Biology subjects

McMahon, S.

Publications and source records attributed to McMahon, S..

At least 19 recordsLinked to original sources

bioRxiv2026

A self-limiting dimeric TIR effector specialised for type III CRISPR-mediated immunity

Antiviral defence systems frequently utilise cyclic nucleotide second messengers. A prominent example is the type III CRISPR-Cas system, which generates cyclic oligoadenylates (cOA) on detecting viral RNA. cOA molecules can bind and activate a wide range of effectors to provide antiviral defence. In both prokaryotes and higher plants, activation of a catalytic Toll/Interleukin Receptor (TIR) domain by multimerization results in degradation of NAD+, limiting cell metabolism and thus viral replication. Here, we describe a CRISPR-associated TIR-containing effector that includes a SAVED (SMODS-associated and fused to various effector domains) domain for nucleotide sensing and a cOA-degrading ring nuclease Crn4 domain. We demonstrate that the TIR-SAVED-Ring nuclease (TSR1) effector binds cA3, resulting in activation of the TIR NADase activity. The Crn4 domain, which imposes an unusual dimeric quaternary structure on the effector, degrades cA3, providing a mechanism to auto-deactivate the effector. TSR1 is thus a highly unusual example of a dimeric and self-limiting TIR effector in antiviral immunity.

microbiology↗

bioRxiv2026

Single-molecule visualisation of human Hsp70-driven conformational remodelling during stress

The Hsp70 chaperone system plays a central role in the folding of nascent polypeptides and in preventing protein misfolding and aggregation during cellular stress. However, the precise mechanism by which the human Hsp70, HspA8, remodels the conformations of individual chemically misfolded clients remains unclear. Moreover, it is unknown whether this mechanism differs from that used by Hsp70 to engage clients during heat stress to preserve native function. To address these questions, we report here the use of single-molecule fluorescence resonance energy transfer (smFRET) to temporally interrogate how the human Hsp70 system regulates the conformation of a heat-sensitive client protein, firefly luciferase (Fluc), following chemical denaturation and during heat stress. We find that Hsp70 recognises both chemically denatured and heat-induced misfolded states of Fluc and resolves them by conformational expansion. Release from a Hsp70-bound state, a process driven by the nucleotide exchange factor, Hsp110, guides Fluc toward productive folding trajectories that would otherwise be unlikely to occur spontaneously following collapse from a conformationally unfolded state. Moreover, we demonstrate that both temperature and the conformational state of misfolded Fluc dictate the ability of HspA8 to meaningfully resolve non-native structure within the protein. Collectively, this work provides direct visualisation of the mechanisms by which Hsp70 modulates client conformations under diverse stress conditions to preserve proteome integrity.

biophysics↗

bioRxiv2026

Non-canonical histone H3.3 and its chaperones HIRA and DAXX participate in the regulation of KSHV latency

Kaposis sarcoma-associated herpesvirus (KSHV), also named HHV-8, is the etiological agent of Kaposi sarcoma (KS), Primary effusion lymphoma (PEL), and Multicentric Castlemans disease. After de novo infection, KSHV genomes rapidly circularize and acquire a chromatin state that favors latency. During latency, the KSHV episome is decorated with distinct epigenetic marks that segregate the viral genome into transcriptionally active and repressed domains, enabling persistent silencing of lytic genes while retaining the capacity for reactivation. Transcription activity of chromatin is regulated at multiple levels, including the incorporation of histone variants such as H3.3, by a specific set of histone chaperones such as HIRA and DAXX. The interaction between LANA and these interphase active chaperones suggests that H3.3 deposition is a critical driver of early chromatinization and the long-term stability of KSHV latency. We detected rapid H3.3 deposition on KSHV episomes and on episomes within long-term infected cells. Moreover, we demonstrated that genetically disrupting the host H3.3 chaperone HIRA pathway by CRISPR/Cas9-mediated knockout impacted the regulation of LANA and maintenance of viral latency that was not altered in DAXX knockout cells. Collectively, these results support a role for HIRA-mediated H3.3 deposition in the regulation of KSHV latency.

molecular biology↗

bioRxiv2026

The CRISPR ring nuclease Csx15 oligomerises on cyclic nucleotide binding to regulate antiviral defence

Prokaryotic type III CRISPR systems signal infection by generating cyclic oligoadenylate (cOA) second messengers, which activate defence proteins allosterically, providing immunity. cOA molecules are typically degraded by extrinsic, stand-alone ring nuclease (RN) enzymes with phosphodiesterase activity, or by the intrinsic RN activity of the effectors themselves. Viruses and plasmids also encode RNs, which can function as anti-CRISPRs (Acr). Eight different families of extrinsic RNs are currently known. Here, we report the structural and biochemical analysis of one of these families: Csx15. We show that Csx15 is a dimeric protein with the ability to bind cyclic tetra-adenylate (cA4) molecules in a shared binding site formed by the head-to-tail stacking of dimers in a filament conformation. Some family members are non-enzymatic, relying on the sequestration (sponging) of cA4 to regulate the host immune response, while others act as canonical RNs, slowly degrading cA4.

biochemistry↗

bioRxiv2026

Experimental evidence for a microbial origin of reduction spots in red beds

Perseverance rover recently discovered sedimentary rocks reddened by ferric oxides and peppered with bleached spots lacking these oxides. Some of these spots are associated with phosphate, iron sulfide minerals, and organic matter, and are regarded as "potential biosignatures", suggestive of microbial iron-and sulfate-reduction and organic matter oxidation. Similar mm-cm-scale "reduction spots" occur in many ancient "red beds" on Earth. Although terrestrial reduction spots are widely considered biogenic, the available evidence is not decisive, and the proposed microbial mechanism of spot formation has not been tested experimentally. Here, we report a successful laboratory demonstration of bleached spot-formation in ferruginous sediment. Mm-cm-scale rounded bleached spots appeared within weeks on the underside of anaerobic sand-ferrihydrite slurries inoculated with mixed microbial communities obtained from the reducing zones of Winogradsky columns. The spatial and temporal distribution of observed bleaching events, which did not occur in sterile controls, are best explained by a microbially induced process, and DNA sequencing confirms that bacteria of iron-reducing genera are abundant in the bleached areas. These results strongly support the longstanding hypothesis that microbial colonies can indeed generate visibly bleached reduction spots in ferruginous sediments and rocks. Further experiments are needed to establish whether and how non-biological processes can mimic these features, and to search for features that disambiguate biogenic and abiogenic reduction spots.

paleontology↗

bioRxiv2025

Small heat shock proteins HspB1 and HspB5 differentially alter the condensation and aggregation of the TDP-43 low complexity domain

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein that regulates processes of mRNA metabolism, during which it undergoes condensation mediated by its C-terminal low complexity domain (TDP-43LCD). TDP-43 aggregation and condensation are associated with neurodegenerative disease. However, the proteostasis mechanisms that regulate these processes remain elusive. Some evidence has shown that the molecular chaperone small heat shock protein HspB1 binds to and regulates the cytoplasmic phase separation of TDP-43, indicating that other small heat shock proteins may have similar effects. Here, we demonstrate divergent behaviours for HspB1 and its homolog HspB5 on TDP-43LCD condensation and aggregation. In addition to inhibiting TDP-43LCD aggregation, HspB1 partitions into TDP-43LCD condensates and increases the dynamic exchange of TDP-43LCD within condensates and with the surrounding solution. These effects of HspB1 are enhanced by mutations that mimic phosphorylation. HspB5 inhibits TDP-43LCD aggregation more effectively than HspB1 and partitions into TDP-43LCD condensates, where it delays pathological transition of the condensate to a gel/solid. We localise the chaperone effects of HspB1 and HspB5 to the N- and C-terminal regions of the protein, emphasising the role of sequence diversity in these regions in defining small heat shock protein function. These findings demonstrate that HspB1 and HspB5 are regulators of TDP-43 phase separation and aggregation and may be potential therapeutic targets in mitigating toxic TDP-43 aggregation in neurodegenerative disease. StatementThis work describes how two small heat shock proteins (proteins that bind to misfolded proteins) impact the condensation and aggregation of TDP-43, a protein implicated in most cases of amyotrophic lateral sclerosis. In doing so, it highlights their divergent behaviours and therapeutic potential.

biochemistry↗

bioRxiv2025

Direct single-molecule visualization of Hsp90-mediated relief of a Hsp70-folding block

Hsp70 and Hsp90 are ubiquitous molecular chaperones that cooperate to promote the correct folding and maturation of client proteins. Despite their central role in proteostasis, the molecular mechanisms by which Hsp90 coordinates with Hsp70 to remodel clients remain poorly understood. In particular, how ATP hydrolysis by Hsp90 is coupled to client engagement and conformational change has been a long-standing question. This gap in understanding is largely due to the challenge of visualizing client conformations during the highly dynamic and heterogeneous interactions with the Hsp70/Hsp90 chaperone machinery. To address this, we used a combination of single-molecule fluorescence resonance energy transfer (smFRET) and total internal reflection fluorescence microscopy to observe individual firefly luciferase client proteins as they are sequentially engaged by Hsp70 and Hsp90. Here, we show that Hsp90 reduces rebinding of Hsp70 to folding intermediates while still allowing engagement to misfolded clients, thereby enabling productive refolding in the presence of typically inhibitory concentrations of Hsp70. Furthermore, Hsp90 uses ATP binding and hydrolysis to actively remodel the conformational landscape of the client, promoting controlled folding through localized compaction across multiple regions. These controlled folding events reduce misfolding and are essential for establishing native interdomain contacts. Using smFRET and kinetic simulations, we further demonstrate that heterogeneous Hsp70 binding generates region-specific folding kinetics and conformational dynamics, which are likely driven by variations in the number of available Hsp70-binding sites. This is then exploited during Hsp70/Hsp90-mediated folding to support localized folding of client subdomains, thereby reducing non-native interactions from distal regions to facilitate proper folding of multi-domain proteins.

biophysics↗

bioRxiv2025

Differential inclusion formation of an aggregation-prone protein reveals differences in the proteostasis capacity of neuronal cell lines

Maintaining proteome integrity is essential for cellular function and survival. Disruptions in proteostasis lead to the aggregation of proteins into inclusions, a process that underlies many neurodegenerative diseases. To quantitatively assess the proteostasis capacity of neuronal cells, we employed an aggregation-prone double mutant form of firefly luciferase (denoted FlucDM) as a reporter protein. We compared two commonly used neuronal cell lines, mouse neuroblastoma cells (Neuro-2a) and a motor neuron-like hybrid line (NSC-34), to evaluate their ability to prevent the aggregation of proteins into intracellular inclusions. We observed a significantly greater propensity of FlucDM to form inclusions in NSC-34 cells compared to Neuro-2a cells. This suggests a reduced capacity of NSC-34 cells for managing aggregation-prone proteins. Proteomic profiling of FlucDM inclusions purified from both cell types revealed cell-type-specific engagement of the proteostasis machinery with aggregation-prone proteins. Comparing the proteomic profiles of key arms of the proteostasis network between these two cell lines revealed that the endoplasmic reticulum (ER) unfolded protein response is differentially expressed. This study establishes a quantitative platform for assessing cellular proteostasis capacity and underscores the importance of cell-type context in proteome maintenance. These insights have implications for understanding the selective vulnerability of neurons in protein misfolding disorders.

cell biology↗

bioRxiv2025

LiDAR and hyperspectral-based structured population models show future forest fire frequency may compromise forest resilience

AbstractForest disturbances are accelerating biodiversity loss and altering tree productivity worldwide. Post-disturbance recovery time is critical for identifying vulnerable areas and targeting conservation but varies with environmental conditions. Monitoring recovery at scale requires tracking tree dynamics, yet traditional ground-based approaches are resource-intensive. We present a pipeline to parameterise integral projection models (IPMs) using LiDAR and hyperspectral data to assess post-fire recovery across large, forested areas. Focusing on the fire-adapted Picea mariana, we model passage times to reproductive heights and life expectancy under different fire regimes as indicators of recovery. To do this, we combined hyperspectral-derived species maps and LiDAR-based crown heights to track individual tree survival and growth at the Caribou-Poker Creek Research Watershed (BONA) from 2017-2023. We incorporated fire history, aspect, slope, elevation, and surrounding canopy height into our models and found partial support for their expected effects on survival and growth. Once accounting for topography and competition, we estimated passage times to reproductive maturity (11-22 years). Life expectancy in the absence of fire is shortest on North-facing slopes with recent fire (579 years). Sensitivity analyses highlight fire history and aspect as key modulators of population resilience, with elevation exerting strong influence on life expectancy across all conditions. Our results demonstrate that remotely sensed IPMs can effectively quantify forest recovery at scale, revealing that in some contexts, stands of P. mariana may not recover between fire disturbances. We discuss the implications of these findings for resilience-based forest management and highlight both the challenges and opportunities of using LiDAR and hyperspectral data to build demographic models for forecasting forest dynamics.

ecology↗

bioRxiv2025

Structure and mechanism of the broad spectrum CRISPR-associated ring nuclease Crn4

Type III CRISPR systems detect the presence of RNA from mobile genetic elements (MGE) in prokaryotes, providing antiviral immunity. On activation, the catalytic Cas10 subunit conjugates ATP to form cyclic oligoadenylate (cOA) signalling molecules that activate ancillary e\ectors, providing an immune response. Cellular ring nucleases degrade cOA to reset the system. Here, we describe the structure and mechanism of a new family of ring nucleases, Crn4, associated with type III-D CRISPR systems. The crystal structure of Crn4 reveals a small homodimeric protein with a fold unrelated to any known ring nuclease or, indeed, any known protein structure. Crn4 degrades a range of cOA species to linear oligoadenylates in vitro and ameliorates type III CRISPR immunity in vivo. Phage and plasmids also encode Crn4 orthologues that may function as anti-CRISPRs. These observations expand our understanding of ring nucleases and reveal a new protein fold for cyclic nucleotide recognition.

biochemistry↗

bioRxiv2025

A viral SAVED protein with ring nuclease activity degrades the CRISPR second messenger cA4

Type III CRISPR systems typically generate cyclic oligoadenylate (cOA) second messengers such as cyclic tetra-adenylate (cA4) on detection of foreign RNA, activating ancillary effector proteins which elicit a diverse range of immune responses. The CalpLTS system elicits a transcriptional response to infection when CalpL binds cA4 in its SAVED (SMODS associated and fused to various effectors domain) sensor domain, resulting in filament formation and activation of the Lon protease domain, which cleaves the anti-Sigma factor CalpT, releasing the CalpS Sigma factor for transcriptional remodelling. Here, we show that thermophilic viruses have appropriated the SAVED domain of CalpL as an anti-CRISPR, AcrIII-2, which they use to degrade cA4. AcrIII-2 dimers sandwich cA4, degrading it in a shared active site to short linear products, using a mechanism highly reminiscent of CalpL. This results in inhibition of a range of cA4 activated effectors in vitro. This is the first example of a virally-encoded SAVED domain with ring nuclease activity, highlighting the complex interplay between viruses and cellular defences.

biochemistry↗

bioRxiv2025

Competing chaperone pathways in α-synuclein disaggregation and aggregation dynamics

The aggregation of the protein -synuclein into amyloid fibrils and their subsequent deposition into large proteinaceous inclusions is a pathological hallmark of several neurodegenerative diseases, including Parkinsons disease. Molecular chaperones, including the small heat shock proteins (sHsps) and the Hsp70 chaperone system, are known to interact with -synuclein fibrils, preventing further aggregation and disaggregating fibrillar species respectively. However, it remains unclear if sHsps co-operate with the Hsp70 chaperones to potentially improve the kinetics or effectiveness of Hsp70-mediated disaggregation and how disaggregation kinetics are influenced by aggregation-prone -synuclein monomers. Using thioflavin-T assays, we demonstrate that the sHsps Hsp27 (HSPB1) and B-crystallin (HSPB5) do not synergise with the Hsp70 chaperones during -synuclein seed fibril disaggregation. Moreover, the addition of monomeric -synuclein with fibril seeds results in increased aggregation that overwhelms Hsp70-mediated disaggregation. Upon addition of sHsps to this system, antagonism between the two chaperone classes is observed, likely due to these chaperones competing for productive binding to the ends of -synuclein fibrils. Overall, these results suggest that while Hsp70 and sHsp chaperones are independently capable of binding to and inhibiting fibril elongation, they do not have synergistic effects on disaggregation. Furthermore, Hsp70-mediated disaggregation is ineffectual in the presence of physiological concentrations of -synuclein monomers, conditions that actually lead to further -synuclein aggregation. Overall, these data may offer insight into factors that lead to the failure of the Hsp70 chaperones to clear cells of -synuclein aggregates that leads to neurodegenerative disease.

biochemistry↗

bioRxiv2025

SAM-AMP lyases in CRISPR defence and anti-defence

Type III CRISPR systems detect non-self RNA and activate the enzymatic Cas10 subunit, which generates nucleotide second messengers for activation of ancillary effectors. Although most signal via cyclic oligoadenylate (cOA), an alternative class of signalling molecule SAM-AMP, formed by conjugating ATP and S-adenosyl methionine, was described recently. SAM-AMP activates a trans-membrane effector of the CorA magnesium transporter family to provide anti-phage defence. Intriguingly, immunity also requires SAM-AMP degradation by means of a specialised CRISPR-encoded NrN family phosphodiesterase in Bacteroides fragilis. In Clostridium botulinum, the nrn gene is replaced by a gene encoding a SAM-AMP lyase. Here, we investigate the structure and activity of C. botulinum SAM-AMP lyase, which can substitute for the nrn gene to provide CorA-mediated immunity in Escherichia coli. The structure of SAM-AMP lyase bound to its reaction product methylthioadenosine-AMP (MTA-AMP) reveals key details of substrate binding and turnover by this PII superfamily protein. Bioinformatic analyses reveal candidate phage-encoded SAM-AMP lyases and we demonstrate that one, hereafter named AcrIIIB4, degrades SAM-AMP efficiently in vitro.

biochemistry↗

bioRxiv2025

Prototaxites was an extinct lineage of multicellular terrestrial eukaryotes

Prototaxites was the first giant organism to live on the terrestrial surface, reaching sizes of 8 metres in the Early Devonian. However, its taxonomic assignment has been debated for over 165 years1-7. Tentative assignments to groups of multicellular algae or land plants1,2,8-11 have been repeatedly ruled out based on anatomy and chemistry5,7,11-16 resulting in two major alternatives: Prototaxites was either a fungus5,6,17-22 or a now entirely extinct lineage 7,16,23. Recent studies have converged on a fungal affinity5-7,17-20,22. Here we test this by contrasting the anatomy and molecular composition of Prototaxites with contemporary fungi from the 407-million-year-old Rhynie chert. We report that Prototaxites taiti was the largest organism in the Rhynie ecosystem and its anatomy was fundamentally distinct from all known extant or extinct fungi. Furthermore, our molecular composition analysis indicates that cell walls of P. taiti include aliphatic, aromatic, and phenolic components most similar to fossilisation products of lignin, but no fossilisation products characteristic of chitin or chitosan, which are diagnostic of all groups of extant and extinct fungi, including those preserved in the Rhynie chert. We therefore conclude that Prototaxites was not a fungus, and instead propose it is best assigned to a now entirely extinct terrestrial lineage.

paleontology↗

bioRxiv2025

Modelling forest dynamics using integral projection models (IPMs) and repeat LiDAR

O_LIEstimating the life histories and population dynamics of trees is important for predicting how forests respond to climate change and disturbances. To do so, ecologist often use stage-structured population models, which explicitly account for individual heterogeneity. However, the data needed to parameterise these models is typically constrained by time- and labour-intensive field methods. C_LIO_LIDespite growing access to remote sensing imagery that could potentially satiate these data-hungry population models, these data remain underutilised in population ecology. Here, we demonstrate a pipeline that integrates repeat-LiDAR data with a type of structured demographic model (Integral Projection Model, IPM) to examine forest-wide dynamics in response to environmental drivers. Using Australias Great Western Woodlands as a case study, we model the survival and growth of [~]40,000 eucalypt trees over a decade to estimate their life expectancy and characterise multiple stages of growth (height growth and crown expansion). C_LIO_LIOur results indicate distinct responses of small trees (where vital rates are predicted by height) and large trees (which invested predominantly in expanding crown area) to proxies for competition and soil moisture (local canopy density and topographic wetness index, respectively). Parameterising structured population models using LiDAR data therefore offers a step-change in perspective towards more ecologically meaningful forest dynamics. C_LIO_LITo broaden the application of this pipeline, we highlight three priorities: (1) Application to more complex systems such as mixed species stands and dense, multilayered canopies); (2) Incorporating complete life histories, including reproduction and early life stages; and (3) Using in long-term or comparative studies through improved availability of high-quality, comparable, repeat-LiDAR surveys. By combining remote sensing data with detailed insights from field- based studies, this pipeline represents a scalable framework for guiding forest management and conservation decisions. C_LI

ecology↗

bioRxiv2024

ARID1A-BAF coordinates ZIC2 genomic occupancy for epithelial to mesenchymal transition in cranial neural crest lineage commitment

The BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris Syndrome (CSS), a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of CSS patients carry variants in one of the mutually exclusive BAF subunits, ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes, little is known about the role of ARID1A in CNCC specification. Using CSS patient-derived ARID1A+/- iPSCs to model CNCC specification, we discovered ARID1A-haploinsufficiency impairs epithelial to mesenchymal transition (EMT), a process necessary for CNCC delamination and migration from the neural tube. Furthermore, wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level, these EMT enhancers contain binding motifs for ZIC2, and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers, ZIC2 relocates to neuronal enhancers, triggering aberrant neuronal gene activation. In mice, deletion of Zic2 impairs NCC delamination, while ZIC2 overexpression in chick embryos at pre-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal a novel ARID1A-ZIC2 axis essential for EMT and CNCC delamination.

developmental biology↗

bioRxiv2023

CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23

CRISPR provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using a N-terminal transmembrane -helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.

microbiology↗

bioRxiv2023

Adaptive introgression of a visual preference gene

Visual preferences are important drivers of mate choice and sexual selection, but little is known of how they evolve at the genetic level. Here we take advantage of the diversity of bright warning patterns displayed by Heliconius butterflies, which are also used during mate choice. We show that two Heliconius species have evolved the same visual mating preferences for females with red patterns by exchanging genetic material through hybridization. Extensive behavioral experiments reveal that male preferences are associated with a genomic region of increased admixture between these two species. Variation in neural expression of regucalcin1, located within this introgressed region, correlates with visual preference across populations, and disruption of regucalcin1 with CRISPR/Cas9 impairs courtship towards conspecific females, proving a direct link between gene and behavior. Our results support a role for hybridization during behavioral evolution, and show how visually-guided behaviors contributing to adaptation and speciation are encoded within the genome.

evolutionary biology↗