an ability to influence multiple targets, or an entire process, will have an advantage over specific signal transduction inhibitors. To counter the metastatic threat it is essential to target cellular components central to the processes of cancer cell migration and adaptation. Our previous work on a novel triterpenoid, AECHL-1, explored its anti-cancer potential, and linked it to elevated ER stress in cancer cells, while its anti-angiogenic potential was credited for its ability to manipulate the cytoskeleton. Here, we broaden its range of action by showing that it curbs the metastatic ability of TNBC cells, both in vitro in MDA-MB-231 cell line and in vivo, in mouse models of metastasis. AECHL-1 does so by disrupting the cytoskeletal network, and also suppressing NF-κB and β-Catenin mediated key molecular pathways. These activities also contributed to AECHL-1 mediated suppression of TGF-β/TNF-α induced Epithelial to Mesenchymal Transition (EMT) and cancer stem cell characteristic. Thus, we present AECHL-1 as a promising therapeutic inhibitor of metastatic disease.