Biology subjects

Moriel-Carretero, M.

Publications and source records attributed to Moriel-Carretero, M..

10 recordsLinked to original sources

bioRxiv2026

The DNA Damage Response kinase ATM restricts Golgi extension

The master kinases of the DNA damage response (DDR), ATR, ATM and DNA-PK, become active in response to DNA damage and orchestrate a downstream wave of phosphorylations contributing to DNA damage repair and preservation of cellular homeostasis. Of them, we recently demonstrated that ATM binds the pool of the lipid phosphatidyl-inositol-4-phosphate (PI4P) situated at the Golgi membrane. Depending on PI4P availability at Golgi membranes, ATM is more or less titrated away from the nucleus, which translates into responses to nuclear DNA damage of matching intensity. Building on this knowledge, in this work we asked if, beyond the Golgi merely serving as a docking platform that retains ATM away from the nucleus, ATM does exert any role important for Golgi biology. We found that ATM maintains Golgi morphology by counteracting its excessive deployment. This occurs both by its mere presence (likely antagonizing the Golgi-stretching action of the protein GOLPH3) and by phosphorylating Golgi-resident substrates. Of relevance, we also report that the morphological alterations caused to the Golgi without ATM affect the biology of a model Golgi cargo. Our findings nourish the growing evidence that kinases of ATMs family display functional interactions with membranes and highlights an underappreciated crosstalk between the Golgi and the nucleus.

cell biology↗

bioRxiv2025

Compromised DNA replication in gut cells underlies tardigrade sensitivity to genotoxic stress

Tardigrades withstand severe DNA insults, including extreme doses of ionizing radiation, through unique protective proteins and strong upregulation of canonical DNA repair pathways. Yet, these extremophile animals are not immortal, and the cellular and organismal processes that ultimately fail under sustained genotoxic stress have not been characterized. Here, we identify DNA replication as the key vulnerability in the tardigrade Hypsibius exemplaris. Using the radiomimetic drug zeocin to induce DNA breaks, we show that continuous exposure progressively kills tardigrades, accompanied by striking body shrinkage and lipid depletion. DNA synthesis labeling reveals that zeocin disrupts replication and triggers de novo reparative synthesis in select non-dividing tissues. Pulse-wash experiments demonstrate that even transient damage to dividing gut cells irreversibly exhausts their replicative capacity, leading to midgut failure and animal death, despite systemic induction of DNA repair genes. Germ cells and embryos, with their high proliferation rates, show heightened sensitivity. Cross-phyla survival assays in the eutelic nematode Caenorhabditis elegans and neoblast-rich planarian Schmidtea mediterranea further link proliferative activity to mortality kinetics under DNA damage. Collectively, our findings pinpoint DNA replication as an Achilles heel of organismal survival under genotoxic stress, even in animals renowned for their extraordinary DNA damage tolerance.

cell biology↗

bioRxiv2024

SARS-CoV-2 predation of Golgi-bound PI4P primes the massive activation of the DNA Damage Response kinase ATM in the cytoplasm

Like all viruses, SARS-CoV-2, the causative agent of COVID-19, relies on host cell resources to replicate. Our study reveals that, among these resources, SARS-CoV-2 hijacks the oxysterol-binding protein 1 (OSBP1) transporter to exploit the Golgi-bound phosphatidylinositol-4-phosphate (PI4P) pool. This leads to a depletion of Golgi-resident PI4P, triggering the activation of the ATM DNA Damage Response (DDR) kinase in the cytoplasm. As such, ATM, typically anchored to PI4P at the Golgi in an inactive state, undergoes auto-phosphorylation and cytoplasmic release upon SARS-CoV-2-induced PI4P depletion. Conversely, pharmacological inhibition of ATM auto-phosphorylation, which stabilizes its interaction with PI4P, significantly impairs SARS-CoV-2 replication. The requirement for PI4P and impact of ATM inhibition might be conserved across coronaviruses, as similar effects were observed with HCoV-229E. Finally, SARS-CoV-2-induced, cytoplasmic ATM pre-activation primes cells for an accelerated response to DNA damage, which might contribute to the severe outcomes of COVID-19 observed in cancer patients undergoing chemo- or radiotherapy. Therefore, this study uncovers a DNA damage-independent mode of ATM activation and highlights the potential of ATM inhibitors as therapeutic agents against COVID-19.

microbiology↗

bioRxiv2024

Neutral lipids restrict the mobility of broken DNA molecules during comet assays

One widespread technique to assess in relative terms the amount of broken DNA present in the genome of individual cells consists in immobilizing the cells nucleus under an agarose pad (called the nucleoid) and subject the whole genome to electrophoresis to force broken DNA molecules out of it. Since the migrating DNA molecules create a tail behind the nucleoid, this technique is named the comet assay. While performing comet assays regularly, we systematically observed circular regions devoid of DNA within the nucleoid region. We characterize here that these correspond to clusters of neutral (apolar) lipids, since they could be labelled with neutral lipid-dying molecules, increased when cells were fed with oleic acid, and were irresponsive to the electrophoretic field. Of relevance, de-lipidation assays either in vivo, or in vitro, show that these neutral lipids within the nucleoid limit the ability of broken DNA molecules to migrate into the comet tail. From a technical point of view, we show that de-lipidation permits a wider range for the detection of broken DNA molecules. Biologically, we put forward the notion that neutral lipids in contact with DNA may locally exert regulatory functions within the cells nucleus.

cell biology↗

bioRxiv2023

Storage cell proliferation during somatic growth establishes that tardigrades are not eutelic organisms

Tardigrades, microscopic ecdysozoans renowned for their resilience to extreme environments, have long been thought to maintain a constant cell number after completing embryonic development, a phenomenon known as eutely. However, sporadic reports of dividing cells have raised questions about this assumption. In this study, we investigated whether tardigrades truly exhibit a fixed cell number during somatic growth using the model species Hypsibius exemplaris. Comparing hatchlings to adults, we observed an overall increase in the number of storage cells, a tardigrade cell type involved in nutrient storage. To assess cell proliferation, we monitored DNA replication via the incorporation of the thymidine analog 5-ethynyl-2-deoxyuridine (EdU). A significantly higher number of storage cells incorporated EdU while animals were still growing. Starvation halted both animal growth and storage cell proliferation, linking the two processes. Additionally, we found that EdU incorporation in storage cells is associated with molting, a critical process in tardigrade post-embryonic development, since it involves cuticle renewal to enable further growth. Finally, we show that hydroxyurea, a drug that slows down DNA replication progression, strongly reduces the number of EdU+ cells and results in molting-related fatalities. Our data not only provide a comprehensive picture of replication events during tardigrade growth but also highlight the critical role of proper DNA replication in tardigrade molting and survival. This study definitively challenges the notion of eutely in tardigrades, offering promising avenues for exploring cell cycle, replication stress, and DNA damage management in these remarkable creatures as genetic manipulation techniques emerge within the tardigrade research field. SIGNIFICANCETardigrades, microscopic invertebrate animals renowned for their resilience in extreme conditions, have traditionally been considered eutelic, implying little to no somatic cell proliferation during their growth. However, a few isolated reports challenged this notion. In this study, using the emerging model Hypsibius exemplaris, we provide unequivocal molecular evidence of DNA replication and proliferation in a specific tardigrade cell type called storage cells, primarily involved in nutrient storage, throughout the animals growth. Furthermore, we demonstrate that this proliferation is associated with the timing of cuticle molting, and we highlight the critical role of proper DNA replication in tardigrade molting and survival. Our research definitively resolves the long-standing controversy surrounding tardigrade eutely, opening up uncharted territories in tardigrade research.

developmental biology↗

bioRxiv2022

A Sterol-PI(4)P exchanger controls the Tel1/ATM axis of the DNA Damage Response

Upon DNA damage, cells activate the DNA Damage Response (DDR) to coordinate proliferation and DNA repair. Dietary, metabolic, and environmental inputs are emerging as modulators of how DNA surveillance and repair take place. Lipids hold potential to convey these cues, although little is known about how. We observed that lipid droplet (LD) number specifically increased in response to DNA breaks. We show that the selective storage of sterols into these LD concomitantly stabilizes phosphatidyl-4-inositol (PI(4)P) at the Golgi, where it binds the DDR kinase ATM. In turn, this titration attenuates the initial nuclear ATM-driven response to DNA breaks, thus allowing processive repair. Further, manipulating this loop impacts the kinetics of DNA damage signaling and repair in a predictable manner. Thus, our findings have major implications for tackling genetic instability pathologies through dietary and pharmacological interventions.

cell biology↗

bioRxiv2021

A CRISPR-Cas9-based system for the dose-dependent study of DNA double strand breaks sensing and repair

The integrity of DNA is put at risk by different lesions, among which double strand breaks (DSBs) occur at low frequency, yet remain one of the most life-threatening harms. The study of DSB repair requests tools provoking their accumulation, and include the use of chemical genotoxins, ionizing radiations or the expression of sequence-specific nucleases. While genotoxins and irradiation allow for dose-dependent studies, nuclease expression permits assessments at precise locations. In this work, we have exploited the repetitiveness of the Ty transposon elements in the genome of Saccharomyces cerevisiae and the cutting activity of the RNA-guided Cas9 nuclease to create a tool that combines sequence specificity and dose-dependency. In particular, we can achieve the controlled induction of 0, 1, 15 or 59 DSBs in cells with an otherwise identical genetic background. We make the first application of this tool to better understand the behavior of the apical kinase of the DNA damage response Tel1 in the nuclear space. We found that Tel1 is capable of forming nuclear foci, which are clustered by condensing when DSBs occur in Ty elements. In striking contrast with other DSB-related protein foci, Tel1 foci are in tight contact with the nuclear periphery, therefore suggesting a role for the nuclear membrane in their congregation.

genetics↗

bioRxiv2021

Negative curvature-promoting lipids instruct nuclear ingression of low autophagic potential vacuoles

Membrane contact sites are functional nodes at which organelles exchange information through moving ions, proteins and lipids, thus driving the reorganization of metabolic pathways and the adaptation to changing cues. The nuclear-vacuole junction of Saccharomyces cerevisiae is among the most extensive and better-known organelle contact sites, described to expand in response to various metabolic stresses. While using genotoxins with unrelated purposes, we serendipitously discovered a phenomenon that we describe as the most extreme and intimate contact ever reported between nuclei and vacuoles: the vacuole becomes completely internalized in the nucleus. We define lipids supporting negative curvature, such as phosphatidic acid and sterols, as bona-fide drivers of this event. Functionally, we purport that internalized vacuoles are low efficiency ones whose removal from the cytoplasm optimizes cargo interaction with functional vacuoles. Thus, our findings also point to nucleus-vacuole interactions as important for metabolic adaptation. Yet, rather than by inter-organelle exchanges, the underlying mechanism literally concurs with vacuolar sequestration.

cell biology↗

bioRxiv2020

PickPocket : Pocket binding prediction for specific ligands family using neural networks.

Most of the protein biological functions occur through contacts with other proteins or ligands. The residues that constitute the contact surface of a ligand-binding pocket are usually located far away within its sequence. Therefore, the identification of such motifs is more challenging than the linear protein domains. To discover new binding sites, we developed a tool called PickPocket that focuses on a small set of user-defined ligands and uses neural networks to train a ligand-binding prediction model. We tested PickPocket on fatty acid-like ligands due to their structural similarities and their under-representation in the ligand-pocket binding literature. Our results show that for fatty acid-like molecules, pocket descriptors and secondary structures are enough to obtain predictions with accuracy >90% using a dataset of 1740 manually curated ligand-binding pockets. The trained model could also successfully predict the ligand-binding pockets using unseen structural data of two recently reported fatty acid-binding proteins. We think that the PickPocket tool can help to discover new protein functions by investigating the binding sites of specific ligand families. The source code and all datasets contained in this work are freely available at https://github.com/benjaminviart/PickPocket. Author SummaryMost of the protein biological functions are defined by its interactions with other proteins or ligands. The cavity of the protein structure that receives a ligand, also called a pocket, is made of residues that are usually located far away within its sequence. Therefore understanding the omplementarity of pocket and ligand is a real challenge. To discover new binding sites, we developed a tool called PickPocket that focuses on a small set of user-defined ligands to train a prediction model. Our results show that for fatty acid-like molecules, pocket descriptors (such as volume, shape, hydrophobicity...) and secondary structures are enough to obtain predictions with accuracy >90% using a dataset of 1740 manually curated ligand-binding pockets. The trained model could also successfully predict the ligand-binding pockets using unseen structural data of two recently reported fatty acid-binding proteins. We think that the PickPocket tool can help to discover new protein functions by investigating the binding sites of specific ligand families.

bioinformatics↗

bioRxiv2019

A new role of the Mus81 nuclease for replication completion after fork restart

Impediments to DNA replication threaten genome stability. The homologous recombination (HR) pathway is involved in the restart of blocked replication forks. Here, we used a new method to study at the molecular level the restart of replication in response to DNA topoisomerase I poisoning by camptothecin (CPT). We show that HR-mediated restart at the global genomic level occurs by a BIR-like mechanism that requires Rad52, Rad51 and Pol32. The Mus81 endonuclease, previously proposed to cleave blocked forks, is not required for replication restart in S phase but appears to be essential to resolve fork-associated recombination intermediates in G2/M as a step necessary to complete replication. We confirmed our results using an independent system that allowed us to conclude that this mechanism is independent of the accumulation of DNA supercoiling and DNA-protein crosslinks normally caused by CPT. Thus, we describe here a specific function for Mus81 in the processing of HR-restarted forks required to complete DNA replication.

molecular biology↗