Biology subjects

Dushek, O.

Publications and source records attributed to Dushek, O..

3 recordsLinked to original sources

bioRxiv2018

The Influence of Molecular Reach and Diffusivity on the Efficacy of Membrane-Confined Reactions

Signalling by surface receptors often relies on tethered reactions whereby an enzyme bound to the cytoplasmic tail f a receptor catalyses reactions on substrates within reach. The overall length and stiffness of the receptor tail, the enzyme, and the substrate determine a biophysical parameter termed the molecular reach of the reaction. This parameter determines the probability that the receptor-tethered-enzyme will contact the substrate, in the volume proximal to the membrane, when separated by different distances within the membrane plane. Here, we develop particle-based stochastic reaction-diffusion models to study the interplay between molecular reach and diffusion. We find that increasing the molecular reach can increase reaction efficacy when diffusion is slow, but when diffusion is fast, increasing molecular reach reduces reaction efficacy. This switching is lost if reactions are forced to take place within the 2D plasma membrane instead of the 3D volume proximal to it, or if molecules diffuse in 3D. We show results in the context of immune receptors (PD-1 dephosphorylating CD28), a standard opposing kinase-phosphatase reaction, and a minimal two-particle model. The work highlights the 3D nature of many 2D membrane-confined interactions, illustrating a role for molecular reach in controlling biochemical reactions.

biophysics↗

bioRxiv2018

Molecular flexibility of DNA as a major determinant of RAD51 recruitment

The timely activation of homologous recombination is essential for the maintenance of genome stability, in which the RAD51 recombinase plays a central role. Biochemically, human RAD51 polymerises faster on single-stranded DNA (ssDNA) compared to double-stranded DNA (dsDNA), raising a key conceptual question: how does it discriminate between them? In this study, we tackled this problem by systematically assessing RAD51 binding kinetics on ssDNA and dsDNA differing in length and flexibility using surface plasmon resonance. By fitting detailed polymerisation models informed by our experimental datasets, we show that RAD51 is a mechano-sensor that exhibits a larger polymerisation rate constant on flexible ssDNA compared to rigid ssDNA or dsDNA. This model presents a new general framework suggesting that the flexibility of DNA, which may increase locally as a result of DNA damage, plays an important role in rapidly recruiting repair factors that multimerise at sites of DNA damage.

biochemistry↗

bioRxiv2017

Constraining CD45 exclusion at close-contacts provides a mechanism for discriminatory T-cell receptor signalling

The T-cell receptor (TCR) triggers the elimination of pathogens and tumors by T lymphocytes. In order for this to avoid damage to the host, the receptor has to discriminate between thousands of peptide ligands presented by each host cell. Exactly how the TCR does this is unknown. In resting T-cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over kinases expressed at the cell surface. When agonist peptides are presented to the TCR by major histocompatibility complex (MHC) proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering occurs, leading to TCR phosphorylation. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we develop and test a quantitative treatment of receptor triggering reliant only upon TCR dwell-time in phosphatase-depleted cell-cell contacts constrained in area by cell topography. Using the model and experimentally-derived parameters, we find that ligand discrimination is possible but that it depends crucially on individual contacts being 400 nm in diameter or smaller, i.e. the size generated by microvilli. The model not only correctly predicts the relative signaling potencies of known agonists and non-agonists, but achieves this in the absence of conventional, multi-step kinetic proof-reading. Our work provides a simple, quantitative and predictive molecular framework for understanding why TCR triggering is so selective and fast, and reveals that for some receptors, cell topography crucially influences signaling outcomes.\n\nSignificance statementOne approach to testing biological theories is to determine if they are predictive. A simple, theoretical treatment of TCR triggering suggests that ligand discrimination by the receptor relies on just two physical principles: (1) the time TCRs spend in cell-cell contacts depleted of large tyrosine phosphatases; and (2) constraints on contact size imposed by T cells using finger-like protrusions to interrogate their targets. The theory not only allows agonistic and non-agonistic TCR ligands to be distinguished but predicts the relative signalling potencies of agonists with remarkable accuracy. This suggests that the theory captures the essential features of receptor triggering.

immunology↗