Biology subjects

Richards, L.

Publications and source records attributed to Richards, L..

13 recordsLinked to original sources

bioRxiv2025

Molecular kinetics dictate population dynamics in CRISPR-based plasmid defense

Understanding and manipulating mobile genetic element (MGE) spread within bacterial communities represents a great challenge with wide-ranging potential benefits. It requires a detailed understanding of spatial effects and cell-to-cell variability in defence systems such as CRISPR-Cas. Here, we report a time-lapse imaging-based assay with simultaneous fluorescent labeling of CRISPR-Cascade complexes and the conjugative plasmid RP4, enabling direct, single-cell-resolution observation of conjugation and CRISPR interference. We find that Cascade, under wild-type-like expression scenarios, provides a nudge towards plasmid clearance rather than full immunity, and can be counteracted by the ParDE plasmid addiction system. These limitations could be overcome by higher Cascade expression, yet by quantifying single-cell variability, we observe a tradeoff between plasmid clearance and cell growth. We synthesize these measurements into a spatially-resolved, agent-based model of plasmid population dynamics. The imaging and analysis techniques used here will facilitate disentanglement of how single-cell molecular events result in community-wide plasmid dynamics.

molecular biology↗

bioRxiv2025

C12ORF57: a novel principal regulator of synaptic AMPA currents and excitatory neuronal homeostasis

ObjectiveExcitatory neuronal homeostasis is crucial for neuronal survival, circuit function, and plasticity. Disruptions in this form of homeostasis are believed to underpin a variety of neuronal conditions including intellectual disability, epilepsy, and autism. However, the underlying genetic and molecular mechanisms maintaining this homeostasis remain poorly understood. Biallelic recurrent loss of function mutations in C12ORF57, an evolutionarily conserved X amino acid novel open reading frame, underlie Temtamy syndrome (TS)--a neurodevelopmental disorder characterized by epilepsy, dysgenesis of the corpus callosum, and severe intellectual disability. MethodsThrough multiple lines of inquiry, we establish that C12ORF57/GRCC10 plays an unexpected central role in synaptic homeostatic downscaling in response to elevated activity, uncovering a novel mechanism for neuronal excitatory homeostasis. To probe these mechanisms, we developed a new knockout (KO) mouse model of the genes murine ortholog, Grcc10 as well as cellular and in vitro assays. ResultsGrcc10 KO mice exhibit the characteristic phenotypic features seen in human TS patients, including increased epileptiform activity. Corresponding with the enhanced seizure susceptibility, hippocampal neurons in these mice exhibited significantly increased AMPA receptor expression levels and higher amplitude of miniature excitatory postsynaptic currents (mEPSCs). We further found that GRCC10/C12ORF57 modulates the activity of calcium/calmodulin dependent kinase 4 (CAMK4) and thereby regulates the expression of CREB and ARC. InterpretationOur study suggests through this novel mechanism, deletion of Grcc10 disrupts the characteristic synaptic AMPA receptor downscaling that accompanies increased activity in glutamatergic neurons.

neuroscience↗

bioRxiv2024

Ammonia leakage can underpin nitrogen-sharing among soil microbes

AbstractSoil microbial communities host a large number of microbial species that support important ecological functions such as biogeochemical cycling and plant nutrition. The extent and stability of these functions are affected by inter-species interactions among soil microbes, yet the different mechanisms underpinning microbial interactions in the soil are not fully understood. Here, we study the extent of nutrient-based interactions among two model, plant-supporting soil microbes, the fungi Serendipita indica and the bacteria Bacillus subtilis. We find that S. indica is unable to grow with nitrate - a common nitrogen source in the soil - but this inability can be rescued, and growth restored in the presence of B. subtilis. We demonstrate that this effect is due to B. subtilis utilising nitrate and releasing ammonia, which can be used by S. indica. We refer to this type of mechanism as ammonia mediated nitrogen sharing (N-sharing). Using a mathematical model, we demonstrate that the pH dependent equilibrium between ammonia (NH3) and ammonium (NH4+) results in an inherent cellular leakiness, and that reduced amonnium uptake or assimilation rates can result in higher levels of leaked ammonia. In line with this model, a mutant B. subtilis - devoid of ammonia uptake - shows higher S. indica growth support in nitrate media. These findings highlight that ammonia based N-sharing can be a previously under-appreciated mechanism underpinning interaction among soil microbes and could be influenced by microbial or abiotic alteration of pH in microenvironments. Significance statementSoil microbial communities are an important factor in environmental nutrient cycling and sub-sequently plant health. S. indica is a well-studied plant growth promoting soil fungus but its inability to use nitrate, a major component of both agricultural/natural soils and crop fertilisers, may have important implications for agriculture and microbial ecology. We have demonstrated that S. indica is dependant on external sources of nitrogen in nitrate-only environments and these can be produced by B. subtilis, another common soil microbe. We then demonstrate that this nitrogen sharing interaction is likely mediated by leaked ammonia and that ammonia leakage is influenced by environmental pH. Ammonia leakage and sharing represent currently unexplored and potentially vital components of nutrient interactions between microbes in soil communities, with profound implications for microbiome community structure and subsequent consequences for soil biogeochemical cycling and crop health.

microbiology↗

bioRxiv2023

Emergence of synchronised growth oscillations in filamentous fungi

Soil fungi are important decomposers of organic matter and play crucial roles in the biogeochemical cycles in the soil. Many species of fungi grow in the form of branched networks. While there have been investigations on the growth and architecture of the fungal networks, their growth dynamics in space and time is still not fully understood. In this study, we monitor the growth dynamics of the plant-promoting filamentous fungus Serendipita indica for several days in a controlled environment within a microfluidic channel. We find that this species displays synchronized growth oscillations with the onset of sporulation and at a period of 3 hours. Quantifying this experimental synchronisation of oscillatory dynamics, we show that the synchronisation can be captured by the nearest neighbour Kuramoto model. Our analysis suggested the existence of millimetre-scale cell-cell communication across the fungi network. The microfluidic setup presented in this work may aid the future characterization of the molecular mechanisms of the cell-cell communication, which could in turn be exploited in order to control fungi growth and reproductive sporulation in soil and plant health management.

biophysics↗

bioRxiv2023

Connecting species-specific extents of genome reduction in mitochondria and plastids

Mitochondria and plastids have both dramatically reduced their genomes since the endosymbiotic events that created them. The similarities and differences in the evolution of the two organelle genome types has been the target of discussion and investigation for decades. Ongoing work has suggested that similar mechanisms may modulate the reductive evolution of the two organelles in a given species, but quantitative data and statistical analyses exploring this picture remain limited outside of some specific cases like parasitism. Here, we use cross-eukaryote organelle genome data to explore evidence for coevolution of mitochondrial and chloroplast genome reduction. Controlling for differences between clades and pseudoreplication due to relatedness, we find that mtDNA and ptDNA gene retention are related across taxa, in a generally positive correlation that appears to differ quantitatively across eukaryotes, for example, between algal and non-algal species. We find limited evidence for coevolution of specific mtDNA and ptDNA gene pairs, suggesting that the similarities between the two organelle types may be due mainly to independent responses to consistent evolutionary drivers.

evolutionary biology↗

bioRxiv2023

Ecological predictors of organelle genome evolution: Phylogenetic correlations with taxonomically broad, sparse, unsystematized data

Comparative analysis of variables across phylogenetically linked observations can reveal mechanisms and insights in evolutionary biology. As the taxonomic breadth of the sample of interest increases, challenges of data sparsity, poor phylogenetic resolution, and complicated evolutionary dynamics emerge. Here, we investigate a cross-eukaryotic question where all these problems exist: which organismal ecology features are correlated with gene retention in mitochondrial and chloroplast DNA (organelle DNA or oDNA). Through a wide palette of synthetic control studies, we characterize the specificity and sensitivity of a collection of parametric and non-parametric phylogenetic comparative approaches to identify relationships in the face of such sparse and awkward datasets. We combine and curate ecological data coupled to oDNA genome information across eukaryotes, including a new semi-automated approach for gathering data on organismal traits from less systematized open-access resources including encyclopedia articles on species and taxa. Combining this unique dataset with our benchmarked comparative approaches, we confirm support for several known links between organismal ecology and organelle gene retention, identify several previously unidentified relationships constituting possible ecological contributors to oDNA genome evolution, and provide support for a recently hypothesized link between environmental demand and oDNA retention. We, with caution, discuss the implications of these findings for organelle evolution and of this pipeline for broad comparative analyses in other fields.

evolutionary biology↗

bioRxiv2022

Nucleoporins facilitate ORC loading onto chromatin

The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we performed immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we found that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis revealed that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, results in decreased ORC2 loading onto chromatin. Depleting Elys also sensitized cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a new connection between ORC, replication initiation and nucleoporins, highlighting a previously unrecognized function of nucleoporins in metazoan replication initiation.

molecular biology↗

bioRxiv2021

Universal features shaping organelle gene retention

Mitochondria and plastids power complex life, and retain their own organelle DNA (oDNA) genomes, with highly reduced gene contents compared to their endosymbiont ancestors. Why some protein-coding genes are retained in oDNA and some lost remains a debated question. Here we harness over 15k oDNA sequences and over 300 whole genome sequences with tools from structural biology, bioinformatics, machine learning, and Bayesian model selection to reveal the properties of genes, and associated underlying mechanisms, that shape oDNA evolution. Striking symmetry exists between the two organelle types: gene retention patterns in both are predicted by the hydrophobicity of a protein product and its energetic centrality within its protein complex, with additional influences of nucleic acid and amino acid biochemistry. Remarkably, retention principles from one organelle type successfully and quantitatively predict retention in the other, supporting this universality; these principles also distinguish gene profiles in independent endosymbiotic relationships. The identification of these features shaping organelle gene retention both provides quantitative support for several existing evolutionary hypotheses, and suggests new biochemical and biophysical mechanisms influencing organelle genome evolution.

evolutionary biology↗

bioRxiv2021

Fragment-based ab initio phasing of peptidic nanocrystals by MicroED

Microcrystal electron diffraction (MicroED) is transforming the visualization of molecules from nanocrystals, rendering their three-dimensional atomic structures from previously unamenable samples. Peptidic structures determined by MicroED include naturally occurring peptides, synthetic protein fragments and peptide-based natural products. However, as a diffraction method, MicroED is beholden to the phase problem, and its de novo determination of structures remains a challenge. ARCIMBOLDO, an automated, fragment-based approach to structure determination. It eliminates the need for atomic resolution, instead enforcing stereochemical constraints through libraries of small model fragments, and discerning congruent motifs in solution space to ensure validation. This approach expands the reach of MicroED to presently inaccessible peptidic structures including segments of human amyloids, and yeast and mammalian prions, and portends a more general phasing solution while limiting model bias for a wider set of chemical structures.

biochemistry↗

bioRxiv2020

The white matter is a pro-differentiative microenvironment for glioblastoma

Glioblastomas are hierarchically organised tumours driven by glioma stem cells that retain partial differentiation potential. Glioma stem cells are maintained in specialised microenvironments, but how they undergo lineage progression outside of these niches remains unclear. Here we identify the white matter as a differentiative niche for glioblastomas with oligodendrocyte lineage competency. Tumour cells in contact with white matter acquire pre-oligodendrocyte-like fate, resulting in decreased proliferation and invasion. Differentiation is a response to white matter injury, which is caused by tumour infiltration itself in a tumoursuppressive feedback loop. Mechanistically, tumour cell differentiation is driven by selective white matter upregulation of SOX10, a master regulator of normal oligodendrogenesis. SOX10 overexpression or treatment with myelination-promoting agents that upregulate endogenous SOX10, mimic this response, leading to white matter-independent pre-oligodendrocyte-like differentiation and tumour suppression in vivo. Thus, glioblastoma recapitulates an injury response and exploiting this latent programme may offer treatment opportunities for a subset of patients.

cancer biology↗

bioRxiv2020

CReSCENT: CanceR Single Cell ExpressioN Toolkit

CReSCENTCanceR Single Cell ExpressioN Toolkit (https://crescent.cloud), is an intuitive and scalable web portal incorporating a containerized pipeline execution engine for standardized analysis of single-cell RNA sequencing (scRNA-seq) data. While scRNA-seq data for tumour specimens are readily generated, subsequent analysis requires high-performance computing infrastructure and user expertise to build analysis pipelines and tailor interpretation for cancer biology. CReSCENT uses public data sets and preconfigured pipelines that are accessible to computational biology non-experts and are user-editable to allow optimization, comparison, and reanalysis for specific experiments. Users can also upload their own scRNA-seq data for analysis and results can be kept private or shared with other users.

bioinformatics↗

bioRxiv2019

Simulating groundcover community assembly in a frequently burned ecosystem using a simple neutral model

Fire is a global process that drives patterns of biodiversity. In frequently burned fire-dependent ecosystems, surface fire regimes allow for the coexistence of high plant diversity at fine-scales even where soils are uniform. The mechanisms on how fire impacts groundcover community dynamics are however, poorly understood. Because fire can act as a stochastic agent of mortality, we hypothesized that a neutral mechanism might be responsible for maintaining plant diversity. We used the demographic parameters of the Unified Neutral Theory of Biodiversity (UNTB) as a foundation to model groundcover species richness, using a southeastern U.S. pine woodland as an example. We followed the fate of over 7,000 individuals of 123 plant species for four years and two prescribed burns in frequently burned Pinus palustris sites in NW FL, USA. Using these empirical data and UNTB-based assumptions, we developed two parsimonious autonomous agent models, which were distinct by spatially explicit and implicit local recruitment processes. Using a parameter sensitivity test, we examined how empirical estimates, input species frequency distributions, and community size affected output species richness. We found that dispersal limitation was the most influential parameter, followed by mortality and birth, and that these parameters varied based on scale of the frequency distributions. Overall, these nominal parameters were useful for simulating fine-scale groundcover communities, although further empirical analysis of richness patterns, particularly related to fine-scale burn severity is needed. This modeling framework can be utilized to examine our premise that localized groundcover assemblages are neutral communities at high fire frequencies, as well as examine the extent to which niche-based dynamics determine community dynamics when fire frequency is altered and spatial fire intensity patterns differ.

ecology↗

bioRxiv2019

Scale dependent patterns in interaction diversity maintain resiliency in a frequently disturbed ecosystem

Frequently disturbed ecosystems are characterized by resilience to ecological disturbances. For example, longleaf pine ecosystems are exposed to frequent fire disturbance, and this feature sustains biodiversity. We examined how fire frequency maintains beta diversity of multi-trophic interactions, as this community parameter provides a measure of functional redundancy of an ecosystem. We found that turnover in interaction diversity at small local scales is highest in the most frequently burned stands, conferring immediate resiliency to disturbance by fire. Interactions become more specialized and less resilient as fire frequency decreases. Local scale patterns of interaction diversity contribute to broader scale patterns and confer long-term ecosystem resiliency. Such natural disturbances are likely to be important for maintaining regional diversity of interactions for a broad range of ecosystems.

ecology↗