Biology subjects

Vitale, N.

Publications and source records attributed to Vitale, N..

11 recordsLinked to original sources

bioRxiv2026

Profilin 1 maintains cell cycle fidelity to prevent unscheduled genome doubling and polyploidy in cancer

Whole-genome doubling (WGD) represents a major route to genome instability and therapeutic resistance in cancer; yet, the mechanisms enabling genome duplication in p53-proficient cells remain poorly understood. Here, we identify Profilin 1 (PFN1) loss as a driver of WGD through impaired mitotic entry. Using FUCCI live-cell imaging and single-cell genomic profiling, we show that PFN1-deficient cells bypass mitosis and undergo endoreplication, generating tetraploid cells. Rather than undergoing stable arrest after mitotic bypass, these genome-doubled cells retain proliferative capacity and proceed through aberrant mitotic divisions, thereby amplifying genomic instability. Proteomic analyses reveal coordinated attenuation of late cell-cycle programs, including downregulation of key mitotic regulators such as CDK1, PLK1 and CKS2, consistent with impaired G2/M transition. Despite accumulating polyploidy, PFN1-knockout cells fail to activate an effective p53 tetraploidy checkpoint and display increased nuclear MDM2, promoting cell-cycle arrest evasion and chemotherapeutic resistance. We supported clinical relevance by an orthotopic osteosarcoma xenograft model, in which PFN1-deficient SaOS2 cells showed enhanced metastatic dissemination, and by pan-cancer TCGA analyses confirming a recurrent association between PFN1 loss and WGD. Together, these findings identify Profilin 1 as a safeguard of cell-cycle fidelity whose loss enables genome doubling, cellular plasticity and therapy tolerance.

cancer biology↗

bioRxiv2026

Endoplasmic reticulum membrane contact sites coordinate exocytic site assembly and activity in neuroendocrine cells

Membrane contact sites (MCS) between intracellular organelles regulate lipid exchange, organelles dynamics and spatial organization of signaling pathways, yet their contribution to regulated exocytosis remains poorly understood. Here, we investigated the role of endoplasmic reticulum (ER) MCS in calcium-regulated exocytosis using primary bovine chromaffin cells. Combining electron microscopy on plasma membrane (PM) sheets with immunogold labeling, we identified ER structures contacting docked secretory granules and classified three types of MCS: ER-PM, ER-granule and tripartite ER-PM-granule contacts. These contacts are enriched at exocytic sites and contain Orai1 and STIM1, both known for mediating store-operated calcium release. Functional perturbation of the Orai/STIM pathway revealed that constitutive STIM activation or pharmacological inhibition of Orai1 reduced the number of exocytotic events, slowed catecholamine release and disrupted actin organization at granule docking sites. Together, our findings revealed a previously unrecognized role for ER MCS in organizing exocytic sites and controlling secretion efficiency in neuroendocrine cells.

cell biology↗

bioRxiv2025

Designing new natural-mimetic phosphatidic acid: aversatile and innovative synthetic strategy forglycerophospholipid research

Glycerophospholipids (GPLs) play important roles in cellular compartmentalization and signaling. Among them, phosphatidic acids (PA) exist as many distinct species depending on acyl chain composition, each one potentially displaying unique signaling function. Although the signaling functions of PA have already been demonstrated in multiple cellular processes, the specific roles of individual PA species remain obscure due to a lack of appropriate tools. Indeed, current synthetic PA analogues fail to preserve all the functions of natural PA. To circumvent these limitations, we developed a novel synthetic approach to produce PA analogues without compromising structural integrity of acyl chains. Moreover, addition of a clickable moiety allowed flexible grafting of different molecules to PA analogues for various biological applications. Hence, this innovation also provides powerful tools to investigate specific biological activities of individual PA species, with potential applications in unraveling complex GPL-mediated signaling pathways.

neuroscience↗

bioRxiv2024

Next-generation Cu(II) selective peptide shuttles prevent Cu(Aβ)-induced toxicity and microglial activation in organotypic hippocampal slices

Alzheimers disease (AD) remains the most prevalent neurodegenerative disease characterized by intracellular neurofibrillary tangles of Tau protein and extracellular senile plaques build on Amyloid-{beta} (A{beta}) peptides. The latter result from an abnormal processing of Amyloid Precursor Protein (APP) leading to its accumulation in plaques. Ex vivo analyses of AD patients brains show an abnormally elevated concentration of metals including Cu, Zn and Fe within these plaques. Altered Cu levels have also been reported in brain regions most affected in AD. These modifications are often accompanied by reduced neuronal Cu levels and by an increased pool of extracellular labile Cu, which in turn promotes reactive oxygen species (ROS) formation. To counteract this Cu dyshomeostasis and limit Cu-A{beta}-induced extracellular ROS generation, we designed and synthesized two Cu(II)-selective peptide shuttles based on kinetically optimized ATCUN sequences for fast Cu(II) extraction out of A{beta}: DapHH-R5W4NBD and HDapH-R5W4NBD. They were also equipped with a fluorophore that showed a very strong response to Cu(II)-binding and release. Interestingly, these two Cu(II) shuttles displayed a dual mode of action. They promptly retrieve Cu from extracellular A{beta}, stop the associated ROS formation, and hence protect both cell culture models and organotypic hippocampal slices (OHSCs) from Cu(A{beta})-induced neurotoxicity. Moreover, these shuttles import and redistribute bioavailable Cu inside cells with a sequence-dependent kinetics.

neuroscience↗

bioRxiv2023

Phospholipid Scramblase-1 is required for efficient neurotransmission and synaptic vesicle retrieval at cerebellar synapses

Structural phospholipids are asymmetrically distributed at the plasma membrane, with phosphatidylethanolamine and phosphatidylserine (PS) virtually absent from the outer leaflet. This asymmetric lipid distribution is transiently altered during specific biological processes including calcium-regulated exocytosis. However, the impact of this transient remodeling of membrane asymmetry on presynaptic function remains unknown. PhosphoLipid SCRamblase 1 (PLSCR1), a protein that randomizes phospholipid distribution between the two leaflets of the plasma membrane in response to calcium activation is an ideal candidate to alter this asymmetry. We therefore set out to determine the role of PLSCR1 in both neurotransmitter release and synaptic vesicle recycling by combining electron microscopy, optical live cell imaging of pHluorin probes and electrophysiology in cerebellar granule cells (GrC) from Plscr1 knock-out mice (Plscr1-/-). We report that PLSCR1 is expressed in GrCs and that PLSCR1-dependent PS egress occurred at synapses in response to neuron stimulation. Furthermore, synaptic transmission is impaired at GrC Plscr1-/- synapses and both PS egress and synaptic vesicle endocytosis are inhibited in Plscr1-/- cultured neurons, demonstrating that PLSCR1 controls phospholipid asymmetry remodeling and synaptic vesicle retrieval following neurotransmitter release. Altogether, our data reveal a key role for PLSCR1 in synaptic vesicle recycling and provide the first evidence that phospholipid scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.

cell biology↗

bioRxiv2021

ORP5 AND ORP8 ORCHESTRATE LIPID DROPLET BIOGENESIS AND MAINTENANCE AT ER-MITOCHONDRIA CONTACT SITES

Lipid droplets (LDs) are the primary organelles of lipid storage, buffering energy fluctuations of the cell. They store neutral lipids in their core that is surrounded by a protein-decorated phospholipid monolayer. LDs arise from the Endoplasmic Reticulum (ER). The ER-protein seipin, localizing at ER-LD junctions, controls LD nucleation and growth. However, how LD biogenesis is spatially and temporally coordinated remains elusive. Here, we show that the lipid transfer proteins ORP5 and ORP8 control LD biogenesis at Mitochondria-Associated ER Membrane (MAM) subdomains, enriched in phosphatidic acid. We found that ORP5/8 regulate seipin recruitment to these MAM-LD contacts, and their loss impairs LD biogenesis. Importantly, the integrity of ER-mitochondria contact sites is crucial for the ORP5/8 function in regulating seipin-mediated LD biogenesis. Our study uncovers an unprecedented ORP5/8 role in orchestrating LD biogenesis at MAMs and brings novel insights into the metabolic crosstalk between mitochondria, ER, and LDs at membrane contact sites. HIGHLIGHTSORP5 and ORP8 localize at MAM subdomains where LDs originate. Phosphatidic acid is enriched in MAM subdomains that are the birthplace of LDs. ORP5 and ORP8 knockdown impairs LD biogenesis. ORP5 and ORP8 regulate seipin recruitment to MAM-LD contact sites.

cell biology↗

bioRxiv2021

Dysfunction of exocytosis causes catecholamine hypersecretion in patient with pheochromocytoma

Pheochromocytoma (Pheo) is a neuroendocrine tumor that develops from chromaffin cells of the adrenal medulla, and is responsible of an excess of catecholamines secretion leading to severe clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Surprisingly, hypersecretory activity of Pheo has never been explored at the cellular and molecular levels from individual tumor cells. In the present study, we have combined catecholamine secretion measurement by carbon fiber amperometry on human tumor cells directly cultured from freshly resected Pheo, with the analysis by mass spectrometry of the exocytotic proteins differentially expressed between the tumor and the matched adjacent non-tumor tissue. Catecholamine secretion recordings from individual Pheo cells obtained from most patients reveal a higher number of exocytic events per cell associated with faster kinetic parameters. Accordingly, we unravel significant tumor-associated modifications in the expression of key proteins involved in different steps of the calcium-regulated exocytic pathway. Altogether, our findings indicate that dysfunction of the calcium-regulated exocytosis at the level of individual Pheo cell is a cause of the tumor-associated hypersecretion of catecholamines.

cancer biology↗

bioRxiv2021

Design considerations of a wearable electronic-skin for mental health and wellness: balancing biosignals and human factors

Chronic stress has been associated with a variety of pathophysiological risks including developing mental illness. Conversely, appropriate stress management, can be used to foster mental wellness proactively. Yet, there is no existing method that accurately and objectively monitors stress. With recent advances in electronic-skin (e-skin) and wearable technologies, it is possible to design devices that continuously measure physiological parameters linked to chronic stress and other mental health and wellness conditions. However, the design approach should be different from conventional wearables due to considerations like signal-to-noise ratio and the risk of stigmatization. Here, we present a multi-part study that combines user-centered design with engineering-centered data collection to inform future design efforts. To assess human factors, we conducted an n=24 participant design probe study that examined perceptions of an e-skin for mental health and wellness as well as preferred wear locations. We complement this with an n=10 and n=16 participant data collection study to measure physiological signals at several potential wear locations. By balancing human factors and biosignals, we conclude that the upper arm and forearm are optimal wear locations.

bioengineering↗

bioRxiv2020

The Synergism Between DHODH Inhibitors and Dipyridamole Leads to Synthetic Lethality in Acute Myeloid Leukemia

BackgroundDihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition was recently found to induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors were previously investigated in solid tumors, where they showed promising antiproliferative activity, both in vitro and in vivo. However, their effectiveness was not confirmed in clinical trials, probably due to the pyrimidine salvage pathway that cancer cells could exploit to survive. In this study we investigated the pro-apoptotic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we challenged our model mimicking in vivo conditions, and looked for synergic combination to boost apoptosis. MethodsWe evaluated the apoptotic rate of multiple AML cell lines and AML primary cells treated with MEDS433 or other DHODH inhibitors, alone and in combination with classical antileukemic drugs or with dipyridamole, a blocker of the pyrimidine salvage pathway. Experiments were also performed mimicking in vivo conditions, i.e., in the presence of physiological uridine plasma levels (5 M). ResultsMEDS433 showed a strong apoptotic effect against multiple AML cell lines, which was at least partially independent from the differentiation process. Its combination with classical antileukemic agents resulted in a further increase of the apoptotic rate. However, when MEDS433 was tested in the presence of 5 M uridine and/or in primary AML cells, results were less impressive. On the contrary, the combination of MEDS433 with dipyridamole resulted in an outstanding synergistic effect, with a dramatic increase of the apoptotic rate both in AML cell lines and AML primary cells, which was unaffected by physiological uridine concentrations. Preliminary analyses show that the toxicity of this treatment should be limited to proliferating cells. ConclusionsThe combination of a DHODH inhibitor and dipyridamole is characterized by differentiating and pro-apoptotic features and induces metabolic lethality on a wide variety of AMLs with different genetic backgrounds.

cancer biology↗

bioRxiv2020

Parkin coordinates mitochondrial lipid remodeling to execute mitophagy

Mitochondrial failure caused by Parkin mutations contributes to Parkinsons disease. Parkin binds, ubiquitinates, and targets impaired mitochondria for autophagic destruction. Robust mitophagy involves peri-nuclear concentration of Parkin-tagged mitochondria, followed by dissemination of juxtanuclear mitochondrial aggregates, and efficient sequestration of individualized mitochondria by autophagosomes. Here, we report that the execution of complex mitophagic events requires active mitochondrial lipid remodeling. Parkin recruits phospholipase D2 to the depolarized mitochondria and generate phosphatidic acid (PA). Mitochondrial PA is subsequently converted to diacylglycerol (DAG) by Lipin-1 phosphatase-a process that further requires mitochondrial ubiquitination and ubiquitin-binding autophagic receptors, NDP52 and Optineurin. We show that Optineurin transports, via Golgi-derived vesicles, a PA-binding factor EndoB1 to ubiquitinated mitochondria, thereby facilitating DAG production. Mitochondrial DAG activates both F-actin assembly to drive mitochondrial individualization, and autophagosome biogenesis to efficiently restrict impaired mitochondria. Thus Parkin, autophagic receptors and the Golgi complex orchestrate mitochondrial lipid remodeling to execute robust mitophagy.

cell biology↗

bioRxiv2019

The atypical Rho GTPase RhoU interacts with Intersectin-2 to regulate endosomal recycling pathways

Rho GTPases play a key role in various membrane trafficking processes. RhoU is an atypical small Rho GTPase related to Rac/Cdc42 which possesses unique N- and C-terminal domains that regulate its function and its subcellular localization. RhoU localized at the plasma membrane, on endosomes and in cell adhesion structures where it governs cell signalling, differentiation and migration. However, despite its endomembrane localization, RhoU function in vesicular trafficking has been unexplored. Here, we identified intersectins (ITSNs) as new binding partners for RhoU and showed that the second PxxP motif at the N-terminus of RhoU mediated interactions with SH3 domains of ITSNs. To evaluate the function of RhoU and ITSNs in vesicular trafficking, we used fluorescent transferrin as a cargo for uptake experiments. We showed that silencing of either RhoU or ITSN2, but not ITSN1 increased transferrin accumulation in early endosomes resulting from defect in fast vesicle recycling. Concomitantly, RhoU and ITSN2 colocalized to a subset of Rab4-positive vesicles suggesting that RhoU-ITSN2 interaction may occur on fast recycling endosomes to regulate the fate of vesicular cargos.

cell biology↗