Biology subjects

Boroni-Rueda, F.

Publications and source records attributed to Boroni-Rueda, F..

4 recordsLinked to original sources

bioRxiv2025

Non-synaptic exocytosis along the axon shaft and its regulation by the submembrane periodic skeleton

Neuronal communication relies on signaling molecules transferred via exo- and endocytosis throughout the brain. Historically, studies have focused on vesicle exo- and endocytosis and their release machinery at synapses, and much less is known about non-synaptic exocytosis. If and how vesicles can access the plasma membrane along the axonal shaft, overcoming the insulating layer of the membrane-associated periodic scaffold, remains unclear. Here, we used fast live-cell imaging of mature cultured hippocampal neurons expressing the vamp2-pHluorin reporter to map sponta-neous exocytosis along axons. We detected non-synaptic exocytic events along the axon shaft that concentrated at the axon initial segment. Perturbation of the membrane-associated actin-spectrin skeleton revealed its role in regulating shaft exocytosis, similarly to its recently demonstrated role in shaping axonal endocytosis. To visualize the nanoscale arrangement of exocytic locations, we developed a novel correlative live-cell/two - color, single-molecule localization microscopy (SMLM) approach. We observed that regions of exocytosis are devoid of the submembrane spectrin mesh, with these spectrin-free areas being spatially separated from the spectrin clearings that contain clathrin-coated pits. Overall, our work reveals a new process of spontaneous exocytosis along the axon shaft, and how the axonal submem-brane skeleton shapes a heterogeneous landscape that uniquely segregates vesicular trafficking events.

cell biology↗

bioRxiv2023

The actin-spectrin submembrane scaffold restricts endocytosis along proximal axons

Neuronal clathrin-mediated endocytosis has unique features in compartments such as dendrites and presynaptic boutons, but how membrane and extracellular components are internalized along the axon shaft remains poorly known. Here we focused on clathrin-coated structures and endocytosis along the axon initial segment (AIS), and their relationship to the periodic actin-spectrin scaffold that lines the axonal plasma membrane. Super-resolution optical microscopy, platinum replica electron microscopy, and their correlative combination on cultured hippocampal neurons reveal that in the AIS, clathrin-coated pits form on bare membrane patches, [~]300 nm circular areas devoid of spectrin mesh and lined by actin filaments we termed "clearings". In fibroblasts and the proximal axon of neurons, spectrin depletion and drug-induced scaffold disorganization increase clathrin-coated pit formation. However, the presence of clathrin-coated pits at the AIS is not directly linked to actual endocytosis: using cargo uptake and live-cell imaging experiments, we find that most AIS clathrin-coated pits are long-lived and immobile within the spectrin mesh clearings. Direct perturbation of the spectrin scaffold as well as elevated neuronal activity could induce endocytosis downstream of clathrin pit formation, showing that spectrin clearings are structures responsible for regulated endocytosis at the AIS.

cell biology↗

bioRxiv2022

Distinct nano-structures support a multifunctional role of actin at presynapses

Synapses are the nexus of signal transmission in the nervous system. Despite decades of work, the architecture of the actin cytoskeleton that concentrates at presynapses remain poorly known, hindering our comprehensive understanding of its roles in presynaptic physiology. In this work, we take advantage of a validated model of bead-induced presynapses to measure and visualize isolated presynaptic actin by diffraction-limited and super-resolution microscopy. We first identify a major population of actin-enriched presynapses that concentrates more presynaptic components, and shows higher synaptic vesicle cycling than their non-enriched counterparts. Using pharmacological perturbations, we determine that an optimal amount of actin is necessary for this effect of actin enrichment. Modulation of this effect by actin nucleation inhibitors indicates its dependance on distinct presynaptic actin assemblies. Using Single Molecule Localization Microscopy (SMLM), we directly visualize these nano-structures in isolated presynapses, defining an actin mesh at the active zone, actin rails between the active zone and deeper reserve pools, and actin corrals around the whole presynaptic compartment. We finally show that these three types of presynaptic actin nano-structures are differentially affected by actin nucleation inhibitors, consistent with their effect on presynaptic component concentration and on synaptic vesicle cycling.

neuroscience↗

bioRxiv2018

Automating multimodal microscopy with NanoJ-Fluidics

Fluorescence microscopy can reveal all aspects of cellular mechanisms, from molecular details to dynamics, thanks to approaches such as super-resolution and live-cell imaging. Each of its modalities requires specific sample preparation and imaging conditions to obtain high-quality, artefact-free images, ultimately providing complementary information. Combining and multiplexing microscopy approaches is crucial to understand cellular events, but requires elaborate workflows involving multiple sample preparation steps. We present a robust fluidics approach to automate complex sequences of treatment, labelling and imaging of live and fixed cells. Our open-source NanoJ-Fluidics system is based on low-cost LEGO hardware controlled by ImageJ-based software and can be directly adapted to any microscope, providing easy-to-implement high-content, multimodal imaging with high reproducibility. We demonstrate its capacity to carry out complex sequences of experiments such as super-resolved live-to-fixed imaging to study actin dynamics; highly-multiplexed STORM and DNA-PAINT acquisitions of multiple targets; and event-driven fixation microscopy to study the role of adhesion contacts in mitosis.

cell biology↗