Biology subjects

Hosy, E.

Publications and source records attributed to Hosy, E..

8 recordsLinked to original sources

bioRxiv2025

Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during microglial activation

ATP-gated purinergic P2X7 receptors are crucial ion channels involved in inflammation. They sense abnormal ATP release during stress or injury and are considered promising clinical targets for therapeutic intervention. However, despite their predominant expression in immune cells such as microglia, there is limited information on P2X7 membrane expression and regulation during inflammation at the single-molecule level, necessitating new labeling approaches to visualize P2X7 in native cells. Here, we present X7-uP, an unbiased, affinity-guided P2X7 chemical labeling reagent that selectively and covalently biotinylates endogenous P2X7 in BV2 cells, a murine microglial cell line, allowing subsequent labeling with streptavidin-Alexa 647 tailored for super-resolution imaging. We uncovered a nanoscale microglial P2X7 redistribution mechanism where evenly spaced individual receptors in quiescent cells undergo upregulation and clustering in response to the pro-inflammatory agent lipopolysaccharide and ATP, leading to synergistic interleukin-1{beta} release. Our method thus offers a new approach to revealing endogenous P2X7 expression at the single-molecule level. Impact statementAn affinity-guided chemical strategy enabling highly specific biotinylation of P2X7 receptors reveals, by super-resolution microscopy, how the nanoscale organization of endogenous P2X7 in BV2 microglial cells dynamically changes upon activation.

neuroscience↗

bioRxiv2022

Imaging minimal bacteria at the nanoscale: a reliable and versatile process to perform Single Molecule Localization Microscopy in mycoplasmas

Mycoplasmas are the smallest free-living organisms. These bacteria are important models for both fundamental and Synthetic Biology, owing to their highly reduced genomes. They are also relevant in the medical and veterinary fields, as they are pathogenic of both humans and most livestock species. Mycoplasma cells have minute sizes, often in the 300-800 nanometers range. As these dimensions are close to the diffraction limit of visible light, fluorescence imaging in mycoplasmas is often poorly informative. Recently developed "Super-Resolution Imaging" techniques can break this diffraction limit, improving the imaging resolution by an order of magnitude and offering a new nanoscale vision of the organization of these bacteria. These techniques have however not been applied to mycoplasmas before. Here, we describe an efficient and reliable protocol to perform Single-Molecule Localization Microscopy (SMLM) imaging in mycoplasmas. We provide a polyvalent transposon-based system to express the photo-convertible fluorescent protein mEos3.2, enabling Photo-Activated Localization Microscopy (PALM) in most Mycoplasma species. We also describe the application of direct STochastic Optical Reconstruction Microscopy (dSTORM). We showcase the potential of these techniques by studying the subcellular localization of two proteins of interest. Our work highlights the benefits of state-of-the-art microscopy techniques for mycoplasmology and provides an incentive to further the development SMLM strategies to study these organisms in the future. ImportanceMycoplasmas are important models in biology, as well as highly problematic pathogens in the medical and veterinary fields. The very small size of these bacteria, well below the micron, limits the usefulness of traditional fluorescence imaging methods as their resolution limit is similar to the dimensions of the cells. Here, to bypass this issue, we established a set of state-of-the-art "Super-Resolution Microscopy" techniques in a wide range of Mycoplasma species. We describe two strategies: PALM, based on the expression of a specific photo-convertible fluorescent protein; and dSTORM, based on fluorophore-coupled antibody labeling. With these methods, we successfully performed single-molecule imaging of proteins of interest at the surface of the cells and in the cytoplasm, at lateral resolutions well below 50 nanometers. Our work paves the way toward a better understanding of mycoplasmas biology through imaging of subcellular structures at the nanometer scale.

microbiology↗

bioRxiv2021

High-resolution imaging and manipulation of endogenous AMPA receptor surface mobility during synaptic plasticity and learning

Regulation of synaptic neurotransmitter receptor content is a fundamental mechanism for tuning synaptic efficacy during experience-dependent plasticity and behavioral adaptation. However, experimental approaches to track and modify receptor movements in integrated experimental systems are limited. Exploiting AMPA-type glutamate receptors (AMPAR) as a model, we generated a knock-in mouse expressing the biotin acceptor peptide (AP) tag on the GluA2 extracellular N-terminus. Cell-specific introduction of biotin ligase allows the use of monovalent or tetravalent avidin variants to respectively monitor or manipulate the surface mobility of endogenous AMPAR containing biotinylated AP-GluA2 in neuronal subsets. AMPAR immobilization precluded the expression of long-term potentiation and formation of contextual fear memory, allowing for target-specific control of the expression of synaptic plasticity and animal behavior. The AP tag knock-in model offers unprecedented access to resolve and control the spatiotemporal dynamics of endogenous receptors, and opens new avenues to study the molecular mechanisms of synaptic plasticity and learning.

neuroscience↗

bioRxiv2021

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

neuroscience↗

bioRxiv2020

CaMKII activation triggers persistent formation and segregation of postsynaptic liquid phase

Transient information input to brain leads to persistent changes in synaptic circuit, thereby forming memory engrams. Synapse undergoes coordinated functional and structural changes during this process but how such changes are achieved by its component molecules still largely remain enigmatic. We found that activated CaMKII, the central player of synaptic plasticity, undergoes liquid-liquid phase separation (LLPS) with NMDAR subunit GluN2B. Due to CaMKII autophosphorylation, the condensate stably persists even after Ca2+ is removed. The selective binding of activated CaMKII with GluN2B co-segregates AMPAR/neuroligin (NLGN) into a phase-in-phase assembly. Because postsynaptic NLGN clusters presynaptic neurexin and other active zone proteins thereby increasing the release probability of synaptic vesicles, this ensures efficient synaptic transmission. In this way, Ca2+-induced and persistent formation of LLPS by CaMKII serves as molecular basis of memory by functioning as an activity-dependent crosslinker for postsynaptic proteins and segregating trans-synaptic nanocolumns.

neuroscience↗

bioRxiv2020

Release sites are positioned to activate NMDA receptors

Neurotransmitter is released synchronously and asynchronously following an action potential. The release sites of these two phases are segregated within an active zone, with asynchronous release sites enriched near the center. Here we demonstrate that synchronous and asynchronous release sites are aligned with AMPA receptor and NMDA receptor clusters, respectively. Computational simulations indicate that this spatial and temporal arrangement of release ensures maximal membrane depolarization through AMPA receptors, alleviating the pore-blocking magnesium leading to greater activation of NMDA receptors. Together, these results suggest that release sites are organized to efficiently activate NMDA receptors.

neuroscience↗

bioRxiv2019

Specific nanoscale synaptic reshuffling and control of short-term plasticity following NMDAR- and P2XR-dependent Long-Term Depression

Long-Term Potentiation (LTP) and Long-Term Depression (LTD) of excitatory synaptic transmission are considered as cellular basis of learning and memory. These two forms of synaptic plasticity have been mainly attributed to global changes in the number of synaptic AMPA-type glutamate receptor (AMPAR) through a regulation of the diffusion/trapping balance at the PSD, exocytosis and endocytosis. While the precise molecular mechanisms at the base of LTP have been intensively investigated, the ones involved in LTD remains elusive. Here we combined super-resolution imaging technique, electrophysiology and modeling to describe the various modifications of AMPAR nanoscale organization and their effect on synaptic transmission in response to two different LTD protocols, based on the activation of either NMDA receptors or P2X receptors. While both type of LTD are associated with a decrease in synaptic AMPAR clustering, only NMDAR-dependent LTD is associated with a reorganization of PSD-95 at the nanoscale. This change increases the pool of diffusive AMPAR improving synaptic short-term facilitation through a post-synaptic mechanism. These results demonstrate that specific dynamic reorganization of synapses at the nanoscale during specific LTD paradigm allows to improve the responsiveness of depressed synapses.

neuroscience↗

bioRxiv2017

Pre-post synaptic alignment through neuroligin tunes synaptic transmission efficiency

The nanoscale organization of neurotransmitter receptors relative to pre-synaptic release sites is a fundamental determinant of both the amplitude and reliability of synaptic transmission. How modifications in the alignment between pre- and post-synaptic machineries affect synaptic current properties has only been addressed with computer modeling, and therefore remains hypothetical. Using dual-color single molecule based super-resolution microscopy, we found a strong spatial correlation between AMPA receptor (AMPAR) nanodomains and the post-synaptic adhesion protein neuroligin-1 (NLG1). Expression of a C-terminal truncated form of NLG1 disrupted this correlation without affecting the intrinsic organization of AMPAR nanodomains. Moreover, this NLG1 dominant-negative mutant significantly shifted the pre-synaptic release machinery from AMPAR synaptic clusters. Electrophysiology and computer modeling show that this physical shift is sufficient to induce a significant decrease in synaptic transmission. Thus, our results suggest the necessity for synapses to release glutamate in front of AMPAR nanodomains, to maintain a high efficiency of synaptic responses.

neuroscience↗