Biology subjects

Bernardi, P.

Publications and source records attributed to Bernardi, P..

8 recordsLinked to original sources

bioRxiv2026

Label-free real-time imaging of mitochondrial matrix volume changes and permeability transition in living cells

Along with the membrane potential and respiration, mitochondrial matrix volume is a critical parameter that determines mitochondrial function. Mitochondria undergo constant changes in matrix volume and cristae dynamics, and in processes that are critical for normal metabolic rates and pathophysiological responses. Changes in matrix volume cannot be easily measured by conventional fluorescence imaging techniques due to the size of the sub-organellar structures, which are below resolution. This challenge was successfully resolved in studies of isolated mitochondria with the use of scattered light. Here we use dark-field imaging, which relies on scattered light contrast, to measure matrix volume dynamics in living cells. We demonstrate that mitochondrial volume changes can be easily detected as changes in intensity of the scattered light following matrix volume modulation with K+ ionophores or by onset of the permeability transition. Specifically, we found that stimulation of K+ influx leads to increase of mitochondrial matrix volume while stimulation of K+ efflux leads to matrix shrinkage, and that activation of the permeability transition leads to high-amplitude mitochondrial swelling in wild-type but not in cells lacking subunit c of ATP synthase. These results directly demonstrate the dynamic nature of mitochondrial matrix volume and its link to physiological and pathological ion transport.

cell biology↗

bioRxiv2026

Novel KIF22 Variants Disrupt Mitosis in Human Chondrocytes and Expand SEMDJL2 Mechanisms

Spondyloepimetaphyseal dysplasia with joint laxity, type 2 (SEMDJL2) is a rare skeletal disorder caused by pathogenic variants in KIF22, a mitotic chromokinesin that generates polar ejection forces (PEF) to ensure proper chromosome alignment and segregation. Although prior work showed that SEMDJL2-associated KIF22 hotspot variants impair chromosome segregation in epithelial cells, how these variants affect chondrocyte mitosis remains incompletely understood. Here, we analyzed the effects of the hotspot variant R149Q, a recently reported recessive variant R49Q, and two newly identified heterozygous variants, P144T and E222Q, in human chondrocytes. Both novel variants were identified in individuals with classic SEMDJL2 features. P144T and E222Q retained PEF-generating activity, whereas R49Q displayed reduced PEFs, consistent with their respective inheritance patterns. Live cell imaging revealed that all variants disrupted mitosis. The heterozygous variants (P144T, E222Q, R149Q) dominantly impeded anaphase chromosome segregation and spindle pole separation, supporting reclassification of P144T and E222Q as likely pathogenic. In contrast, R49Q caused milder, partially penetrant segregation defects, consistent with reduced and dysregulated motor activity. Together, our results define two mechanistic classes of KIF22 dysregulation: constitutive activation in heterozygous variants, which fail to down-regulate KIF22 at anaphase onset, and mixed-state dysregulation in the recessive R49Q variant, which exhibits partial loss of polar ejection force activity coupled with incomplete inactivation during anaphase. These findings broaden the mechanistic framework for how KIF22 variants perturb mitosis in chondrocytes and expand the genotypic landscape associated with SEMDJL2.

cell biology↗

bioRxiv2024

Sociality shapes mitochondrial adaptations supporting hypoxia tolerance

Oxygen deprivation or hypoxia is poorly dealt with by most terrestrial species and often leads to permanent tissue damage and death. One prominent exception is the naked mole-rat (Heterocephalus glaber) which is remarkably adapted to withstand prolonged periods ([~]18 mins) of severe hypoxia, a trait likely driven by its crowded underground lifestyle. Other African mole-rat species are less social or entirely solitary like the Cape mole-rat (Georychus capensis). Here, we asked whether cellular and molecular adaptations to hypoxia map to social traits. We discovered that at the cellular level naked mole-rat fibroblasts survive >30 hours in 1% oxygen, while fibroblasts from terrestrial or non-social mole-rat species (human, mouse and Cape mole-rat) die rapidly under hypoxic conditions. We further show that naked mole-rat mitochondria have evolved morphological, functional and proteomic adaptations crucial for hypoxia resistance, remaining unaffected after long periods of severe hypoxia. We identify the mitochondrial protein Optic Atrophy 1 (OPA1) as a key player in naked mole-rat hypoxia resilience. Naked mole-rat mitochondria not only express more protective forms of OPA1, but also harbor a structurally unique isoform that likely protects cells from hypoxic damage. We show that evolutionary changes including the functionalization of a unique Opa1 exon support mitochondrial mediated cellular protection. Indeed, knockdown of OPA1 in naked mole-rat cells is sufficient to render them equally susceptible to hypoxia as human cells or cells from non-social African species. Our study demonstrates how molecular evolution drives unique adaptations that enable cells to achieve unprecedented resistance to hypoxic damage. We also show that molecular changes at the level of mitochondria are crucial in conferring hypoxia resistance. Our results thus chart a novel molecular path to understand how robust cellular hypoxia resistance can be achieved. Such knowledge may eventually inspire novel strategies to circumvent the consequences of hypoxic-damage in humans.

physiology↗

bioRxiv2024

Screening great ape museum specimens for DNA viruses

Natural history museum collections harbour a record of wild species from the past centuries, providing a unique opportunity to study animals as well as their infectious agents. Thousands of great ape specimens are kept in these collections, and could become an important resource for studying the evolution of DNA viruses. Their genetic material is likely to be preserved in dry museum specimens, as reported previously for monkeypox virus genomes from historical orangutan specimens. Here, we screened 209 great ape museum specimens for 99 different DNA viruses, using hybridization capture coupled with short-read high-throughput sequencing. We determined the presence of multiple viruses within this dataset from historical specimens and obtained several near-complete viral genomes. In particular, we report high-coverage (>18-fold) hepatitis B virus genomes from one gorilla and two chimpanzee individuals, which are phylogenetically placed within clades infecting the respective host species.

genomics↗

bioRxiv2021

TRAP1 and cyclophilin D compete at OSCP subunit to regulate enzymatic activity and permeability transition pore opening by F-ATP synthase

Binding of the mitochondrial chaperone TRAP1 to client proteins shapes cell bioenergetic and proteostatic adaptations, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Moreover, TRAP1 inhibits opening of the permeability transition pore (PTP) formed by F-ATP synthase and effectively antagonizes the PTP-inducing effect of CyPD, which elicits mitochondrial depolarization and cell death. Consistently, electrophysiological measurements indicate that TRAP1 and CyPD compete in the modulation of channel activity of purified F-ATP synthase, resulting in PTP inhibition and activation, respectively, and outcompeting each other effect on the channel. Moreover, TRAP1 counteracts PTP induction by CyPD, whereas CyPD reverses TRAP1-mediated PTP inhibition. Our data identify TRAP1 as a F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

cell biology↗

bioRxiv2020

HIF1a-dependent induction of the mitochondrial chaperone TRAP1 regulates bioenergetic adaptations to hypoxia

The mitochondrial paralog of the Hsp90 chaperone family TRAP1 is often induced in tumors, but the mechanisms controlling its expression, as well as its physiological functions remain poorly understood. Here, we find that TRAP1 is highly expressed in the early stages of Zebrafish development, and its ablation delays embryogenesis while increasing mitochondrial respiration of fish larvae. TRAP1 expression is enhanced by hypoxic conditions both in developing embryos and in cancer models of Zebrafish and mammals. The TRAP1 promoter contains evolutionary conserved hypoxic responsive elements, and HIF1 stabilization increases TRAP1 levels. TRAP1 inhibition by selective compounds or by genetic knock-out maintains a high level of respiration in Zebrafish embryos after exposure to hypoxia. Our data identify TRAP1 as a primary regulator of mitochondrial bioenergetics in highly proliferating cells following reductions in oxygen tension and HIF1 stabilization.

biochemistry↗

bioRxiv2019

Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca2+-dependent death of cancer cells

Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (Mitochondria-Associated Membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+-dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signalling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.

cancer biology↗

bioRxiv2018

Lipid utilization in skeletal muscle cells is modulated in vitro and in vivo by specific miRNAs

Skeletal muscle is composed by different myofiber types that can preferentially use glycolysis or lipids for ATP production. How fuel preference is specified in these post-mitotic cells is unknown. Here we show that miRNAs are important players in defining the myofiber metabolic profile. mRNA and miRNA signatures of all myofiber types obtained at single cell level unveiled fiber-specific regulatory networks and identified two master miRNAs that coordinately control myofiber fuel preference and mitochondrial morphology. Our work provides a complete and integrated myofiber type-specific catalogue of genes and miRNAs expressed and establishes miR-27a-3p and miR-142-3p as key regulators of lipid utilization in skeletal muscle.\n\nHIGHLIGHTSO_LITranscriptional networking in single cells distinguished myofibers based on glycolytic or oxidative metabolism, regulated by specific miRNAs\nC_LIO_LImiR-27a-3p and -142-3p influence mitochondrial morphology\nC_LIO_LImiR-27a-3p improves lipid utilization and increases glycogen storage both in vitro and in vivo\nC_LIO_LImiR-142-3p reduces lipid utilization both in vitro and in vivo\nC_LI

genomics↗