Biology subjects

Hugelier, S.

Publications and source records attributed to Hugelier, S..

10 recordsLinked to original sources

bioRxiv2025

Sparse delivery of Synaptobrevin-2 to neurons using extracellular vesicles

Synaptobrevin-2 (Syb2) is an essential SNARE protein for neurotransmitter release and communication in the nervous system. We previously showed that Syb2 is also exchanged among neurons via extracellular vesicles (EVs). Host neurons can rapidly incorporate exogenous Syb2 into their synaptic vesicle cycle to support neurotransmitter release, however the endocytic mechanism of Syb2-containing EVs is unknown. Here, we use a fusion of Syb2 with the pH-sensitive GFP (Syb2-pHluorin) to track the incorporation of Syb2-containing EVs into neurons and the trafficking of exogenous Syb2 to synaptic vesicles at synapses. We determined that Syb2-containing EVs are endocytosed via Clathrin- and Dynamin-independent pathways. Moreover, Syb2-containing EVs are directly uptaken by axons and are rapidly incorporated into functional synaptic vesicles. These Syb2-pHluorin positive synaptic vesicles are endocytosed with either ultrafast (<1s) or fast ([~]1-3s) kinetics during synaptic transmission, suggesting limited diffusion and high fidelity in the fast retrieval of synaptic vesicle molecules immediately after fusion. This work introduces a novel application of EVs as vehicles to deliver fluorescent molecules in a neuron-specific, targeted manner to investigate protein transport mechanisms without overexpression artifacts. Our findings expand our understanding of the mechanisms EVs use to enter neurons.

cell biology↗

bioRxiv2024

Super-Resolution Imaging Uncovers Nanoscale Tau Aggregate Hyperphosphorylation Patterns in Human Alzheimers Disease Brain Tissue

Tau aggregation plays a critical role in Alzheimers Disease (AD), where tau neurofibrillary tangles (NFTs) are a key pathological hallmark. While much attention has been given to NFTs, emerging evidence underscores nano-sized pre-NFT tau aggregates as potentially toxic entities in AD. By leveraging DNA-PAINT super-resolution microscopy, we visualized and quantified nanoscale tau aggregates (nano-aggregates) in human postmortem brain tissues from intermediate and advanced AD, and Primary Age-Related Tauopathy (PART). Nano-aggregates were predominant across cases, with AD exhibiting a higher burden compared to PART. Hyperphosphorylated tau residues (p-T231, p-T181, and p-S202/T205) were present within nano-aggregates across all AD Braak stages and PART. Moreover, nano-aggregates displayed morphological differences between PART and AD, and exhibited distinct hyperphosphorylation patterns in advanced AD. These findings suggest that changes in nano-aggregate morphology and hyperphosphorylation patterns may exacerbate tau aggregation and AD progression. The ability to detect and profile nanoscale tau aggregates in human brain tissue opens new avenues for studying the molecular underpinnings of tauopathies.

biophysics↗

bioRxiv2024

Multiplexed DNA-PAINT Imaging of the Heterogeneity of Late Endosome/Lysosome Protein Composition

Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins, however, whether these proteins are uniformly present on each LEL, or if there are cell-type dependent LEL sub-populations with unique protein compositions is unclear. We employed a quantitative, multiplexed DNA-PAINT super-resolution approach to examine the distribution of six key LEL proteins (LAMP1, LAMP2, CD63, TMEM192, NPC1 and LAMTOR4) on individual LELs. While LAMP1 and LAMP2 were abundant across LELs, marking a common population, most analyzed proteins were associated with specific LEL subpopulations. Our multiplexed imaging approach identified up to eight different LEL subpopulations based on their unique membrane protein composition. Additionally, our analysis of the spatial relationships between these subpopulations and mitochondria revealed a cell-type specific tendency for NPC1-positive LELs to be closely positioned to mitochondria. Our approach will be broadly applicable to determining organelle heterogeneity with single organelle resolution in many biological contexts. SummaryThis study develops a multiplexed and quantitative DNA-PAINT super-resolution imaging pipeline to investigate the distribution of late endosomal/lysosomal (LEL) proteins across individual LELs, revealing cell-type specific LEL sub-populations with unique protein compositions, offering insights into organelle heterogeneity at single-organelle resolution.

cell biology↗

bioRxiv2023

ECLiPSE: A Versatile Classification Technique for Structural and Morphological Analysis of Super-Resolution Microscopy Data

We introduce a new automated machine learning analysis pipeline to precisely classify cellular structures captured through single molecule localization microscopy, which we call ECLiPSE (Enhanced Classification of Localized Pointclouds by Shape Extraction). ECLiPSE leverages 67 comprehensive shape descriptors encompassing geometric, boundary, skeleton and other properties, the majority of which are directly extracted from the localizations to accurately characterize individual structures. We validate ECLiPSE through unsupervised and supervised classification on a dataset featuring five distinct cellular structures, achieving exceptionally high classification accuracies nearing 100%. Moreover, we demonstrate the versatility of our approach by applying it to two novel biological applications: quantifying the clearance of tau protein aggregates, a critical marker for neurodegenerative diseases, and differentiating between two distinct morphological features (morphotypes) of TAR DNA-binding protein 43 proteinopathy, potentially associated to different TDP-43 strains, each exhibiting unique seeding and spreading properties. We anticipate that this versatile approach will significantly enhance the way we study cellular structures across various biological contexts, elucidating their roles in disease development and progression.

biophysics↗

bioRxiv2022

Simultaneous multicolor fluorescence imaging using duplication-based PSF engineering

We present a way to encode more information in fluorescence imaging by splitting the emission into copies of the original point-spread function (PSF), which offers broadband operation and compatibility with other PSF engineering modalities and existing analysis tools. We demonstrate the approach using the Circulator, an add-on that encodes the fluorophore emission band into the PSF, enabling simultaneous multicolor super-resolution and single-molecule microscopy using essentially the full field of view.

biophysics↗

bioRxiv2022

Detyrosination enrichment on microtubule subsets is established by the interplay between a stochastically-acting enzyme and microtubule stability

Microtubules in cells consist of functionally diverse subpopulations carrying distinct post-translational modifications (PTMs). Akin to the histone code, the tubulin code regulates a myriad of microtubule functions ranging from intracellular transport to chromosome segregation. Yet, how individual PTMs only occur on subsets of microtubules to contribute to microtubule specialization is not well understood. In particular, microtubule detyrosination, which is the removal of the C-terminal tyrosine on -tubulin subunits, marks the stable population of microtubules and modifies how microtubules interact with other microtubule-associated proteins to regulate a wide range of cellular processes. Previously, we found that, in certain cell types, only a small subpopulation of microtubules is highly enriched with the detyrosination mark ([~]30%) and that detyrosination spans most of the length of a microtubule, often adjacent to a completely tyrosinated microtubule. How the activity of a cytosolic detyrosinase, Vasohibin (VASH) leads to only a small subpopulation of highly detyrosinated microtubules is unclear. Here, using quantitative super-resolution microscopy, we visualized nascent microtubule detyrosination events in cells consisting of 1-3 detyrosinated -tubulin subunits after Nocodazole washout. Microtubule detyrosination accumulates slowly and in a disperse pattern across the microtubule length. By visualizing single molecules of VASH in live cells, we found that VASH engages with microtubules stochastically on a short time scale suggesting limited removal of tyrosine per interaction, consistent with the super-resolution results. Combining these quantitative imaging results with simulations incorporating parameters from our experiments, we propose a stochastic model for cells to establish a subset of detyrosinated microtubules via a detyrosination-stabilization feedback mechanism.

cell biology↗

bioRxiv2021

Self-contained and modular structured illumination microscope

We present a modular implementation of structured illumination microscopy (SIM) that is fast, largely self-contained and that can be added onto existing fluorescence microscopes. Our instrument, which we call HIT-SIM, can theoretically deliver well over 50 super-resolved images per second and is readily compatible with existing acquisition software packages. We provide a full technical package consisting of schematics, a list of components and an alignment scheme that provides detailed specifications and assembly instructions. We illustrate the performance of the instrument by imaging optically large samples containing sequence-specifically stained DNA fragments.

biophysics↗

bioRxiv2021

Simultaneous readout of multiple FRET pairs using photochromism

Forster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral band-width required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra. We develop the theory underlying this method and validate our approach using numerical simulations. To apply our system, we develop rsAKARev, a photochromic biosensor for cAMP-dependent kinase (PKA), and combine it with the spectrally-identical biosensor EKARev, a reporter for ERK kinase activity, to deliver simultaneous readout of both activities in the same cell. We further perform multiplexed PKA, ERK, and calcium measurements by including a third, spectrally-shifted biosensor. Our work demonstrates that exploiting donor photochromism in FRET can be a powerful approach to simultaneously read out multiple associations within living cells.

biophysics↗

bioRxiv2020

Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins

Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

biophysics↗

bioRxiv2018

Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of significantly longer tracks in sptPALM with minimum effort.

biophysics↗