Biology subjects

Domart, F.

Publications and source records attributed to Domart, F..

5 recordsLinked to original sources

bioRxiv2025

3D MINFLUX combined with DNA-PAINT resolves the arrangement of Bassoon at active zones

Neurotransmitter release and membrane retrieval at active zones require precise spatial and temporal coordination relying on an intricate molecular machinery. However, the exact nano-structural organization of this machinery is not yet fully elucidated. Here, we used 3D MINFLUX combined with both spectral demixing and DNA-PAINT to analyze the positioning of the scaffolding protein Bassoon at presynaptic active zones of glutamatergic spine synapses of hippocampal neurons, achieving a localization precision of 5 nm in 3D. This approach allowed us to visualize directly the distribution of N-terminal and the C-terminal regions of Bassoon, and demonstrates that Bassoon exhibits an orientation at the active zone, where the C-terminal region is directed toward the synaptic cleft and the N-terminal region towards synaptic vesicles. Having demonstrated this spatial configuration for endogenous and recombinant Bassoon molecules, our study paves the way towards molecular-scale resolution analysis of other prominent proteins of the presynaptic release machinery.

neuroscience↗

bioRxiv2025

A correlative workflow streamlines synaptic imaging by cryo-electron tomography

Despite decades of intense research, the molecular organization of the synapse is not well understood. To address this issue, we sought to develop a method for systematic imaging of synapses by cryo-electron tomography (cryo-ET), a technology capable of mapping cellular architecture at molecular resolution. Thinning of cellular samples by cryo-focused ion beam milling is a prerequisite for high-quality cryo-ET imaging, but this process needs to be guided to the structures of interest. To allow robust synaptic targeting, we established a correlative cryo-light/electron microscopy approach by which synapses are fluorescently labeled in a minimally invasive manner, using a synthetic binder of the postsynaptic scaffold PSD-95 and antibodies against the presynaptic protein Synaptotagmin-1. Cryo-ET imaging at sites of colocalization consistently revealed excitatory synapses. Our method allows structural studies of synaptic protein complexes in situ, facilitating investigations of the molecular architecture of synapses.

neuroscience↗

bioRxiv2022

The synaptic vesicle protein Mover/TPRG1L is associated with lipid droplets in astrocytes

Crucial brain functions such as neurotransmission, myelination, and signaling pose a high demand for lipids. Lipid dysregulation is associated with neuroinflammation and neurodegeneration. Astrocytes protect neurons from lipid induced damage by accumulating and metabolizing toxic lipids in organelles called lipid droplets (LDs). LDs have long been considered as lipid storage compartments in adipocytes, but less is known about their biogenesis and composition in the brain. In particular, proteins covering the LD surface are not yet fully identified. Here, we report that the synaptic vesicle protein Mover/TPRG1L, which regulates the probability of neurotransmitter release in neurons, is a component of the LD coat in astrocytes. Using conventional and super resolution microscopy, we demonstrate that Mover surrounds naive and oleic acid induced astrocytic LDs. We confirm the identity of astrocytic LDs using the neutral lipid stains Bodipy and LipidTox, as well as immunofluorescence for perilipin-2, a known component of the LD coat. In astrocytes, recombinant Mover was sufficient to induce an accumulation of LDs. Furthermore, we identified point mutations that abolish targeting to LDs and show similarities in the required binding sequences for association to the presynapse and LDs. Our results show that Mover is not only a presynaptic protein but also a candidate for LD regulation. This highlights the dual role of Mover in synaptic transmission and regulation of astrocytic LDs, which may be particularly important in the context of lipid-related neurological disorders. Main points- Mover is a lipid droplet protein - Overexpression of Mover drives lipid droplet accumulation - The F206 is important for LD targeting

neuroscience↗

bioRxiv2020

Cytoplasmic aggregation of uranium in human dopaminergic cells after continuous exposure to soluble uranyl at non-cytotoxic concentrations

Uranium exposure can lead to neurobehavioral alterations in particular of the monoaminergic system, even at non-cytotoxic concentrations. However, the mechanisms of uranium neurotoxicity after non-cytotoxic exposure are still poorly understood. In particular, imaging uranium in neurons at low intracellular concentration is still very challenging. We investigated uranium intracellular localization by means of synchrotron X-ray fluorescence imaging with high spatial resolution (< 300 nm) and high analytical sensitivity (< 1 g.g-1 per 300 nm pixel). Neuron-like SH-SY5Y human cells differentiated into a dopaminergic phenotype were continuously exposed, for seven days, to a non-cytotoxic concentration (10 M) of soluble natural uranyl. Cytoplasmic submicron uranium aggregates were observed accounting on average for 62% of the intracellular uranium content. In some aggregates, uranium and iron were co-localized suggesting common metabolic pathways between uranium and iron storage. Uranium aggregates contained no calcium or phosphorous indicating that detoxification mechanisms in neuron-like cells are different from those described in bone or kidney cells. Uranium intracellular distribution was compared to fluorescently labeled organelles (lysosomes, early and late endosomes) and to fetuin-A, a high affinity uranium-binding protein. A strict correlation could not be evidenced between uranium and the labelled organelles, or with vesicles containing fetuin-A. Our results indicate a new mechanism of uranium cytoplasmic aggregation after non-cytotoxic uranyl exposure that could be involved in neuronal defense through uranium sequestration into less reactive species. The remaining soluble fraction of uranium would be responsible for protein binding and the resulting neurotoxic effects.

pharmacology and toxicology↗

bioRxiv2019

Correlating STED and synchrotron XRF nano-imaging unveils the co-segregation of metals and cytoskeleton proteins in dendrites

Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecular mechanisms behind these processes are still elusive due in part to the difficulty of imaging trace metals together with proteins at the synaptic level. We correlate stimulated emission depletion (STED) microscopy of proteins and synchrotron X-ray fluorescence (SXRF) imaging of trace metals, both performed with 40 nm spatial resolution, on primary rat hippocampal neurons. We achieve a detection limit for zinc of 14 zeptogram (10-21 g) per pixel. We reveal the co-localization at the nanoscale of zinc and tubulin in dendrites with a molecular ratio of about one zinc atom per tubulin-{beta} dimer. We observe the co-segregation of copper and F-actin within the nano-architecture of dendritic protrusions. In addition, zinc chelation causes a decrease in the expression of cytoskeleton proteins in dendrites and spines. Overall, these results indicate new functions for zinc and copper in the modulation of the cytoskeleton morphology in dendrites, a mechanism associated to neuronal plasticity and memory formation.

neuroscience↗