Biology subjects

Perego, E.

Publications and source records attributed to Perego, E..

10 recordsLinked to original sources

bioRxiv2026

Mapping the diffusional landscape of short NEAT1 in living cells

The long non-coding RNA NEAT1 is a fundamental architect of nuclear condensates, specifically paraspeckles. While the scaffold-essential isoform NEAT1-2 has been extensively characterized, the function and dynamics of its shorter isoform, NEAT1-1, remain poorly understood. Investigating NEAT1-1 in live cells has been historically hindered by its genomic overlap with NEAT1-2. Traditional visualization study designs require either the genetic ablation of NEAT1-2, which disrupts paraspeckle integrity, or the use of bulky tandem tagging arrays, which can sterically hinder RNA folding and partitioning. Here, we implemented a non-invasive imaging strategy and performed diffusivity analysis of NEAT1-1 using the fluorescence light-up aptamer biRhoBAST. This small, high-affinity RNA tag enables high-contrast visualization of NEAT1-1 while preserving the structural integrity of both isoforms and their associated nuclear bodies. By combining imaging and fluorescence fluctuation spectroscopy, we provide characterization of NEAT1-1 within intact micro-and para-speckles. Our results reveal that NEAT1-1 is not purely sequestered within visible condensates; rather, a fraction exists in a distinct diffusive state within the nucleoplasm, likely as nanoscale complexes. These findings suggest that NEAT1-1 possesses a previously unrecognized regulatory role independent of the primary paraspeckle scaffold, offering new insights into the functional diversity of the lncRNA isoforms.

biophysics↗

bioRxiv2026

BrightEyes-FFS: an open-source platform for comprehensive analysis of fluorescence fluctuation spectroscopy experiments with small detector arrays

Fluorescence fluctuation spectroscopy (FFS) is an ensemble of techniques for quantitative measurement of molecular dynamics and interactions. Recently, the introduction of small-format array detectors has opened up a new range of spatiotemporal information, allowing for more detailed analysis of system kinetics. However, there is currently no open-source software available for analyzing the high-dimensional FFS data sets. We present BrightEyes-FFS, an open-source Python-based environment for FFS analysis with array detectors. The environment includes a Python package for reading raw FFS data, computing auto- and cross-correlations using various algorithms, and fitting the correlations to several models. A graphical user interface (GUI), available as a standalone executable, makes the analysis fast and user-friendly. An automated Jupyter Notebook writing tool enables transition from the GUI to Jupyter Notebook for custom analysis. We believe that BrightEyes-FFS will enable a wider community to study diffusion, flow, and interaction dynamics.

bioinformatics↗

bioRxiv2024

The role of RNA in the nanoscale organization of alpha-synuclein phase separation

The cellular accumulation of alpha-synuclein (aS) aggregates is a hallmark of several neurodegenerative diseases. Recent studies suggest that the aberrant transition of monomeric aS into solid-like aggregates may occur through an intermediate liquid-like state, where the protein partitions between dense and dilute phases. Although aS is not typically recognized as an RNA-binding protein, it can bind RNA under aggregation conditions, but its impact on aS liquid-like phases remains unexplored. Employing a combination of fluorescence spectroscopy techniques, we investigated aS dynamics in both phases in the presence of RNA. Our analysis revealed the formation of nanoclusters involved in initiating phase separation and uncovered heterogeneity within the dense phase, discovering that aS molecules exist in two distinct mobility states. Additionally, we demonstrated that RNA induces morphological changes and promotes the liquid-to-solid transition of aS dense phase. These findings underscore the active role of RNA in modulating aS phase transitions.

biophysics↗

bioRxiv2024

CONDENSATES OF SYNAPTIC VESICLES AND SYNAPSIN ARE MOLECULAR BEACONS FOR ACTIN SEQUESTERING AND POLYMERIZATION

Neuronal communication relies on precisely maintained synaptic vesicle (SV) clusters, which assemble via liquid-liquid phase separation (LLPS). This process requires synapsins, the major synaptic phosphoproteins, which are known to bind actin. The reorganization of SVs, synapsins and actin is a hallmark of synaptic activity, but their interplay is still unclear. Here, we combined the reconstitution approaches, expansion microscopy, super-resolution imaging and cryo-electron tomography to dissect the roles of synapsin-SV condensates in the organization of the presynaptic actin cytoskeleton. Our data indicate that LLPS of synapsin initiates actin polymerization, allowing for SV:synapsin:actin assemblies to facilitate the mesoscale organization of SV clusters along axons mimicking the native presynaptic organization in both lamprey and mammalian synapses. Understanding the relationship between the actin network and synapsin-SVs condensates is an essential building block on a roadmap to unravel how coordinated neurotransmission along the axon enables circuit function and behavior.

cell biology↗

bioRxiv2023

M6A reduction relieves FUS-associated ALS granules

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motorneurons (MN) degeneration1. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by mutant protein aggregation, among which the RNA binding protein FUS2. In this work we show that such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished as a consequence of the m6A writer METTL3 knock-down. These effects were observed both in neuronal cell lines and in iPSC-derived human motor neurons expressing mutant FUS. Importantly, stress granules formed in ALS condition showed a distinctive transcriptome with respect to control cells; interestingly, after METTL3 downregulation, it reverted to similar to control. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, a well characterized inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.

molecular biology↗

bioRxiv2023

ALS-associated FUS mutation reshapes the RNA and protein composition and dynamic of Stress Granules.

Stress Granules (SG) formation is a cellular protection mechanism, constituting a storage for untranslated mRNAs and RNA-binding proteins (RBPs); however, these condensates can turn into pathological aggregates, related to the onset of neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS). This transition towards cytotoxic inclusions is triggered by ALS-causative mutations in the RBP FUS, which lead to its cytoplasmic mis-localization and accumulation in SG. Here, we describe the SG transcriptome in a neural context and describe several features for RNA recruitment in SG. We demonstrate that SG dynamics and RNA content are strongly modified by the incorporation of mutant FUS, switching to a more unstructured, AU-rich SG transcriptome. Moreover, we show that mutant FUS, together with its protein interactors and their target RNAs, are responsible for the reshaping of the mutant SG transcriptome with alterations that can be linked to neurodegeneration. Therefore, our data give a comprehensive view of the molecular differences between physiological and pathological SG in ALS conditions, showing how FUS mutations impact the RNA and protein population of these condensates.

molecular biology↗

bioRxiv2023

4D Single-Particle Tracking with Asynchronous Read-Out SPAD-Array Detector

Single-particle tracking (SPT) techniques are essential for investigating the com-plex functions and interactions of individual, specifically labelled particles in biological environments. Many SPT techniques exist, each optimised towards a different balance between spatiotemporal resolution and range, technical com-plexity, and information content. This bargain is exemplified by the contrast between wide-field camera-based and real-time SPT approaches, with the latter being generally more advanced but at the cost of high complexity. Further-more, the fluorescence lifetime, a powerful tool for investigating the particles interactions and nano-environment, has yet to be measured consistently. To overcome these limitations, we propose a novel real-time three-dimensional SPT technique based on a hybrid approach. In our implementation, we equip a confocal laser-scanning microscope with an asynchronous read-out single-photon avalanche diode (SPAD) array detector and few other optics. Each sensitive detector element acts as a confocal pinhole, and the recorded intensity distribu-tion reflects the particles position in three dimensions relative to the excitation volume. This localization is used in a real-time feedback system to keep the par-ticle in the centre of the excitation volume. Importantly, as each pixel is an independent single-photon detector, SPT is combined with fluorescence lifetime measurement. Our system achieves a localization precision of up to 30 nm with 100 photons and microsecond time resolution, while also performing fluorescence lifetime mea-surements. First, we validated the technique by tracking fluorescent particles in artificial environments. Secondly, as further validation, we investigated the move-ment of lysosomes in living SK-N-BE cells and measured the fluorescence lifetime of the GFP marker expressed on a membrane protein. We observed an unprece-dented correlation between the changes in fluorescence lifetime and the motion state of the lysosomes. Thanks to its simplicity and the great momentum of confocal microscopy based on SPAD array detector, we expect that this implementation will open to many information-rich correlative imaging and tracking studies.

biophysics↗

bioRxiv2023

Content-enriched fluorescence lifetime fluctuation spectroscopy to study bio-molecular condensate formation

Quantitative fluorescence microscopy is experiencing an important revolution thanks to single-photon array detectors. These detectors provide users with so far inaccessible specimen information: The distribution of the specimens fluorescence emission at single-photon level and high spatiotemporal sampling. In laser-scanning microscopy, this photon-resolved measurement has enabled robust fluorescence lifetime imaging at sub-diffraction spatial resolution, thus opening new perspectives for structural and functional imaging. Despite these significant advances in imaging, studying the time evolution of biological processes remains a considerable challenge. Here we present a com-prehensive framework of live-cell spectroscopy methodologies - compatible with imaging - to investigate bio-molecular processes at various spatiotemporal scales. We use photon-resolved spatial and temporal measurements granted by a single-photon array detector to boost the information content of a unified fluorescence fluctuation spectroscopy and fluorescence lifetime experiment. To demonstrate the potential of this approach, we investigate the phase transition of liquid-like condensates during oxidative stress inside living cells. These condensates are generally found in several cellular processes and exhibit substantial variations in molecular composition, size, and kinetics, posing a significant challenge for quantifying their underlying molecular dynamics. This study demonstrates how the pro-posed approach reveals the mutual dynamics of different RNA-binding proteins involved in the stress granules formation - inaccessible to imaging alone. We observe condensate formation by performing time-lapse super-resolved imaging of the cellular macro-environment while simultaneously monitoring the molecular mobility, the sub-diffraction environment organization, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. We are confident that our framework offers a versatile toolkit for investigating a broad range of bio-molecular processes - not limited to liquid-liquid phase transition - and we anticipate their widespread application in future life-science research.

biophysics↗

bioRxiv2021

The BrightEyes-TTM: an Open-Source Time-Tagging Module for Single-Photon Microscopy

Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to the introduction of new asynchronous read-out single-photon (SP) array detectors. These detectors give access to an entirely new set of single-photon information typically lost in conventional fluorescence LSM, thus triggering a new imaging/spectroscopy paradigm - the so-called singlephoton LSM (SP-LSM). The revolutions outcomes are, from one side, the blooming of new SP-LSM techniques and tailored SP array detectors; from the other side, the need for data-acquisition (DAQ) systems effectively supporting such innovations. In particular, there is a growing need for DAQ systems capable of handling the high throughput and high temporal resolution information generated by the single-photon detectors. To fill this gap, we developed an open-source multi-channel timetagging module (TTM) based on a field-programmable-gatearray (FPGA), that can temporally tag single-photon events - with 30 ps precision - and synchronisation events - with 4 ns precision. Furthermore, being an open-access project, the TTM can be upgraded, modified, and customized by the microscopy-makers. We connected the TTM to a fluorescence LSM equipped with a single-photon avalanche diode (SPAD) bi-dimensional array detector, and we implemented fluorescence lifetime image scanning microscopy (FLISM) and, for the first time, fluorescence lifetime fluctuation spectroscopy (FLFS). We expect that our BrigthEyes-TTM will support the microscopy community to spread SP-LSM in many life science labs.

bioengineering↗

bioRxiv2021

Cooled SPAD array detector for low light-dose fluorescence laser scanning microscopy

The single-photon timing and sensitivity performance and the imaging ability of asynchronous-readout single-photon avalanche diode (SPAD) array detectors have opened up enormous perspectives in fluorescence (lifetime) laser scanning microscopy (FLSM), such as super-resolution image scanning microscopy and high-information content fluorescence fluctuation spectroscopy (FFS). However, the strengths of these FLSM techniques depend on the many different characteristics of the detector, such as dark-noise, photon-detection efficiency, after-pulsing probability, and optical-cross talk, whose overall optimization is typically a trade-off between these characteristics. To mitigate this trade-off, we present a novel SPAD array detector with an active cooling system, which substantially reduces the dark-noise without significantly deteriorating any other detector characteristics. In particular, we show that lowering the temperature of the sensor to -15{degrees}C significantly improves the signal-to-noise ratio due to a 10-fold decrease in the dark-count rate compared to room temperature. As a result, for imaging, the laser power can be decreased by more than a factor of three, which is particularly beneficial for live-cell super-resolution imaging, as demonstrated in fixed and living cells expressing GFP-tagged proteins. For FFS, together with the benefit of the reduced laser power, we show that cooling the detector is necessary to remove artifacts in the correlation function, such as spurious negative correlations observed in the hot elements of the detector, i.e., elements whose dark-noise is substantially higher than the median value. Overall, this detector represents a further step towards the integration of SPAD array detectors in any FLSM system. SIGNIFICANCESingle-photon avalanche diode (SPAD) array detectors are revolutionizing fluorescence laser-scanning microscopy (FLSM). Thanks to their single-photon timing and sensitivity ability and their imaging faculty, a SPAD array detector transforms any FLSM into a super-resolution microscope, and opens a whole range of possibilities for the study of sample dynamics by means of fluorescence fluctuation spectroscopy (FFS). However, dark-noise can be a severe problem for both imaging and FFS. For imaging, the signal overcomes noise only for a relatively high illumination intensity, which can be detrimental for live-cell experiments. For FFS, the noise leads to artifacts in the correlation curves, potentially leading to wrong conclusions about the sample. We show that lowering the temperature of the detector to -15{degrees}C solves both problems

biophysics↗