Biology subjects

Clark, G.

Publications and source records attributed to Clark, G..

At least 19 recordsLinked to original sources

bioRxiv2026

RNF213 M1-ubiquitinates damaged mitochondria to activate pro-inflammatory NF-κB signaling

Through mitochondrial outer membrane permeabilization (MOMP), mitochondria are central to apoptosis. MOMP is pro-inflammatory, impacting diverse processes including anti-tumor immunity and cellular senescence. Despite this, how damaged mitochondria activate inflammation remains unclear. Recent studies have shown that following MOMP, mitochondria are extensively ubiquitinated activating pro-inflammatory NF-{kappa}B signaling. We identify the E3 ligase, RNF213, as essential for pro-inflammatory mitochondrial ubiquitination following MOMP. RNF213 has an established role in pro- inflammatory, cell-autonomous immunity to bacteria and other pathogens. We discover that RNF213 is required for mitochondrial M1-linked (linear) ubiquitination. Interestingly, this was found to be independent of the canonical M1-ubiquitin ligase complex LUBAC. RNF213 can directly catalyze M1-linked ubiquitination. By promoting mitochondrial M1-linked ubiquitination, RNF213 initiates the recruitment of the essential NF{kappa}B adaptor molecule, NEMO, and subsequently activates pro-inflammatory NF-{kappa}B signaling. Collectively, our findings highlight striking similarities between how cells detect damaged mitochondria and intracellular pathogens, leading to inflammation.

cell biology↗

bioRxiv2026

A conserved mechanism for regulation of mtDNA copy number in eukaryotes

Mitochondrial mass and mitochondrial DNA (mtDNA) copy number are coupled to metabolic demand at the cellular, tissue and organismal level, however, the molecular basis for homeostatic regulation of mtDNA is not understood. Here we show that mitochondria and mtDNA copy number are regulated by compartmentalisation of iron-sulfur (Fe-S) clusters, glutathione and cysteine, a mechanism we exemplify in model systems ranging from plants to human cells. Using genome-wide CRISPR screens we discovered that the mitochondrial ABC-family transporter, ABCB7, is a negative regulator of mtDNA copy number. Partial silencing of ABCB7 in human cells increased mtDNA 2-3 fold, enhancing mitochondrial mass and function. ABCB7 silencing compelled co-incident mitochondrial accumulation and cytosolic depletion of Fe-S clusters, simultaneously engaging the cellular iron starvation response and stabilising the mitochondrial glutathione transporter, SLC25A39. Transport of glutathione from the cytosol into mitochondria was co-incident with mitochondrial cysteine accumulation and cytosolic cysteine depletion, which was necessary and sufficient to increase mtDNA copy number in an integrated stress response-dependent fashion, with induction of PGC1{beta} and ERR. Silencing or partial loss of function mutations in the ABCB7 homologs of D.melanogaster, S.cerevisiae and A. thaliana elicited similar increases of mtDNA within these organisms. These data reveal a fundamental metabolic logic coupling compartmentalisation of redox co-factors to organellar genome content; a conserved axis across eukaryotes that pre-dates several elements of the mtDNA replication machinery.

cell biology↗

bioRxiv2026

Phenotype-dependent subtyping exposes high MYC activity as a targetable dependency in LuAd

C-MYC (MYC) occupies a critical nexus of oncogenic signalling and deregulated expression of MYC is widespread across most human cancer types, suggesting that MYC should be an attractive target for therapeutic intervention. Although 30-40% of human Non-Small Cell lung cancers show low level amplification of c-MYC and genetic evidence has shown that c-Myc is a key downstream effector of KRas-driven lung tumourigenesis in mouse models, the functional contribution of MYC to human lung cancer remains unclear. We applied a phenotype-based classifier to the TCGA Lung Adenocarcinoma (LuAd) cohort and found that high MYC transcriptional activity identifies a subset of LuAd with significantly reduced survival. Application of the same methodology to a panel of genetically engineered mouse models identified multiple genotypes that give rise to the high MYC activity phenotype, disease positioning such models as reflective of a distinct subset of human LuAd. We show that high MYC activity predicts sensitivity to a small molecule dual-inhibitor of the MYC co-factors, EZH2 and G9A, HKMTi-1-005, and that treatment with HKMTi-1-005 strongly reduced MYC protein expression, induced B cell-mediated immune surveillance and suppressed growth of autochthonous KRasG12D-driven lung tumours. Statement of significanceThis work establishes the principle of indirectly targeting MYC in LuAd, via inhibition of associated enzymatic cofactors, EZH2 and G9A, and identifies a large subset of aggressive human LuAd with a high MYC activity signature that may benefit from this approach.

cancer biology↗

bioRxiv2025

Exploring the Evolution of the Cariogenic Oral Pathobiont Streptococcus mutans Using Ancient DNA

The oral pathobiont Streptococcus mutans can contribute to dental caries development through metabolism of dietary carbohydrates. Adoption of carbohydrate-rich agricultural diets is associated with increased prevalence of dental caries in archaeological populations; however, the evolutionary impact of changing subsistence strategies on cariogenic microbes like S. mutans remains to be explored. Here, we use a novel hybridization capture reagent to generate genome-wide ancient DNA data from a global set of 75 S. mutans strains spanning the last 8,000 years. Most virulence-associated genes predate the origins of agriculture; however, we highlight loci regulating genetic competence, bacteriocin production, and biofilm formation which are absent in 5 strains from pre-agricultural ancient hunter-gatherers, suggesting that their acquisition may have been associated with adaptation to carbohydrate-rich agricultural diets. Together, our study highlights ancient DNA as a promising tool for exploring the dynamic interplay between subsistence strategy, microbes, and dental pathology in human populations through time.

evolutionary biology↗

bioRxiv2025

Genomic and morphometric evidence for Austronesian-mediated pig translocation in the Pacific

Human mediated translocation of non-native pig species (genus Sus) to the islands of Wallacea and Oceania has significantly altered local ecosystems. To investigate the timing and trajectory of these introductions, we conducted both genomic analyses of 576 pig nuclear genomes and a geometric morphometric analysis of 714 modern and ancient dental remains. Our analyses demonstrate that feral and domestic pigs in Wallacea and Oceania possess diverse ancestries resulting from the introduction of multiple, sequential pig populations followed by gene flow. Despite the variability in their genomic ancestry these pigs all possess a distinct tooth morphology, and a genetic link to the Chinese domestic pig populations that accompanied the dispersal of Austronesian language speakers [~]4,000-3,000 years ago via Taiwan and the Philippines.

evolutionary biology↗

bioRxiv2025

CDH-3/Cadherin, YAP-1/YAP and EGL-44/TEAD promote SYX-2/Syntaxin and EFF-1 fusogen-mediated phagosome closure

Physical interactions between cells, such as cell-cell junctions, can profoundly impact cell fate. A vital cell fate for normal development and homeostasis is programmed cell death. Cells fated to die must be efficiently cleared away via phagocytosis, and defects are associated with a variety of diseased states. Whether cell-cell physical associations affect programmed cell elimination has not been well-explored. Here we describe, in vivo, a cell-cell adhesion-driven signaling pathway that ensures compartment-specific cell clearance during development. We previously described the specialized cell death program "Compartmentalized Cell Elimination" (CCE) in the C. elegans embryo. During CCE, the tail-spike cell (TSC), a polarized epithelial cell, undergoes a tripartite, ordered, and organized death sequence, allowing for the study of three distinct death modalities in a single cell setting. Prior to its demise, the TSC serves as a scaffold for the tail tip, formed by the hyp10 epithelial cell which develops along the TSC process. The hyp10 cell in turn also serves as the phagocyte for the dying TSC process. Here we present data suggesting that the physical association between the dying TSC and hyp10 phagocyte via CDH-3/cadherin mediates function of the mechanosensitive transcriptional coactivator YAP-1/YAP and its partner EGL-44/TEAD in the hyp10 phagocyte to promote localization of hyp10 SYX-2/Syntaxin around the dying TSC remnant. This pathway facilitates the phagocytic function of EFF-1/fusogen, which we have previously shown to be required for phagosome sealing during CCE. Our work sheds additional light on a poorly understood step of phagocytosis and implicates adhesive forces and signaling between cells as important in cell uptake.

cell biology↗

bioRxiv2025

Saturation mapping of MUTYH variant effects using DNA repair reporters

Variants of uncertain significance (VUS) limit the actionability of genetic testing. A prominent example is MUTYH, a base excision repair factor associated with polyposis and colorectal cancer, which has a pathogenic variant carrier rate approaching 1 in 50 individuals in some populations. To systematically interrogate variant function in MUTYH, we coupled deep mutational scanning with a DNA repair reporter containing its lesion substrate, 8OG:A. Our variant-to-function map covers >97% of all possible MUTYH point variants (n=10,941) and achieves 100% accuracy classifying the pathogenicity of known clinical variants (n=247). Leveraging a large clinical registry, we observe significant associations with colorectal polyps and cancer, with more severely impaired missense variants conferring greater risk. We recapitulate known functional differences between pathogenic founder alleles, and highlight sites of complete missense intolerance, including residues that intercalate DNA and coordinate essential Zn2+ or Fe-S clusters. This map provides a resource to resolve the 1,032 existing missense VUS and 90 variants with conflicting interpretations in MUTYH, and demonstrates a scalable strategy to interrogate other clinically relevant DNA repair factors.

genomics↗

bioRxiv2025

Mitochondria transported by Kinesin 3 prevent localized calcium spiking to inhibit caspase-dependent specialized cell death

Polarized cells (such as neurons), defined by distinct compartments are, like many cell types, subject to developmental elimination, as in neurite pruning. The molecular mechanism behind specialized elimination remains a largely open question. We previously introduced the "tri-partite" embryonic cell death program Compartmentalized Cell Elimination (CCE) in the scaffolding tail-spike epithelial cell and sex-specific CEM neurons of C. elegans. CCE is stereotyped and ordered, with three distinct programs eliminating three cell compartments, and bearing morphological hallmarks reminiscent of neurite pruning. Here we report first, that, prior to CCE onset, mitochondria undergo UNC-116/Kinesin 1-dependent irreversible retrograde transport; and second, that the caspase protease CED-3 promotes the completion of the proximal nicking event of CCE by helping inhibit the kinesin 3 homolog UNC-104. While known canonically to carry only synaptic vesicles, UNC-104/Kinesin 3, in the CCE context, and in the absence of CED-3/Caspase, can transport mitochondria anterogradely to the severing site. We observe both caspase activity and Ca2+ spiking at this site prior to nicking. Mitochondria appear to protect against nicking in the absence of CED-3 via their MCU-1 uniporter-dependent Ca2+ uptake capacity. Our study sheds light on the molecular machinery of specialized cell elimination and pruning, highlighting involvement of region-specific Ca2+ signaling, an anti-death function of mitochondria via local Ca2+ uptake, mitochondrial transport as a regulatory strategy, with UNC-116/kinesin 1-mediated retrograde transport important for cell elimination priming and UNC-104/kinesin 3 in a previously undescribed role as a non-canonical anterograde mitochondrial motor.

cell biology↗

bioRxiv2024

EOR-1/PLZF-regulated WAH-1/AIF sequentially promotes early and late stages of non-apoptotic corpse removal

AbstractProgrammed cell death (PCD) is a crucial, genetically-encoded, and evolutionarily-conserved process required for development and homeostasis. We previously identified a genetically non-apoptotic, highly ordered, and stereotyped killing program called Compartmentalized Cell Elimination (CCE) in the C. elegans tail-spike epithelial cell (TSC). Here we identify the transcription factor EOR-1/PLZF as an important coordinator of CCE. Loss of EOR-1 results in a large, persisting, un-engulfed soma with enlarged nuclei. We find that EOR-1 and its partners positively regulate the transcription of the Apoptosis Inducing Factor AIF homolog, WAH-1/AIF. We report stereotyped and sequential spatiotemporal dynamics of WAH-1/AIF1 during phagocytosis, with defined roles acting early and late, within the dying cells. Mitochondria to plasma membrane translocation within the TSC soma is required its internalization by its phagocyte, and plasma membrane to nuclear translocation is required for DNA degradation and ultimately, corpse resolution. Our study suggests that EOR-1 serves as a master regulator for the transcriptional control of DNA degradation is essential for changes in nuclear morphology required for cellular dismantling and infers that tight spatiotemporal regulation of WAH-1/AIF is required for this function. Summary StatementThis work describes the genetic control and cellular dynamics of a factor linked to cancer, metabolic and degenerative disease acting in developmentally dying cells to instruct their own removal.

cell biology↗

bioRxiv2023

Exploring archaeogenetic studies of dental calculus to shed light on past human migrations in Oceania

The Pacific islands have experienced multiple waves of human migrations, providing a case study for exploring the potential of using the microbiome to study human migration. We performed a metagenomic study of archaeological dental calculus from 103 ancient individuals, originating from 12 Pacific islands and spanning a time range of [~]3000 years. Oral microbiome DNA preservation in calculus is far higher than that of human DNA in archaeological bone from the Pacific, and comparable to that seen in calculus from temperate regions. Variation in the microbial community composition was minimally driven by time period and geography within the Pacific, while comparison with samples from Europe, Africa, and Asia reveal the microbial communities of Pacific calculus samples to be distinctive. Phylogenies of individual bacterial species in Pacific calculus reflect geography. Archaeological dental calculus shows potential to yield information about past human migrations, complementing studies of the human genome.

microbiology↗

bioRxiv2023

FYN regulates aqueous humor outflow and IOP through the phosphorylation of VE-cadherin

The exact sites and molecules that determine resistance to aqueous humor drainage and control intraocular pressure (IOP) need further elaboration. Proposed sites include the inner wall of Schlemmss canal and the juxtacanalicular trabecular meshwork ocular drainage tissues. The adherens junctions (AJs) of Schlemms canal endothelial cells (SECs) must both preserve the blood-aqueous humor (AQH) barrier and be conducive to AQH drainage. How homeostatic control of AJ permeability in SC occurs and how such control impacts IOP is unclear. We hypothesized that mechano-responsive phosphorylation of the junctional molecule VE-CADHERIN (VEC) by SRC family kinases (SFKs) regulates the permeability of SEC AJs. We tested this by clamping IOP at either 16 mmHg, 25 mmHg, or 45 mmHg in mice and then measuring AJ permeability and VEC phosphorylation. We found that with increasing IOP: 1) SEC AJ permeability increased, 2) VEC phosphorylation was increased at tyrosine-658, and 3) SFKs were activated at the AJ. Among the two SFKs known to phosphorylate VEC, FYN, but not SRC, localizes to the SC. Furthermore, FYN mutant mice had decreased phosphorylation of VEC at SEC AJs, dysregulated IOP, and reduced AQH outflow. Together, our data demonstrate that increased IOP activates FYN in the inner wall of SC, leading to increased phosphorylation of AJ VEC and, thus, decreased resistance to AQH outflow. These findings support a crucial role of mechanotransduction signaling in IOP homeostasis within SC in response to IOP. These data strongly suggest that the inner wall of SC partially contributes to outflow resistance.

cell biology↗

bioRxiv2023

Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states

Schlemms canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemms canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel, and lymphatic endothelial cells from limbal tissue ([~]4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics. Significance statementSchlemms canal is a critical ocular drainage structure that maintains intraocular pressure with important consequences for glaucoma. Despite this, Schlemms canal has been relatively understudied with the molecular process that mediate its function, maintain its health, or that are compromised in glaucoma needing much deeper characterization. This study is important as it provides the most comprehensive molecular characterization of Schlemms canal cells to date providing a wealth of molecular details. In addition to highlighting important biological pathways, this information will guide studies to develop new treatments against glaucoma.

genomics↗

bioRxiv2023

Role of Apyrase in Mobilization of Phosphate from Extracellular Nucleotides and in Regulating Phosphate Uptake in Arabidopsis

Apyrase (nucleotide triphosphate diphosphohydrolase, NTPDase; EC 3.6.1.5) functions in a variety of plant growth and developmental processes and responses to pathogens, in part, by regulating extracellular ATP (eATP) concentrations. In this study, we investigated possible roles for apyrase in the recruitment of phosphate (Pi) from extracellular nucleotides in Arabidopsis thaliana seedlings that constitutively express apyrase 1 (APY1). Under Pi limitation, both WT and APY1 seedlings had decreased Pi contents and a characteristic remodeling of root system architecture (RSA). This phosphate starvation response (PSR) was prevented by uptake of Pi released by metabolism of extracellular NTP, which supported normal seedling growth. In Pi-sufficient media, Pi contents of APY1 seedlings were higher than in WT seedlings. Addition of NTP reduced the number of LR and decreased Pi contents of WT seedlings but markedly increased both LR and root hair (RH) formation and Pi contents of APY1 seedlings. Genome-wide expression profiling revealed the repression of PSR genes in APY1 seedlings, relative to WT seedlings, consistent with their elevated Pi contents. The expanded RSA of APY1 seedlings was correlated with induction of >100 genes involved in regulation of auxin homeostasis, signaling and transport, which previous studies have shown to be increased when APY1 is overexpressed. APY1 regulation of [eNTP] and purinergic signaling may play a role in regulating this auxin response, resulting in enhanced uptake of Pi from the medium, including Pi released by eNTP metabolism.

plant biology↗

bioRxiv2023

ERBB signalling contributes to immune evasion in KRAS-driven lung adenocarcinoma

Immunotherapy is increasingly viewed as treatment of choice for lung cancer, however, clinical responses to immune checkpoint blockade remain highly unpredictable and are largely transient. A deeper mechanistic understanding of the dynamics of tumour:immune interactions is needed to drive rational development of improved treatment strategies. Progress is hampered by a paucity of autochthonous model systems in which to interrogate the 2-way interactions of immune responses to evolving tumours and vice-versa. Specifically, commonly used genetically engineered mouse models typically lack the genetic diversity needed to drive an adaptive immune response. APOBEC mutagenesis signatures are prominent in lung cancer and APOBEC activity is predicted to drive immune visibility through Cytidine deaminase activity, coupled with inaccurate DNA-repair responses. We therefore generated a CRE-inducible APOBEC3B allele, interbred with multiple oncogenic drivers of lung adenocarcinoma, and used the resulting mice to investigate the response to PD1 blockade at single cell resolution. SIGNIFICANCE Using our novel immune-visible model of KRas-driven autochthonous lung adenocarcinoma, we uncovered a surprising increase in tumour-cell expression of EGFR/ERBB ligands following treatment with -PD1 and present evidence that transient ERBB blockade can restore immune surveillance in KRas mutant LuAd and combine effectively with immune checkpoint blockade.

cancer biology↗

bioRxiv2022

The propensity to sign-track is associated with externalizing behaviour and distinct patterns of reward-related brain activation in youth

Externalizing behaviours in childhood often predict impulse control disorders in adulthood; however, the underlying biobehavioural risk factors are incompletely understood. In animals, the propensity to signtrack, or the degree to which incentive motivational value is attributed to reward cues, is associated with externalizing-type behaviours and deficits in executive control. Using a Pavlovian conditioned approach paradigm, we quantified sign-tracking in healthy 9-12-year-olds. We also measured parent-reported externalizing behaviours and anticipatory neural activations to outcome-predicting cues using the monetary incentive delay fMRI task. Sign-tracking was associated with attentional and inhibitory control deficits and the degree of amygdala, but not cortical, activation during reward anticipation. These findings support the hypothesis that youth with a propensity to sign-track are prone to externalizing tendencies, with an over-reliance on subcortical cue-reactive brain systems. This research highlights sign-tracking as a promising experimental approach delineating the behavioural and neural circuitry of individuals at risk for externalizing disorders.

neuroscience↗

bioRxiv2021

Using Oxford Nanopore Technology direct RNA sequencing to identify depurination events induced by ricin and other ribosome inactivating proteins

Depurination is a frequent modification to both DNA and RNA, in DNA causing point mutations through misincorporation, in RNA, disabling ribosomes and halting protein synthesis. Some modifications of nucleic acids can be determined by direct sequencing using Oxford Nanopore Technologies (ONT). However, the identification of modifications is often limited by noise and their variety and number. Ricin is a toxin which enters cells and depurinates an adenine base in the sarcin-ricin loop of the large ribosomal subunit. This leaves only a ribose backbone, thus inhibiting protein translation. In humans, biological threat agents and ribosome inactivating proteins, such as ricin and saporin, depurinate base 4605 on the 28S rRNA providing a single defined target to try and identify. We postulated that the depurination event could be detected using ONT direct RNA sequencing through a change in charge in the ricin loop. A software tool was developed, RIPpore, that quantified the adenine modification from direct RNA sequencing data of ribosomal RNA purified from respiratory epithelial cells exposed to ricin. This provided a novel method of directly identifying ricin exposure and a basis for ONTs utility in detecting lesions in nucleic acids caused by depurination events.

pharmacology and toxicology↗

bioRxiv2021

GLIS1 regulates trabecular meshwork function and intraocular pressure and is associated with glaucoma in humans

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open- angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Kruppel- like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- and extracellular matrix-associated genes as direct transcriptional targets of GLIS1. We also identified a significant association between GLIS1 variant rs941125 and glaucoma in humans (P=4.73x10-6), further supporting a role for GLIS1 into glaucoma etiology. Our study identifies GLIS1 as a critical regulator of TM function and maintenance, AqH dynamics, and IOP.

molecular biology↗

bioRxiv2020

Portable Take-Home System Enables Proportional Control and High-Resolution Data Logging with a Multi-Degree-of-Freedom Bionic Arm

This paper describes a portable, prosthetic control system for at-home use of an advanced bionic arm. The system uses a modified Kalman filter to provide 6 degree-of-freedom, real-time, proportional control. We describe (a) how the system trains motor control algorithms for use with an advanced bionic arm, and (b) the systems ability to record an unprecedented and comprehensive dataset of EMG, hand positions and force sensor values. Intact participants and a transradial amputee used the system to perform activities-of-daily-living, including bi-manual tasks, in the lab and at home. This technology enables at-home dexterous bionic arm use, and provides a high-temporal resolution description of daily use--essential information to determine clinical relevance and improve future research for advanced bionic arms.

bioengineering↗