Biology subjects

Wheeler, A.

Publications and source records attributed to Wheeler, A..

10 recordsLinked to original sources

bioRxiv2026

Tryptophan became part of the universal genetic code post-LUCA

We evaluate whether tryptophan (W), widely thought to be the last of the 20 canonical amino acids added to the genetic code, was already present in the Last Universal Common Ancestor (LUCA). We reconstruct the evolutionary history of tryptophanyl-tRNA synthetase (WRS), the enzyme that attaches W to its tRNA, and the related tyrosyl-tRNA synthetase (YRS). We identify and exclude sequences derived from ancient recombination between archaeal and bacterial YRSs. Diverse rooting methods, including a novel approach exploiting time non-reversible evolution, all place the root between bacterial and archaeal YRS rather than between YRS and WRS. This supports post-LUCA WRS origination in Archaea, followed by its horizontal transfer to Bacteria. However, ancestral sequence reconstruction suggests that Archaea were depleted for W while Bacteria were not, and enzymes essential for W biosynthesis emerged in Bacteria. This suggests that W usage originated in Bacteria, with later WRS emergence in Archaea allowing the archaeal genetic code to converge with the bacterial code. The universality of the genetic code is usually attributed to common descent from LUCA, but the final step making the code universal was instead achieved by horizontal gene transfer. This gives credence to similar mechanisms for earlier steps in genetic code evolution.

evolutionary biology↗

bioRxiv2025

ER-liquid condensate contacts sequester FAM134B/C and RhoA to govern cell morphology

ER-phagy receptors have elusive physiological functions beyond ER remodelling. Here, we use proximity biotinylation to identify their cytoplasmic interactomes. Secondary CRISPR/Cas9 screening reveals regulators of the prototypical FAM134B/C receptors, which include PRKAR1A, canonically known as a subunit of PKA. PRKAR1A directly binds an amphipathic helix in the otherwise disordered cytoplasmic domain of FAM134B. Super-resolution, FRAP and CLEM imaging reveal novel interorganellar contacts between liquid-like condensates of cAMP-bound PRKAR1A and the ER. Condensates promote clustering of ER-embedded FAM134B/C with LC3B, independently of regulation of PKA by PRKAR1A. Proteomics reveal that cytoplasmic RhoA interacts with FAM134B/C clusters and that sequestration of both these molecules occurs within lysosomes embedded within the proximal condensates. This results in reduced actomyosin contractility and ER-condensate interactions thusly determine cell morphology and cancer cell invasion modality. In summary, ER-condensate contacts mediated by FAM134B/C are novel cellular degradation hubs that coordinate ER and cellular remodelling.

cell biology↗

bioRxiv2025

Chronic lysosome damage boosts interferon responses to Palbociclib through a mitochondrial signalling axis

Acute lysosome damage triggers the endolysosome damage response (ELDR) in order to co-ordinate vesicle repair or removal by autophagy (lysophagy). However, it is unclear whether persistent damage, as occurs after chronic challenge to lysosome integrity, triggers wider cellular responses. Here, we show that longitudinal treatment with a lysosomotropic cancer therapeutic, the CDK4/6 inhibitor Palbociclib, invokes chronic lysosome damage in breast and lung cancer cells. Autophagy ameliorates but does not avert this phenotype, which persists over days. Damaged lysosomes form contacts with mitochondria, which correlates with mitochondrial stress and cytosolic efflux of immunostimulatory mitochondrial nucleic acids. Importantly, mitochondrial nucleic acid release is necessary for the anti-cancer interferon response to Palbociclib. In conclusion, chronic lysosome damage rewires cellular signalling responses in a mitochondrion-dependent manner and this effect should be considered when assessing the cellular actions of cancer therapeutics. Summary statementBozic et al suggest that lysosome damage can trigger interferon responses dependent upon mitochondrial release of immunogenic nucleic acid. This is associated with damaged lysosome-mitochondrion contacts and is prevented by autophagy.

cell biology↗

bioRxiv2024

Identifying the Last Universal Common Ancestor's protein domains resolves the order in which the amino acids were recruited into the genetic code

The current "consensus" order in which amino acids were added to the genetic code is based on potentially biased criteria, such as absence of sulfur-containing amino acids from the Urey-Miller experiment which lacked sulfur. More broadly, abiotic abundance might not reflect biotic abundance in the organisms in which the genetic code evolved. Here, we instead identify which protein domains date to the last universal common ancestor (LUCA), then infer the order of recruitment from deviations of their ancestrally reconstructed amino acid frequencies from the still-ancient post-LUCA controls. We find that smaller amino acids were added to the code earlier, with no additional predictive power in the previous "consensus" order. Metal-binding (cysteine and histidine) and sulfur-containing (cysteine and methionine) amino acids were added to the genetic code much earlier than previously thought. Methionine and histidine were added to the code earlier than expected from their molecular weights, and glutamine later. Early methionine availability is compatible with inferred early use of S-adenosylmethionine, and early histidine with its purine-like structure and the demand for metal-binding. Even more ancient protein sequences -- those that had already diversified into multiple distinct copies prior to LUCA -- have significantly higher frequencies of aromatic amino acids (tryptophan, tyrosine, phenylalanine and histidine), and lower frequencies of valine and glutamic acid than single copy LUCA sequences. If at least some of these sequences predate the current code, then their distinct enrichment patterns provide hints about earlier, alternative genetic codes. Significance StatementThe order in which the amino acids were added to the genetic code was previously inferred from consensus among forty metrics. Many of these reflect abiotic abundance on ancient Earth. However, the abundances that matter are those within primitive cells that already had sophisticated RNA and perhaps peptide metabolism. Here, we directly infer the order of recruitment from the relative ancestral amino acid frequencies of ancient protein sequences. Small size predicts ancient amino acid enrichment better than the previous consensus metric does. We place metal-binding and sulfur-containing amino acids earlier than previously thought, highlighting the importance of metal-dependent catalysis and sulfur metabolism to ancient life. Understanding early life has implications for our search for life elsewhere in the universe.

evolutionary biology↗

bioRxiv2022

DNMT3B PWWP mutations cause hypermethylation of heterochromatin

The correct establishment of DNA methylation patterns is vital for mammalian development and is achieved largely by the de novo DNA methyltransferases DNMT3A and DNMT3B. Mutations in DNMT3B can cause immunodeficiency-centromeric instability-facial anomalies type 1 (ICF1) syndrome which is characterised by hypomethylated heterochromatin. However, in the genome, DNMT3B primarily localises to actively transcribing gene bodies through the interaction of its PWWP domain with the histone modification H3K36me3 and it is unclear how it is recruited to heterochromatin. Here we show that in DNMT3B knockout cells, loss of DNA methylation predominantly occurs in heterochromatic domains marked by H3K9me3. We also find that PWWP domain mutations which disrupt DNMT3Bs interaction with H3K36me3 result in striking increases of DNA methylation in H3K9me3-marked heterochromatin. Gains of methylation are also observed when the PWWP domain of DNMT3B is deleted. In contrast, we find that the ICF1 syndrome-causing PWWP mutation, S270P, does not result in hypermethylation of heterochromatin and destabilises the protein. We also show that removal of the N-terminus region of DNMT3B affects its recruitment to chromatin and ability to methylate H3K9me3 marked regions. Our results suggest that DNMT3B is recruited to H3K9me3 marked heterochromatin in a PWWP-independent manner and that this recruitment is facilitated by the proteins N-terminus. More generally, we suggest that DNMT3B plays a role in DNA methylation homeostasis at heterochromatin, a process which is disrupted in ICF syndrome, cancer and aging.

genetics↗

bioRxiv2021

Structural Characteristics and Proton Conductivity of the Gel Within the Electrosensory Organs of Cartilaginous Fishes

The Ampullae of Lorenzini (AoL) of cartilaginous fishes are sensory organs used to detect environmental electric fields. The proximal ends of the organs are externally visible as pores in the skin that lead into gel-filled tubular canals which terminate in rounded chambers filled with highly specialized electrosensory cells. The viscoelastic gel that fills the organs is composed of proteins and polysaccharides that are not yet completely characterized but are thought to play a critical role in the electrosensing mechanism. Although recent studies have identified various components of AoL gel, it has remained unclear how the component molecules are structurally arranged and how their structure influences the overall function of the AoL. Here we present the first microscopic descriptions and x-ray scattering data from AoL gel extracted from spotted ratfish (Hydrolagus colliei). Our results suggest that AoL gel is colloidal in nature and composed of spherical globules that are approximately 10-100 nm in size. We investigated the structural influence of the protein components of the gel specifically by analyzing gel that had been digested in situ via enzymatic proteolysis. By comparing gel before and after digestion using microscopy, x-ray scattering analyses, and proton conductivity measurements, we directly observed the structural and functional influence of proteins in AoL gel. The findings described here represent the first detailed structural analysis of AoL gel and lay the groundwork for more detailed studies into the specific interactions of molecules inside AoL gel at the nanoscale, with particular reference to their mechanistic role in electrosensing.

biophysics↗

bioRxiv2020

Periodic propagating activation waves coordinate Rho GTPase network dynamics at the leading and trailing edges during cell migration

Migrating cells need to coordinate distinct leading and trailing edge dynamics but the underlying mechanisms are unclear. Here, we combine experiments and mathematical modeling to elaborate the minimal autonomous biochemical machinery necessary and sufficient for this dynamic coordination and cell movement. RhoA activates Rac1 via DIA and inhibits Rac1 via ROCK, while Rac1 inhibits RhoA through PAK. Our data suggest that in motile, polarized cells, RhoA-ROCK interactions prevail at the rear whereas RhoA-DIA interactions dominate at the front where Rac1/Rho oscillations drive protrusions and retractions. At the rear, high RhoA and low Rac1 activities are maintained until a wave of oscillatory GTPase activities from the cell front reaches the rear, inducing transient GTPase oscillations and RhoA activity spikes. After the rear retracts, the initial GTPase pattern resumes. Our findings show how periodic, propagating GTPase waves coordinate distinct GTPase patterns at the leading and trailing edge dynamics in moving cells.

systems biology↗

bioRxiv2018

Super-resolution fight club: A broad assessment of 2D & 3D single-molecule localization microscopy software

With the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. To guide researchers on the optimal analytical software for their experiments, we have designed, in a large community effort, a competition to extensively characterise and rank these options. We generated realistic simulated datasets for popular imaging modalities - 2D, astigmatic 3D, biplane 3D, and double helix 3D - and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D single molecule localization microscopy software, provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions, and ultimately provides insight into the current limits of the field.

biophysics↗

bioRxiv2018

Activated BAX/BAK enable mitochondrial inner membrane permeabilisation and mtDNA release during cell death

During apoptosis, pro-apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP. Such caspase-independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS-STING signaling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS-STING signaling pathway. Strikingly, using super-resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP. In a temporal manner, we find that following MOMP, BAX/BAK-mediated mitochondrial outer membrane pores gradually widen over time. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation can occur during cell death in a BAX/BAK-dependent manner. Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase-independent cell death.

cell biology↗

bioRxiv2017

5-Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages

5-Hydroxymethylcytosine (5hmC) is an important mammalian DNA epigenetic modification that has been linked to gene regulation and cancer pathogenesis. Here we explored the diagnostic potential of 5hmC in circulating cell-free DNA (cfDNA) using a sensitive chemical labeling-based low-input shotgun sequencing approach. We sequenced cell-free 5hmC from 49 patients of seven different cancer types and found distinct features that could be used to predict cancer types and stages with high accuracy. Specifically, we discovered that lung cancer leads to a progressive global loss of 5hmC in cfDNA, whereas hepatocellular carcinoma and pancreatic cancer lead to disease-specific changes in the cell-free hydroxymethylome. Our proof of principle results suggest that cell-free 5hmC signatures may potentially be used not only to identify cancer types but also to track tumor stage in some cancers.\n\nOne Sentence SummaryAnalyzing the epigenetic modification 5-hydroxymethylcysoine in circulating cell-free DNA reveals tumor tissue of origin and stages for cancer diagnostics.

genomics↗