Biology subjects

Mougios, N.

Publications and source records attributed to Mougios, N..

9 recordsLinked to original sources

bioRxiv2026

Fatty acid binding protein-8 (FABP8/PMP2) reveals molecular heterogeneity of myelin sheaths in the human CNS

Myelin is classically viewed as a uniform axon-insulating membrane, yet its molecular composition may differ between species and even within one species. Fatty acid binding protein-8 (FABP8/PMP2) was previously identified in CNS myelin of humans but not mice. Here we show that FABP8/PMP2 is a defining feature of CNS myelin in humans and old-world-monkeys, but absent from CNS myelin in other mammals, indicating evolutionary neofunctionalization of this lipid-binding protein in the primate lineage. In the human CNS, FABP8/PMP2 marks a subset of myelin sheaths that preferentially ensheath large-diameter axons, revealing sheath-to-sheath molecular heterogeneity correlated with axonal geometry. Chromatin is accessible at the PMP2/Pmp2 gene locus in oligodendrocytes of humans but not mice. Human oligodendrocytes intrinsically express FABP8/PMP2 when transplanted into mouse brains, demonstrating species-specific competence independent of environmental cues. Humanized transgenic mice expressing FABP8/PMP2 in oligodendrocytes form morphologically normal but developmentally transiently thicker myelin sheaths, and show elevated cholesterol content in purified myelin. Because FABP8/PMP2 binds cholesterol, we propose that its emergence in primate CNS myelin contributes to the cholesterol enrichment of human myelin. Thus, CNS myelin protein composition is evolvable and modular, with relevance for myelin lipids and morphology, and previously unrecognized complexity in neuron-glia co-adaptation. Main points- Fatty acid binding protein 8 (FABP8/PMP2) is present in CNS myelin of humans and old-world monkeys - PMP2 defines sheath-to-sheath heterogeneity in the human CNS - PMP2-immunopositive myelin ensheaths large-diameter axons - Human oligodendrocytes intrinsically express PMP2 upon transplantation into mice - Humanized PMP2-transgenic mice show thicker myelin and altered myelin lipid composition

neuroscience↗

bioRxiv2026

Nanobodies equipped with HaloTag variants enable rapid and straightforward one-step immunofluorescence lifetime multiplexing

Multiplexed fluorescence imaging is limited by spectral overlap, whereas fluorescence lifetime provides an orthogonal encoding dimension. We replace genetically fused HaloTags with recombinant nanobody-HaloTag constructs applied as immunofluorescence reagents and determine a lifetime palette. Integrating lifetime and spectral encoding enables up to eight targets in a single acquisition and supports multiplexed imaging in cells and tissue using a simple OneStep labeling strategy compatible with fluorescent proteins, STED nanoscopy, and any standard laboratory antibody.

cell biology↗

bioRxiv2025

A versatile microfluidics platform for enhanced multi-target super-resolution microscopy

DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is a powerful variant of single-molecule localization microscopy (SMLM) that overcomes the limitations of photobleaching, offers flexible fluorophore selection, and enables fine control of imaging parameters through tunable on- and off-binding kinetics. Its most distinctive feature is the capacity for multiplexing, which is achieved through a process known as Exchange-PAINT. This technique involves assigning orthogonal DNA strands to different targets within a sample and then sequentially adding and removing complementary imager strands that are specific to only one target at a time. However, manual Exchange-PAINT workflows are often inefficient, prone to drift and variability, and lack reproducibility. Here, we introduce a custom compressed-air-driven microfluidics system specifically designed for multiplexed SMLM. Featuring a stackable and modular design that is, in principle, not limited by the number of channels, the system ensures robust, reproducible, and material-efficient buffer exchange with minimal dead volume. It operates in both manual and automated modes and can be readily adapted to a wide range of commercial and custom microscopes, including wide-field, confocal, STED, and MINFLUX platforms. We demonstrate robust 5-plex Exchange-PAINT imaging in cancerous U2OS cells and, importantly, we establish multiplexed nanoscale imaging in fragile primary cardiomyocytes. These applications highlight the unique power of our platform to extend super-resolution multiplexing into physiologically relevant systems, thereby opening new avenues for biomedical research.

biophysics↗

bioRxiv2024

Three-dimensional multi-target super-resolution microscopy of cells using Metal-Induced Energy Transfer and DNA-PAINT

AbsrtactAchieving nanometer precision in 3D remains a major challenge in super-resolution microscopy. DNA-PAINT offers excellent lateral resolution and versatile multiplexing, but its axial localization precision is typically 3-5 times poorer, limiting quantitative 3D imaging. Here, we present MIET-PAINT, which combines DNA-PAINT with Metal-Induced Energy Transfer (MIET) to overcome this limitation. We implement MIET-PAINT on both wide-field fluorescence lifetime and confocal TCSPC platforms. Wide-field MIET-PAINT enables robust, multiplexed imaging of focal adhesion proteins and actin in fixed cells. To address the lateral resolution limits of lifetime cameras, we further developed confocal MIET-PAINT, which leverages optical background rejection and high-efficiency SPAD detection. This modality achieves [~]12 nm lateral precision and resolves the 3D architecture of microtubules, vimentin, and actin with high fidelity. MIET-PAINT thus unites nanometer-scale axial accuracy with the multiplexing versatility of DNA-PAINT, establishing a powerful tool for quantitative 3D cell biology.

biophysics↗

bioRxiv2024

NanoPlex: a universal strategy for fluorescence microscopy multiplexing using nanobodies with erasable signals

Fluorescence microscopy has long been a transformative technique in biological sciences. Nevertheless, most implementations are limited to a few targets, revealed using primary antibodies (1.Abs) and fluorescently conjugated secondary antibodies. Super-resolution techniques such as Exchange-PAINT and, more recently, SUM-PAINT have increased multiplexing capabilities, but they require specialized equipment, software, and knowledge. To enable multiplexing for any imaging technique in any laboratory, we developed NanoPlex, a streamlined method based on conventional 1.Abs revealed by engineered secondary nanobodies (2.Nbs) that allow to selectively erase the fluorescence signals. We developed three complementary signal removal strategies: OptoPlex (light-induced), EnzyPlex (enzymatic), and ChemiPlex (chemical). We showcase NanoPlex reaching 21 targets for 3D confocal analyses and 5-8 targets for dSTORM and STED super-resolution imaging. NanoPlex has the potential to revolutionize multi-target fluorescent imaging methods, potentially redefining the multiplexing capabilities of antibody-based assays.

neuroscience↗

bioRxiv2023

Neuronal activity modulates the incorporation of newly translated PSD-95 into a robust structure as revealed by STED and MINFLUX

The postsynaptic density component PSD-95 undergoes activity-dependent plasticity mechanisms that rely on protein synthesis and structural remodeling. How synaptic activity can influence these dynamics at the single synapse level remains unclear. Here we combine genome-editing, pulse-chase experiments, STED and 3D MINFLUX nanoscopy on hippocampal neuronal cultures to study the integration of newly translated PSD-95 molecules at postsynaptic sites and their rearrangement within individual clusters at near-molecular resolution. We show that the amount of newly translated PSD-95 recruited to individual synapses scales with synaptic size, and modulates in a bidirectional manner, resulting in less new protein following excitatory and more new protein following inhibitory stimulation. Furthermore, we show that within synaptic clusters PSD-95 has a dispersed organization that is largely robust to long-lasting changes in activity. Altogether, this work sheds new light on the mechanisms underlying plasticity at the single synapse level, adding previously inaccessible information.

neuroscience↗

bioRxiv2023

A versatile synaptotagmin-1 nanobody provides perturbation-free live synaptic imaging and low linkage-error in super-resolution microscopy

Imaging of living synapses has relied for over two decades on the overexpression of synaptic proteins fused to fluorescent reporters. This strategy changes the stoichiometry of synaptic components and ultimately affects synapse physiology. To overcome these limitations, here we introduce a nanobody that binds the calcium sensor synaptotagmin-1 (NbSyt1). This nanobody functions in living neurons as an intrabody (iNbSyt1) and is minimally invasive, leaving synaptic transmission almost unaffected, as demonstrated by the crystal structure of the NbSyt1 bound to synaptotagmin-1 and by our physiological data. Its single-domain nature enables the generation of protein-based fluorescent reporters, as we showcase here by measuring spatially-localized presynaptic Ca2+ with an NbSyt1-jGCaMP8 chimera. Moreover, its small size makes the NbSyt1 ideal for various super-resolution imaging methods. Overall, NbSyt1 is a versatile binder that will enable imaging in cellular and molecular neuroscience at a higher precision than possible in the past, over multiple spatiotemporal scales.

neuroscience↗

bioRxiv2022

Expansion microscopy at one nanometer resolution

Fluorescence imaging is one of the most versatile and widely-used tools in biology1. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving2, 3, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context4, 5. Here we report a solution to this problem, in the form of one-step nanoscale expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis6, 7 to enable the description of cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the [~]17 kDa protein calmodulin upon Ca2+ binding were readily observable. We also imaged and classified molecular aggregates in cerebrospinal fluid samples from Parkinsons Disease (PD) patients, which represents a promising new development towards improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

biophysics↗

bioRxiv2022

Heat denaturation enables multicolor X10-STED microscopy at single-digit nanometer resolution

Expansion microscopy (ExM) improves imaging quality by physically enlarging the biological specimens. In principle, combining a large expansion factor with optical super-resolution should provide extremely high imaging precision. However, large expansion factors imply that the expanded specimens are dim and are therefore poorly suited for optical super-resolution. To solve this problem, we present a protocol that ensures the 10-fold expansion of the samples through high-temperature homogenization (X10ht). The resulting gels exhibited relatively high fluorescence intensity, enabling the sample analysis by multicolor stimulated emission depletion (STED) microscopy, for a final resolution of 6-8 nm. X10ht offers a more thorough homogenization than previous X10 protocols based on enzymatic digestion, and thereby enables the expansion of thick samples. The better epitope preservation also enables the use of nanobodies as labeling probes and the implementation of post-expansion signal amplification. We conclude that X10ht is a promising tool for nanoscale resolution in biological samples.

bioengineering↗