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Allard, J.

Publications and source records attributed to Allard, J..

4 recordsLinked to original sources

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

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

Hydrodynamics of transient cell-cell contact: The role of membrane permeability and active protrusion length

In many biological settings, two or more cells come into physical contact to form a cell-cell interface. In some cases, the cell-cell contact must be transient, forming on timescales of seconds. One example is offered by the T cell, an immune cell which must attach to the surface of other cells in order to decipher information about disease. The aspect ratio of these interfaces (tens of nanometers thick and tens of micrometers in diameter) puts them into the thin-layer limit, or \"lubrication limit\", of fluid dynamics. A key question is how the receptors and ligands on opposing cells come into contact. What are the relative roles of thermal undulations of the plasma membrane and deterministic forces from active filopodia? We use a computational fluid dynamics algorithm capable of simulating 10-nanometer-scale fluid-structure interactions with thermal fluctuations up to seconds-and microns-scales. We use this to simulate two opposing membranes, variously including thermal fluctuations, active forces, and membrane permeability. In some regimes dominated by thermal fluctuations, proximity is a rare event, which we capture by computing mean first-passage times using a Weighted Ensemble rare-event computational method. Our results demonstrate that the time-to-contact increases for smaller cell-cell distances (where the thin-layer effect is strongest), leading to an optimal initial cell-cell separation for fastest receptor-ligand binding. We reproduce a previous experimental observation that fluctuation spatial scales are largely unaffected, but timescales are dramatically slowed, by the thin-layer effect. We also find that membrane permeability would need to be above physiological levels to abrogate the thin-layer effect.\n\nAuthor summaryThe elastohydrodynamics of water in and around cells is playing an increasingly recognized role in biology. In this work, we investigate the flow of extracellular fluid in between cells during the formation of a cell-cell contact, to determine whether its necessary evacuation as the cells approach is a rate-limiting step before molecules on either cell can interact. To overcome the computational challenges associated with simulating fluid in this mechanically soft, stochastic and high-aspect-ratio environment, we extend a computational framework where the cell plasma membranes are treated as immersed boundaries in the fluid, and combine this with computational methods for simulating stochastic rare events in which an ensemble of simulations are given weights according to their probability. We find that the internal dynamics of the membranes has speeds in approximately microseconds, but that as the cells approach, a new slow timescale of approximately milliseconds is introduced. Thermal undulations nor typical amounts of membrane permeability can overcome the timescale, but active forces, e.g., from the cytoskeleton, can. Our results suggest an explanation for differences in molecular interactions in live cells compared to in vitro reconstitution experiments.

biophysics

Nucleo-cytoplasmic trafficking regulates nuclear surface area during nuclear organogenesis

Throughout development, nuclei must be assembled following every cell division to establish a functional organelle from compact, mitotic chromatin. During nuclear organogenesis, chromatin expands to establish a nucleus of a given size seperate from the cytoplasm. Determining how nuclear organogenesis is regulated is particularly significant in the context of certain cancers in which scaling relationships between cell and nuclear sizes are not maintained. Controlling cell size in vitro using a microfluidics approach, we determined that neither nuclear volume nor surface area scale directly with cell size. Looking to explain differential nuclear scaling relationships, we developed a simple mechano-chemical mathematical model. In simulating biological perturbations in silico, our model predicted crucial roles for nucleo-cytoplasmic trafficking in regulating nuclear expansion and in restricting the recruitment of a potential nuclear surface area factor. In mammalian tissue culture, inhibiting nuclear export increased nuclear expansion rates and reduced the amount of nuclear lamin, a candidate surface area factor, being recruited to assembling nuclei, supporting our models predictions. Targeting the principal nuclear export component in the Drosophila syncytial embryo, Embargoed, we show that nuclear expansion rates are also increased in this developmental context, consistent with our model. Using the MS2-reporter system in fly embryos, we demonstrate a role for nuclear export in regulating transcription activation timing and dynamics, suggesting that regulating nuclear assembly is crucial for downstream nuclear function. Taken together, we propose a simple model through which nuclear organogenesis is achieved and demonstrate a role for nuclear export in regulating nuclear assembly.

cell biology