Talk 1: Mechanical force regulation of T cell-activation across scales from molecules to cells and tissues
Prof Marco Fritzsche, BPI Laboratory Director – Rosalind Franklin Institute & Kennedy Institute of Rheumatology (Oxford)
Mechanical force is emerging as a critical determinant of T-cell activation. Recent evidence indicates that cells regulate their cell mechanics not downstream of signalling events triggered by ligand receptor binding, but that cells employ a diversity of feedback mechanisms to dynamically adjust their mechanics in response to external stimuli. Quantifying cellular forces has therefore become an contentious challenge across multiple disciplines at the interface of biophysics, cell-biology, and immunology. Mechanical forces are especially important for the activation of immune T cells. Using a suite of advanced quantitative super-resolution imaging and force probing methodologies to analyse resting and activated T cells, we demonstrate activating T cells sequentially rearrange their nanoscale mechanobiology at the immunological synapse (IS). We show evidence that the kinetics of the antigen engaging the T-cell receptor controls the nanoscale actin organisation and mechanics of the IS. Using an engineered T-cell system expressing a specific T-cell receptor and stimulated by a range of antigens, force measurements revealed that the peak force experienced by the T-cell receptor during activation was independent of the kinetics of the stimulating antigen. Conversely, quantification of the actin retrograde flow velocity at the IS revealed a striking dependence on the antigen kinetics. Novel ultra-thin superextended lightsheet technology allowed to correlate early calcium activation signalling, IS formation, and effector function. Taken together, these findings suggest that the dynamics of the actin cytoskeleton actively adjusted to normalise the force experienced by the T-cell receptor in antigen specific manner. Consequently, tuning actin dynamics in response to antigen kinetics may thus be a mechanism that allows T cells to adjust the length- and time-scale of T-cell receptor signalling. In the future, the Biophysical Immunology Laboratory aims to translate the established technologies from single cells to live tissues in the Oxford-ZEISS Centre of Excellence.
Talk 2: From Pore Radius Profiles to Conductance Estimates: Exploring the Influence of Pore Shape on Conductance and Barrier to Permeation
David Seiferth, Biggin Group & Tucker Group, Kavli Institute for Nanoscience Discovery (Oxford)
Recent advances in structural biology have led to a growing number of ion channel structures featuring heteromeric subunit assembly, exemplified by synaptic Glycine receptors (GlyRs) and α4β2 nicotinic receptors. These structures exhibit inherent pore asymmetry, which has raised questions about the role of asymmetry in ion channel function. Furthermore, molecular dynamics simulations performed on symmetrical homomeric channels often lead to thermal distortion that means conformations of the resulting ensemble are also asymmetrical. When functionally annotating ion channels, researchers often rely on minimal constrictions determined through radius-profile calculations such as HOLE [1] or CHAP [2], coupled with an assessment of pore hydrophobicity. However, such tools typically employ spherical probe particles, limiting their ability to accurately capture pore asymmetry. In this study, we introduce an algorithm that employs ellipsoidal probe particles, enabling a more comprehensive characterization of pore asymmetries. A constriction is more asymmetric for a larger difference between the smaller and larger radius of the ellipsoidal probe particle. Our analysis reveals that the use of non-spherical ellipsoids for pore characterization, taking into account both a smaller and a larger radius, provides a more accurate and easily interpretable depiction of conductance, shedding light on the key structural properties governing ion conductance. To quantify the implications of pore shape, we investigate Carbon Nanotubes (CNTs) with varying degrees of pore asymmetry as model systems. The conductance through these channels show surprising effects that would otherwise not be predicted with spherical probes. The results have broad implications not only for the functional annotation of biological ion channels, but also for the design of synthetic channel systems for use in areas such as water filtration.
References:
[1] Smart, O.S., Neduvelil, J.G., Wang, X., Wallace, B.A., Sansom, M.S.P., 1996. J. Mol. Graph. 14, 354–360.
[2] Klesse, G., Rao, S., Sansom, M.S.P., Tucker, S.J., 2019. J. Mol. Bio. 431, 3353–3365.
