Biology subjects

Danglot, L.

Publications and source records attributed to Danglot, L..

10 recordsLinked to original sources

bioRxiv2024

Neuronal fatty acid oxidation fuels memory after intensive learning

Metabolic flexibility allows cells to adapt to different fuel sources, which is particularly important for cells with high metabolic demands. In contrast, neurons, which are major energy consumers, are considered to rely almost solely on glucose and its derivatives to support their metabolism1-3. Here, using Drosophila melanogaster, we show memory formed after intensive massed training is dependent on mitochondrial fatty acid (FA) {beta}-oxidation to produce ATP in neurons of the mushroom bodies (MB), a major integrative center in insects brain. We identify neuronal lipid droplets as the main source of FAs for this type of memory. Furthermore, we demonstrate that this intensive massed training is associated with mitochondria network remodeling in the soma of MB neurons, resulting in increased mitochondrial size. Artificially increasing mitochondria size in adult MB neurons increases ATP production in their soma and, at the behavioral level, strikingly results in improved memory performance after massed training. These findings challenge the prevailing view that neurons are unable to use FAs for energy production, and importantly revealing on the contrary that in vivo neuronal FA oxidation has an essential role in cognitive function, including memory formation.

neuroscience↗

bioRxiv2024

Influenza A virus-induced production of PI4P at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

The genomic RNAs of influenza A viruses (IAVs) are replicated in the nucleus of infected cells in the form of viral ribonucleoproteins (vRNP) before being exported to the cytoplasm. The small GTPase RAB11A is involved in the transport of vRNPs to the sites of viral assembly at the plasma membrane, but the molecular mechanisms involved remain largely unknown. Here we show that IAV infection remodels the architecture of the endoplasmic reticulum (ER) sheets, where vRNPs tend to accumulate in the absence of RAB11A. To decipher the interplay between RAB11A, vRNPs and the ER, we investigated viral-induced perturbations of RAB11A proximity interactome. To this end, we generated cells stably expressing a TurboID-RAB11A fusion protein and performed biotin-based proximity labeling upon viral infection. We found that cellular regulators of phophatidylinositol-4-phosphate (PI4P) homeostasis, including the autophagic and stress response protein ATG16L1, are significantly enriched at the vicinity of RAB11A in infected cells. Infection induces an increase in cellular PI4P levels in an ATG16L1-dependent manner, while ATG16L1 relocalizes to ER membranes upon infection. Depletion of ATG16L1 decreases the co-distribution of vRNPs with PI4P punctae on ER membranes, and reduces the accumulation of vRNPs at the plasma membrane as well as the production of IAV infectious particles. Our data extend to IAVs the notion that viruses can modulate the metabolism and localization of phosphoinositides to control host membrane dynamics and point to the ER as an essential platform for vRNP transport. They provide evidence for a pivotal role of ATG16L1 in regulating the identity of endomembranes and coordinating RAB11A and PI4P-enriched membranes to ensure delivery of vRNPs to the plasma membrane.

microbiology↗

bioRxiv2023

Slitrk/LAR-RPTP and disease-associated variants control neuronal migration in the developing mouse cortex independently of synaptic organizer activity

Slitrks and their ligands LAR-RPTPs are type I transmembrane proteins previously implicated in the etiology of various neuropsychiatric disorders including obsessive-compulsive disorders (OCDs) and schizophrenia. Over the last decade, their functions were extensively studied in hippocampal neurons in vitro and shown to shape synapse organization. Although both protein families are highly expressed prior to synapse formation, their function in earlier steps of cerebral cortex development remains unknown. Here we investigated the role of Slitrk1, Slitrk2, Slitrk3 and LAR-RPTPs (Ptprs and Ptprd) in the embryonic mouse cortex by acute genetic manipulation using in utero electroporation. All genes, except Slitrk3, promoted specific alterations in radial migration of glutamatergic neurons. Slitrk1 and Slitrk2 overexpression was associated with accumulation of neurons in distinct regions of the cortical plate. Using deletion mutants and a series of Slitrk variants associated with neurodevelopmental disorders (NDDs), we showed that distinct domains are crucial for intracellular Slitrk1 distribution and/or density and shape of VAMP2+ presynaptic boutons. Interestingly, bouton alterations did not correlate with the observed migration delays, suggesting that Slitrk1 influence cell migration independently on its synaptogenic function. Furthermore, co-electroporation experiments with LAR-RPTPs, mimicking their co-expression observed by scRNAseq, rescued the migration deficits, suggesting possible cis-interactions between Slitrks and LAR-RPTPs. Together, these data indicate that in the embryonic cerebral cortex Slitrks and LAR-RPTPs cooperate in consecutive steps of radial migration through distinct mechanisms than in synapse organization and support a relevant role of Slitrk/LAR-RPTP dysfunctions in NDDs at earlier stages of cortical development.

neuroscience↗

bioRxiv2023

A polarized cell system amenable to subcellular resolution imaging of influenza virus infection

The life cycle of influenza A viruses (IAV), and notably intracellular trafficking of the viral genome, depend on multiple interactions with the cellular cytoskeleton and endomembrane system. A limitation of the conventional cellular models used for mechanistic study and subcellular imaging of IAV infection is that they are cultured in two dimensions (2D) under non-polarizing conditions, and therefore they do not recapitulate the intracellular organization of the polarized respiratory epithelial cells naturally targeted by IAVs. To overcome this limitation, we developed an IAV-infection assay in a 3D cell culture system which allows imaging along the baso-lateral axis of polarized cells, with subcellular resolution. Here we describe a protocol to grow polarized monolayers of Caco2-TC7 cells on static Cytodex-3 microcarrier beads, infect them with IAV, and subsequently perform immunostaining and confocal imaging, or electron microscopy, on polarized IAV-infected cells. This method can be extended to other pathogens that infect human polarized epithelial cells.

microbiology↗

bioRxiv2023

Targeted Photoconvertible BODIPYs Based on Directed Photooxidation Induced Conversion for Applications in Photoconversion and Live Super Resolution Imaging

Photomodulable fluorescent probes are drawing an increasing attention due to their applications in advanced bioimaging. Whereas photoconvertible probes can be advantageously used in tracking, photoswitchable probes constitute key tools for single molecule localization microscopy to perform super resolution imaging. Herein we shed light on a red and far-red BODIPY, namely BDP-576 and BDP-650 possessing both properties of conversion and switching. Our study demonstrates that theses pyrrolyl-BODIPYs respectively convert towards typical green- and red-emitting BODIPYs that are perfectly adapted to microscopy. We also showed that these pyrrolyl-BODIPYs undergo Directed Photooxidation Induced Conversion, a photoconversion mechanism that we recently introduced and where the pyrrole moiety plays a central role. These unique features were used to develop targeted photoconvertible probes towards different organelles or subcellular units (plasma membrane, mitochondria, nucleus, actin, Golgi apparatus, etc.) using chemical targeting moieties and Halo tag. We notably showed that BDP-650 could be used to track intracellular vesicles over more than 20 minutes in two color imaging with laser scanning confocal microscopy demonstrating its robustness. The switching properties of these photoconverters were studied at the single molecule level and were then successfully used in live Single Molecule Localization Microscopy in epithelial cells and neurons. Both membrane and mitochondria targeted probes could be used to decipher membrane 3D architecture and mitochondria dynamics at the nanoscale. This study builds a bridge between the photoconversion and photoswitching properties of probes undergoing directed photooxidation and shows the versatility and efficacy of this mechanism in live advanced imaging.

cell biology↗

bioRxiv2022

ExoJ: an ImageJ2/Fiji plugin for automated spatiotemporal detection of exocytosis

Exocytosis is a dynamic physiological process that enables the release of biomolecules to the surrounding environment via the fusion of membrane compartments to the plasma membrane. Understanding its mechanisms is crucial, as defects can compromise essential biological functions. The development of pH-sensitive optical reporters alongside fluorescence microscopy enables the assessment of individual vesicle exocytosis events at the cellular level. Manual annotation represents, however, a time-consuming task, prone to selection biases and human operational errors. Here, we introduce ExoJ, an automated plugin based on ImageJ2/Fiji. ExoJ identifies user-defined genuine populations of exocytic events, recording quantitative features including intensity, apparent size and duration. We designed ExoJ to be fully user-configurable, making it suitable to study distinct forms of vesicle exocytosis regardless of the imaging quality. Our plugin demonstrates its capabilities by showcasing distinct exocytic dynamics among tetraspanins and vesicular SNAREs protein reporters. Assessment of performance on synthetic data demonstrated ExoJ is a robust tool, capable to correctly identify exocytosis events independently of signal-to-noise ratio conditions. We propose ExoJ as a standard solution for future comparative and quantitative studies of exocytosis.

bioinformatics↗

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↗

bioRxiv2019

The interaction between non-fusogenic Sec22b-Stx complexes and Extended-Synaptotagmins promotes neurite growth and ramification

Axons and dendrites are long and often ramified neurites that need particularly intense plasma membrane (PM) expansion during the development of the nervous system. Neurite growth depends on non-fusogenic Sec22b-Stx1 SNARE complexes at endoplasmic reticulum (ER)-PM contacts. Here we show that Sec22b interacts with the endoplasmic reticulum lipid transfer proteins Extended-Synaptotagmins (E-Syts) and this interaction depends on the Longin domain of Sec22b. Overexpression of E-Syts stabilizes Sec22b-Stx1 association, whereas silencing of E-Syts has the opposite effect. Overexpression of wild-type E-Syt2, but not mutants unable to transfer lipids or attach to the ER, increase the formation of axonal filopodia and ramification of neurites in developing neurons. This effect is inhibited by a clostridial neurotoxin cleaving Stx1, expression of Sec22b Longin domain and a Sec22b mutant with extended linker between SNARE and transmembrane domains. We conclude that Sec22b-Stx1 ER-PM contact sites contribute to PM expansion by interacting with lipid transfer proteins such as E-Syts.

cell biology↗

bioRxiv2019

Molecular Tuning of Styryl Dyes Leads to Versatile and Efficient Plasma Membrane Probes for Cell and Tissue Imaging

The plasma membrane (PM) plays a major role in many biological processes; therefore its proper fluorescence staining is required in bioimaging. Among the commercially available PM probes, styryl dye FM1-43 is one of the most widely used. In this work, we demonstrated that fine chemical modifications of FM1-43 can dramatically improve the PM staining. The newly developed probes, SP-468 and SQ-535 were found to display enhanced photophysical properties (reduced crosstalk, higher brightness, improved photostability) and unlike FM1-43, provided excellent and immediate PM staining in 5 different mammalian cell lines including neurons (primary culture and tissue imaging). Additionally, we showed that the new probes displayed differences in their internalization pathways compared to their parent FM1-43. Finally, we demonstrated that the modifications made to FM1-43 did not impair the ability of the new probes to stain the PM of plant cells. Overall, this work presents new useful probes for PM imaging in cells and tissues and provides insights on the molecular design of new PM targeting molecules.

molecular biology↗

bioRxiv2018

MemBright: a Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience

The proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright: a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near-infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono and two-photon microscopy. These probes are compatible with long-term live cell imaging and immunostaining. Moreover, MemBright label neurons in a brighter manner than surrounding cells allowing identification of neurons in acute brain tissue section and neuromuscular-junctions without any use of transfection or transgenic animals. At last, MemBright were used in super-resolution imaging to unravel the dendritic spines neck. 3D multicolor dSTORM in combination with immunostaining revealed en-passant synapse displaying endogenous glutamate receptors clustered at the axonal-dendritic contact site. MemBright probes thus constitute a universal toolkit for cell biology and neuroscience biomembrane imaging with a variety of microscopy techniques.

neuroscience↗