Here we use a quantitative FRET approach, specifically developed to probe membrane protein interactions, to study the homo-association of neuropilin 1 (NRP1) in the plasma membrane, as well as its hetero-interactions with vascular endothelial growth factor receptor 2 (VEGFR2). Experiments are performed both in the absence and presence of the soluble ligand vascular endothelial growth factor A (VEGFA), which binds to both VEGFR2 and NRP1. We demonstrate the presence of homo-interactions between NRP1 molecules, as well as hetero-interactions between NRP1 and VEGFR2 molecules, in the plasma membrane. Our results underscore the complex nature of the interactions between self-associating receptors, co-receptors, and their ligands in the plasma membrane. They also highlight the need for new methodologies that capture this complexity, and the need for precise physiological measurements of local receptor surface densities in the membrane of cells. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.

and intermediate filaments (IFs). While the roles of actin-based networks and MT in cell mechanics and cell function have been extensively studied (reviewed, e.g., in refs. [2, 3]), the contribution of IFs to cell mechanics is still relatively opaque, beyond their function in bearing large tensile forces. Of the cytoskeletal proteins, IF constituent proteins have the unique feature of being able to undergo molecular structural changes in response to external loads, at least in vitro, as shown by single-molecule experiments and molecular dynamics simulations. [4-7] This structural polymorphism observed outside of a cellular context has led to the hypothesis that IFs could play a role in intracellular mechanotransduction-relaying information about the cell's mechanical state into biochemical changes that influence cell shape [8-10] and phenotype. [11] IF proteins consist dimeric building blocks ≈45 nm in length that assemble into ≈60 nm antiparallel tetrameters (due to dimer overlap), which laterally associate into unit length filaments that assemble longitudinally to make apolar filaments. These filaments can further bundle into fibers and form networks. [7,12,13] Crystal structures show that IF protein are at least 67% α-helical. [14,15] Individual IFs self-assemble into quite diverse networks with location-specific architectures and composition in cells. [8] IFs proteins can form both ionic and hydrophobic molecular contacts resulting in multiple stabilizing interactions. In addition, proteins such as plectin help bundling and cross-bridging between IFs and to stress fibers and MT. [8,16] Vimentin is one IF protein that forms cytoplasmic IF networks and is particularly interesting because of its important role in cell adhesion, mechanical stability, [17-19] and cell migration, as well as intracellular signaling. [8,20] Specific demonstrations of laser ablation of super-stretched cells showed that the IF network is load bearing; however, this effect was not observed on relaxed cells grown on very soft substrates. [21] Moreover, vimentin has been established as a marker of the epithelial-to-mesenchymal transitions (EMT) in embryogenesis and in tumor metastasis, as the cytoplasmic IF network in epithelial cells mostly consists of keratin that is converted to a vimentin-rich IF network during EMT. [10,22-24] A complex multi-regime response of IF protein structure to deformation has been revealed via computational [7,25,26] and in vitro experimental [4,5] studies of IF protein mechanics.

Background Prior studies have suggested that the interactions occurring between VEGFR2 extracellular domains in the absence of ligand are complex. Here we seek novel insights into these interactions, and into the role of the different Ig-like domains (D1 through D7) in VEGFR2 dimerization. Methods We study the dimerization of a single amino acid mutant and of three deletion mutants in the plasma membrane using two photon microscopy and fully quantified spectral imaging. Results We demonstrate that a set of cooperative interactions between the different Ig-like domains ensure that VEGFR2 dimerizes with a specific affinity instead of forming oligomers. Conclusions The contributions of subunits D7 and D4 seem to be the most critical, as they appear essential for strong lateral interactions and for the formation of dimers, respectively. General Significance This study provides new insights into the mechanism of VEGFR2 dimerization and activation.

Lipid rafts are ordered lipid domains that are enriched in saturated lipids, such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have not been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here, we report that microscopic GM1-enriched domains can form in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2depleted domains. By measuring dissociation constants, we demonstrate that the propensity of EphA2 to oligomerize is similar in EphA2-enriched and -depleted domains. However, surprisingly, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biophysical chemistry of lipid rafts.

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