Biology subjects

Richards, D. M.

Publications and source records attributed to Richards, D. M..

5 recordsLinked to original sources

bioRxiv2026

Systematic Characterization of Thermal Stability Assay Parameters and Application in Discovery of Peptide-Protein Interactions

Thermal proteome profiling (TPP) and its higher-throughput derivative, the proteome integral solubility alteration (PISA) assay, measure changes in protein thermal stability upon ligand binding or other perturbations and have been widely adopted in drug discovery and biomedical research. Though the PISA workflow is straightforward, key parameters, including detergent concentration, methods for removing denatured aggregates, and temperature range selection, vary across studies and can markedly influence assay outcomes. Yet these factors have not been systematically evaluated, limiting rational experimental design and data interpretation. Here, through a combined use of TPP, PISA, tandem mass tag (TMT)-based multiplexing, and computational simulation, we systematically characterize these parameters based on the melting behavior of [~]9,000 proteins. We find that reducing detergent concentration elevates apparent Tm by 1.5-2{degrees}C proteome-wide, and aggregate removal by filtration versus centrifugation further alters measurements. We leverage these observations to characterize how these parameters shape PISA, then apply selected conditions to identify the aminopeptidase NPEPPS as a previously uncharacterized binding partner of angiotensin II, a key vasoactive peptide hormone in blood pressure regulation. Together, this work provides a general framework for assay design and data interpretation, and extends the utility of PISA beyond small molecules to dissecting peptide-protein interactions, an increasingly important modality in drug discovery.

biochemistry↗

bioRxiv2026

How simple physics drives the earliest stages of embryogenesis

The initial stages of mammalian embryo development involve a single fertilised egg that repeatedly divides to create a solid ball of cells called a morula. Despite the apparent simplicity of this process, which involves only one cell type and a few tens of cells, there are still a host of unanswered questions, particularly around the underlying biophysical mechanisms that are at play. To address this, we here develop a novel type of vertex model that includes cortical tension, cell-to-cell adhesion, membrane curvature and cell volume forces, along with a zona pellucida, cell division and the effect of noise. We fit our model to both mouse and human experimental data, which allows us to address a number of key questions including the relative roles of adhesion and tension, how the cortical tension varies around the cell, the purpose of the zona pellucida, and the rules governing the first few cell divisions. We also determine the biophysical effects responsible for compaction and internalisation, including addressing why the morula does not typically decompact during internalisation. Next, we investigate the position-versus-polarisation debate during trophectoderm differentiation, how the division axis is determined during later divisions, and the role of noise. Finally, we compare human and mouse, focussing on the key similarities that may span all mammals. Our use of a force-based computational model allows us to address fundamental questions relating to mammalian development, particularly the underlying biophysical rules governing early embryogenesis, with important applications to stem cell models such as blastoids, conservation efforts of endangered species and embryo grading during IVF. Significance StatementThe very first stages of embryogenesis are a vital but still poorly understood part of development, with important applications to fertility, fertility lifespan and assisted conception such as IVF. Most research in this area has focussed on experimental approaches, ignoring the potential for biophysical modelling. Here, we address this by developing a novel computer simulation of the first thee-to-four cell divisions of the fertilised egg, resulting in a compact bunch of cells called a morula. In particular, we develop a new type of vertex model for the early embryo that for the first time includes contributions from a range of realistic biophysical forces. By analysing real mouse and human embryo data through our model, we reveal how simple physics drives crucial early developmental processes, including compaction, internalisation, cleavage, noise and species-specific differences.

developmental biology↗

bioRxiv2023

AUTOMATIC EXTRACTION OF ACTIN NETWORKS IN PLANTS

AO_SCPLOWBSTRACTC_SCPLOWThe actin cytoskeleton is essential in eukaryotes, not least in the plant kingdom where it plays key roles in cell expansion, cell division, environmental responses and pathogen defence. Yet, the precise structure-function relationships of properties of the actin network in plants are still to be unravelled, including details of how the network configuration depends upon cell type, tissue type and developmental stage. Part of the problem lies in the difficulty of extracting high-quality, three-dimensional, quantitative measures of actin network features from microscopy data. To address this problem, we have developed DRAGoN, a novel image analysis algorithm that can automatically extract the actin network across a range of cell types, providing seventeen different quantitative measures that describe the network at a local level. Using this algorithm, we then studied a number of cases in Arabidopsis thaliana, including several different tissues, a variety of actin-affected mutants, and cells responding to powdery mildew. In many cases we found statistically-significant differences in actin network properties. In addition to these results, our algorithm is designed to be easily adaptable to other tissues, mutants and plants, and so will be a valuable asset for the study and future biological engineering of the actin cytoskeleton in globally-important crops.

plant biology↗

bioRxiv2022

Wheat cells show positional responses to invasive Zymoseptoria tritici

The stomatal complex of grasses consists of two guard cells and two adjacent subsidiary cells that cooperate during stomatal closure. Zymoseptoria tritici, the main causal agent of Septoria tritici blotch in wheat, enters the host via stomata. Here we test the hypothesis that the stomatal complex shows focused sub-cellular responses to invading Z. tritici hyphae. We have combined live-cell transmission light microscopy, immunofluorescence and CRS microscopy to identify cell wall modifications triggered by hyphal invasion. Furthermore, we have used confocal fluorescence microscopy and automated quantitative image analysis to assess whether host cells respond to hyphae through spatial redistribution of organelles. We find that subsidiary cells construct papillae that are accurately aligned with hyphal position even when hyphae are occluded by guard cells. These are distinct from those induced by powdery mildew, with callose restricted to a crust that surrounds content with a high-amplitude Raman signal in the CH-band. Peroxisome populations in subsidiary cells show distributions with modes weakly correlated with hyphal position but do not differ significantly between compatible and incompatible interactions. Our data suggest local changes to cell wall architecture and focal accumulation of organelles in subsidiary cells could play roles in crop defence during host leaf penetration by Z. tritici. Molecular strategies to amplify these responses may provide novel routes for crop protection.

plant biology↗

bioRxiv2020

Mitochondrial fission factor (MFF) is a critical regulator of peroxisome maturation

Peroxisomes are highly dynamic subcellular compartments with important functions in lipid and ROS metabolism. Impaired peroxisomal function can lead to severe metabolic disorders with developmental defects and neurological abnormalities. Recently, a new group of disorders has been identified, characterised by defects in the membrane dynamics and division of peroxisomes rather than by loss of metabolic functions. However, the contribution of impaired peroxisome plasticity to the pathophysiology of those disorders is not well understood. Mitochondrial fission factor (MFF) is a key component of both the peroxisomal and mitochondrial division machinery. Patients with MFF deficiency present with developmental and neurological abnormalities. Peroxisomes (and mitochondria) in patient fibroblasts are highly elongated as a result of impaired organelle division. The majority of studies into MFF-deficiency have focused on mitochondrial dysfunction, but the contribution of peroxisomal alterations to the pathophysiology is largely unknown. Here, we show that MFF deficiency does not cause alterations to overall peroxisomal biochemical function. However, loss of MFF results in reduced import-competency of the peroxisomal compartment and leads to the accumulation of pre-peroxisomal membrane structures. We show that peroxisomes in MFF-deficient cells display alterations in peroxisomal redox state and intra-peroxisomal pH. Removal of elongated peroxisomes through induction of autophagic processes is not impaired. A mathematical model describing key processes involved in peroxisome dynamics sheds further light into the physical processes disturbed in MFF-deficient cells. The consequences of our findings for the pathophysiology of MFF-deficiency and related disorders with impaired peroxisome plasticity are discussed.

cell biology↗