Biology subjects

Ward, R. J.

Publications and source records attributed to Ward, R. J..

3 recordsLinked to original sources

bioRxiv2024

Diffusion and Oligomerization States of the Muscarinic M1 Receptor in Live Cells - The Impact of Ligands and Membrane Disruptors

G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational changes that modulate their interactions with G proteins and arrestins at intracellular sites. High-resolution structures in different liganded states together with spectroscopic studies and molecular dynamics simulations have revealed a rich conformational landscape of GPCRs. However, their supramolecular spatio-temporal distribution is also thought to play a significant role in receptor activation and signaling bias within the native cell membrane environment. Here, we applied single-molecule fluorescence techniques, including single-particle tracking, single-molecule photobleaching and fluorescence correlation spectroscopy, to characterize the diffusion and oligomerization behavior of the muscarinic M1 receptor (M1R) in live cells. Control samples included the monomeric protein CD86 and fixed cells, and experiments performed in the presence of different orthosteric M1R ligands and of several compounds known to change the fluidity and organization of the lipid bilayer. M1 receptors exhibit Brownian diffusion characterized by three diffusion constants: confined/immobile ([~]0.01 m2/s), slow ([~]0.04 m2/s), and fast ([~]0.14 m2/s), whose populations were found to be modulated by both orthosteric ligands and membrane disruptors. The lipid raft disruptor C6-ceramide led to significant changes for CD86 while leaving the diffusion of M1R unchanged, indicating that M1 receptors do not partition in lipid rafts. The extent of receptor oligomerization was found to be promoted by increasing the level of expression, and the binding of orthosteric ligands; in particular the agonist carbachol elicited a large increase in the fraction of M1R oligomers. This study provides new insights into the balance between conformational and environmental factors that define the movement and oligomerization states of GPCRs in live cells under close-to-native conditions.

biophysics↗

bioRxiv2020

Smelling sensations: olfactory crossmodal correspondences

Crossmodal correspondences are the associations between apparently distinct stimuli in different sensory modalities. These associations, albeit surprising, are generally shared in most of the population. Olfaction is ingrained in the fabric of our daily life and constitutes an integral part of our perceptual reality, with olfaction being more commonly used in the entertainment and analytical domains, it is crucial to uncover the robust correspondences underlying common aromatic compounds. Towards this end, we investigated an aggregate of crossmodal correspondences between ten olfactory stimuli and other modalities (angularity of shapes, smoothness of texture, pleasantness, pitch, colours, musical genres and emotional dimensions) using a large sample of 68 observers. We uncover the correspondences between these modalities and extent of these associations with respect to the explicit knowledge of the respective aromatic compound. The results revealed the robustness of prior studies, as well as, contributions towards olfactory integration between an aggregate of other dimensions. The knowledge of an odours identity coupled with the multisensory perception of the odours indicates that these associations, for the most part, are relatively robust and do not rely on explicit knowledge of the odour. Through principal component analysis of the perceptual ratings, new cross-model mediations have been uncovered between odours and their intercorrelated sensory dimensions. Our results demonstrate a collective of associations between olfaction and other dimensions, potential cross modal mediations via exploratory factor analysis and the robustness of these correspondence with respect to the explicit knowledge of an odour. We anticipate the findings reported in this paper could be used as a psychophysical framework aiding in a collective of applications ranging from olfaction enhanced multimedia to marketing.

neuroscience↗

bioRxiv2018

A general method to quantify ligand-driven oligomerization using single- or two-photon excitation microscopy

Current technologies for probing membrane protein association and stability in cells are either very laborious or lack the bandwidth needed for fully quantitative analysis. Here we introduce a platform, termed one- or two-dimensional fluorescence intensity fluctuation spectrometry, for determining the identity, abundance, and stability of oligomers of differing sizes. The sensitivity of this approach is demonstrated by using monomers and oligomers of known sizes in both solutions and cell membranes. The analysis was extended to uncover the oligomeric states and their stability for both the epidermal growth factor receptor, a receptor tyrosine kinase, and the G protein-coupled secretin receptor. In both cases, agonist ligand binding shifted the equilibrium from monomers or dimers to rather large oligomers. Our method can be used in conjunction with a variety of light-based microscopy techniques, is several orders of magnitude faster than current approaches, and is scalable for high-throughput analyses.

biophysics↗