Biology subjects

Pietrancosta, N.

Publications and source records attributed to Pietrancosta, N..

7 recordsLinked to original sources

bioRxiv2026

Temporal tuning of actin nucleation by the repurposed DNA repair complex RAD51/fignl1 encodes distinct axon guidance decisions

Axon guidance is essential for neural circuit assembly and function. While much of the repertoire of guidance cues and receptors is known, its limited diversity cannot fully explain the remarkable complexity of the brain connectome. How temporally ordered, context-dependent intrinsic programs shape the diversity of axonal responses to guidance signals remains poorly understood. We reveal how non-canonical Wnt5a signalling repurposes the DNA repair complex RAD51/fignl1 as a downstream integrator to control retinal ganglion cell axon decision-making at the midline. We demonstrate that differential RAD51/fignl1 expression and interaction dynamics between ipsilateral and contralateral axons tunes the magnitude and temporal dynamics of F-actin nucleation, thereby encoding their trajectory choice. Our findings establish temporal control of cytoskeletal dynamics as a key determinant of axon decision-making and thereby a fundamental layer of circuit wiring. They provide a potential mechanistic framework linking mutations in the DNA repair gene RAD51 to neurodevelopmental disorders with midline guidance defects.

neuroscience↗

bioRxiv2026

Focal drug uncaging for the precise control of endogenous nicotinic receptors in behaving mice

Nicotinic acetylcholine receptors shape diverse brain functions, including reward, aversion, and pain, but dissecting their roles in vivo requires drug delivery with spatiotemporal precision that conventional pharmacology cannot provide. Here we establish in vivo photopharmacology of endogenous nicotinic receptors: two complementary, highly-potent agonists, administered systemically in an inactive caged form, are photoreleased locally in target brain circuits of behaving wild-type mice. Despite irreversible photochemistry, photoactivation can be repeated over many trials on a minute timescale, for hours following a single injection. Local uncaging evokes circuit-specific dopamine release, drives place preference from the ventral tegmental area and aversion from the interpeduncular nucleus, and, in pain circuits, reveals a pronociceptive effect masked when the same agonists are given systemically. By controlling where and when nicotinic signaling acts, this approach exposes circuit-specific drug effects invisible to conventional pharmacology, and outlines a general strategy for on-demand control of endogenous receptors in vivo.

neuroscience↗

bioRxiv2025

NKCC1 as a signaling hub regulating KCC2 stability, chloride homeostasis, and seizure susceptibility

Chloride homeostasis relies on the dynamic balance between the neuronal co-transporters NKCC1 and KCC2. We reveal an unexpected mechanism by which NKCC1 governs KCC2 membrane stability. NKCC1 clusters recruits SPAK and PP1 to dynamically trap KCC2, compensating for its lack of a direct SPAK-binding site. Single-particle tracking shows that these NKCC1-rich assemblies operate as signaling hubs, enabling either SPAK-driven KCC2 phosphorylation and its membrane destabilization or PP1-mediated dephosphorylation of SPAK and KCC2 membrane stabilization. Peptides that activate SPAK by engaging NKCC1s PP1-binding motif lower KCC2 surface levels and reduce chloride extrusion, whereas a SPAK-inhibiting peptide prevents SPAK recruitment to NKCC1, stabilizes KCC2 in membrane clusters, and enhances chloride extrusion. An optimized peptide analog preserves KCC2 clustering under hyperexcitable conditions, reduces seizure frequency and severity in PTZ-induced epilepsy, and suppresses ictal activity in human epileptic tissue. These findings identify NKCC1-KCC2 coupling as a central regulatory axis for inhibitory signaling, and position our peptides as promising therapeutic candidates to restore chloride homeostasis in epilepsy and other disorders marked by impaired KCC2 membrane stability.

neuroscience↗

bioRxiv2025

Exploring the multi-protein assembly of the enzymes of the de novo purine nucleotide biosynthetic pathway from Pseudomonas aeruginosa

Purine nucleotide biosynthesis is a crucial metabolic pathway responsible that produces building blocks essential for a plethora of cellular processes. In bacteria, the de novo purine nucleotide biosynthetic pathway (DNPNB) involves fifteen chemical steps catalysed by fourteen different enzymes. While the mammalian orthologues have been extensively shown to interact and form a metabolon named "purinosome", the possible existence of a prokaryotic equivalent was only recently revealed for the case of Escherichia coli. In this study, we explored the potential conservation of a bacterial purinosome-like complex in Pseudomonas aeruginosa, an opportunistic pathogen known for its high antibiotic resistance. Using a bacterial two-hybrid system, we mapped protein-protein interactions among all tested DNPNB enzymes in P. aeruginosa and revealed a dense interaction network. An in-silico protein-protein docking approach on three core enzymes allowed the structural reconstitution of a complex composed of PurK, PurE and PurC with a 4:8:8 stoichiometry, respectively. Interestingly, a tunnel connecting the different active sites has been revealed, showing a metabolon-like property for possible efficient substrate channelling. These findings support a conserved regulatory organization of purine biosynthesis in bacteria, providing deeper insights into bacterial metabolism and paving the way for potential antibiotic targets.

biochemistry↗

bioRxiv2025

"Smurf Mice": revolutionising our understanding of age-related and end-of-life animal physiology

Living animals reach their end-of-life through a stereotypic set of fascinating but poorly understood processes. The discovery, first in flies and later in nematodes and zebrafish, of the "Smurf phenotype" is a central tool for picking this complex "lock of biology", that one of ageing. Using the Smurfs, we have shown an evolutionarily conserved end-of-life transition across Drosophilids, nematodes and zebrafish. This tool has been key to identify the discontinuous nature of ageing and predict impending death from natural causes as well as from environmental stresses. This phenotype allowed us to discover that ageing is made up of two successive phases : a first phase where individuals are healthy and have no risk of mortality, but show an age-dependent and increasing risk of entering a second phase, characterized by the so-called hallmarks of ageing and a high risk of death. Here, we test whether these two consecutive phases of ageing separated by the Smurf transition are a conserved feature of ageing in the mammals using Mus musculus as a model. We performed a longitudinal longevity study using both males and females from two different mouse genetic backgrounds and by integrating physiological, metabolic and molecular measurements with the life history of approximately 150 mice. We show the existence of a phenotypic signature typical of the last phase of life, observable at any chronological age. Validating the two-phase ageing model in a mammalian organism allows better characterized the high risk of imminent death and would extend its implications to a broader range of species for ageing research.

physiology↗

bioRxiv2024

A tunable and versatile chemogenetic near infrared fluorescent reporter

Near-infrared (NIR) fluorescent reporters provide additional colors for highly multiplexed imaging of cells and organisms, and enable imaging with less toxic light and higher contrast and depth. Here, we present the engineering of nirFAST, a small tunable chemogenetic NIR fluorescent reporter that is brighter than top-performing NIR fluorescent proteins in cultured mammalian cells. nirFAST is a small genetically encoded protein of 14 kDa that binds and stabilizes the fluorescent state of synthetic, highly cell-permeant, fluorogenic chromophores (so-called fluorogens) that are otherwise dark when free. Engineered to emit NIR light, nirFAST can also emit far-red or red lights through change of chromophore. nirFAST allows the imaging of proteins in live cultured mammalian cells, chicken embryo tissues and zebrafish larvae. Its near infrared fluorescence provides an additional color for high spectral multiplexing. We showed that nirFAST is well-suited for stimulated emission depletion (STED) nanoscopy, allowing the efficient imaging of proteins with subdiffraction resolution in live cells. nirFAST enabled the design of a chemogenetic green-NIR fluorescent ubiquitination-based cell cycle indicator (FUCCI) for the monitoring of the different phases of the cell cycle. Finally, bisection of nirFAST allowed the design of a fluorogenic chemically induced dimerization technology with NIR fluorescence readout, enabling the control and visualization of protein proximity.

cell biology↗

bioRxiv2021

An engineered multifunctional protein tag for advanced fluorescence imaging

Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we present the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of live-cell compatible fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. We showed that the ability to tune the fluorescence color and properties through simple molecular modulation provides an unprecedent experimental versatility for imaging proteins in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms. The ability to tune the spectral properties and fluorescence performance enables to match the spectral specifications and requirements of the most advanced imaging techniques, and allowed us to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons.

molecular biology↗