Lydia Danglot

Late endosomal secretion is an unconventional secretion mechanism that depends on the SNARE protein VAMP7. We previously showed that VAMP7 mediates the secretion of the ER protein Reticulon3. However, the functional relevance and molecular mechanism of this secretory pathway remain unclear. Here, we show that VAMP7 knockout cells exhibit impaired secretion of ER- and mitochondrial-derived proteins and signs of ER and mitochondrial stress. In addition, pharmacological induction of organellar stress enhances the VAMP7-dependent secretion. We assess the pathophysiological significance of this mechanism using a preclinical glioblastoma model. VAMP7 knockout glioblastoma cells implanted in male rat brain develop into more necrotic tumors with reduced macrophage infiltration compared to controls, suggesting that VAMP7-dependent late endosomal secretion contributes to the tumor microenvironment and affects macrophage infiltration. Together, our results support a model in which late endosomal secretion functions as an organelle quality-control and stress-communication mechanism, with particular relevance to cancer.

In recent years, advances in microscopy and the development of novel fluorescent probes have significantly improved neuronal imaging. Many neuropsychiatric disorders are characterized by alterations in neuronal arborization, neuronal loss—as seen in Parkinson’s disease—or synaptic loss, as in Alzheimer’s disease. Neurodevelopmental disorders can also impact dendritic spine morphogenesis, as observed in autism spectrum disorders and schizophrenia. In this review, we provide an overview of the various labeling and microscopy techniques available to visualize neuronal structure, including dendritic spines and synapses. Particular attention is given to available fluorescent probes, recent technological advances in super-resolution microscopy (SIM, STED, STORM, MINFLUX), and segmentation methods. Aimed at biologists, this review presents both classical segmentation approaches and recent tools based on deep learning methods, with the goal of remaining accessible to readers without programming expertise.

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 metabolism ^1–3 . Here, using Drosophila melanogaster , we show memory formed after intensive massed training is dependent on mitochondrial fatty acid (FA) β-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.

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.

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 that is prone to selection biases and human operational errors. Here, we introduce ExoJ, an automated plugin based on Fiji/ImageJ2 software. ExoJ identifies user-defined genuine populations of exocytosis events, recording quantitative features including intensity, apparent size and duration. We designed ExoJ to be fully user-configurable, making it suitable for studying distinct forms of vesicle exocytosis regardless of the imaging quality. Our plugin demonstrates its capabilities by showcasing distinct exocytic dynamics among tetraspanins and vesicular SNARE protein reporters. Assessment of performance on synthetic data shows that ExoJ is a robust tool that is capable of correctly identifying exocytosis events independently of signal-to-noise ratio conditions. We propose ExoJ as a standard solution for future comparative and quantitative studies of exocytosis.

Photomodulable fluorescent probes are drawing 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, which possess both properties of conversion and switching. Our study demonstrates that these pyrrolyl-BODIPYs convert into typical green- and red-emitting BODIPYs that are perfectly adapted to microscopy. We also showed that this pyrrolyl-BODIPYs undergo Directed Photooxidation Induced Conversion, a photoconversion mechanism that we recently introduced, where the pyrrole moiety plays a central role. These unique features were used to develop targeted photoconvertible probes toward different organelles or subcellular units (plasma membrane, mitochondria, nucleus, actin, Golgi apparatus, etc.) using chemical targeting moieties and a Halo tag. We notably showed that BDP-650 could be used to track intracellular vesicles over more than 20 min in two-color imagings 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 mitochondrial 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 advanced live imaging.

The development of robust tools for segmenting cellular and sub-cellular neuronal structures lags behind the massive production of high-resolution 3D images of neurons in brain tissue. The challenges are principally related to high neuronal density and low signal-to-noise characteristics in thick samples, as well as the heterogeneity of data acquired with different imaging methods. To address this issue, we design a framework which includes sample preparation for high resolution imaging and image analysis. Specifically, we set up a method for labeling thick samples and develop SENPAI, a scalable algorithm for segmenting neurons at cellular and sub-cellular scales in conventional and super-resolution STimulated Emission Depletion (STED) microscopy images of brain tissues. Further, we propose a validation paradigm for testing segmentation performance when a manual ground-truth may not exhaustively describe neuronal arborization. We show that SENPAI provides accurate multi-scale segmentation, from entire neurons down to spines, outperforming state-of-the-art tools. The framework will empower image processing of complex neuronal circuitries.

Loss of arterial smooth muscle cells (SMCs) and abnormal accumulation of the extracellular domain of the NOTCH3 receptor (Notch3 ^ECD ) are the 2 core features of CADASIL, a common cerebral small vessel disease caused by highly stereotyped dominant mutations in NOTCH3 . Yet the relationship between NOTCH3 receptor activity, Notch3 ^ECD accumulation, and arterial SMC loss has remained elusive, hampering the development of disease-modifying therapies. Using dedicated histopathological and multiscale imaging modalities, we could detect and quantify previously undetectable CADASIL-driven arterial SMC loss in the CNS of mice expressing the archetypal Arg169Cys mutation. We found that arterial pathology was more severe and Notch3 ^ECD accumulation greater in transgenic mice overexpressing the mutation on a wild-type Notch3 background (Tg Notch3 ^R169C ) than in knockin Notch3 ^R170C/R170C mice expressing this mutation without a wild-type Notch3 copy. Notably, expression of Notch3 -regulated genes was essentially unchanged in Tg Notch3 ^R169C arteries. We further showed that wild-type Notch3 ^ECD coaggregated with mutant Notch3 ^ECD and that elimination of 1 copy of wild-type Notch3 in Tg Notch3 ^R169C was sufficient to attenuate Notch3 ^ECD accumulation and arterial pathology. These findings suggest that Notch3 ^ECD accumulation, involving mutant and wild-type NOTCH3, is a major driver of arterial SMC loss in CADASIL, paving the way for NOTCH3-lowering therapeutic strategies.

Imaging neuronal architecture has been a recurrent challenge over the years. The use of Golgi technique by Ramón y Cajal paved the way for the first characterization of neuronal architecture using microscopy on fixed brains. Indeed, metallic impregnation with silver salts provided an opportunity to see and reconstruct the dendritic architecture of various types of neurons in the depth of the nervous tissue. Although Golgi staining is still used in widefield microscopy, its use in confocal microscopy remains limited for 3D analysis. The use of fluorescent labeling in conjunction with 3D microscopy led to the production of large amounts of published data that are on the way to being classified and accessible through various infrastructures or free repositories (eBrains, Zenodo, etc.). This huge amount of data and their accessibility raise the question of potential new automated, unbiased statistical analysis.

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.