Biology subjects

Diaspro, A.

Publications and source records attributed to Diaspro, A..

At least 19 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↗

bioRxiv2023

DEK oncoprotein participates in heterochromatin replication via SUMO-dependent nuclear bodies

The correct inheritance of chromatin structure is key for maintaining genome function and preventing cellular transformation. DEK, a conserved chromatin protein, has recognized tumor-promoting properties, its overexpression being associated with poor prognosis in various cancer types. At the cellular level, DEK displays pleiotropic functions, influencing differentiation, apoptosis, and stemness, but a characteristic oncogenic mechanism remains elusive. Here, we report the identification of DEK bodies, focal assemblies of DEK occurring at specific, yet unidentified sites of heterochromatin replication. In these bodies, DEK localizes in direct proximity to active replisomes suggesting a function in the early maturation of heterochromatin. A high-throughput siRNA screen identifies SUMO as a major regulator of DEK body formation, linking DEK to the SUMO network that controls chromatin states and cell fate. This work combines and refines our previous data on DEK as a factor essential for heterochromatin integrity and facilitating replication under stress and delineates an avenue of further study for unraveling DEKs contribution to cancer development.

cell biology↗

bioRxiv2023

Super-resolution microscopy reveals the nanoscale organization of the DEK protein cancer biomarker

The chromatin architectural factor DEK is involved in regulating the chromatin organization and is highly overexpressed in numerous forms of cancer. DEK is traditionally described as uniformly distributed within the nucleus by diffraction-limited microscopy studies, but super-resolution advent highlighted the formation of cluster-like DEK structures. Still, a characterization of the DEK protein cellular distribution and its role in cancer and cell proliferation is missing. In this work, we used single-molecule localization microscopy (SMLM) to investigate the nanoscale characteristics of DEK organization in normal-like and aggressive types of breast cancer cell lines, characterizing the number of localizations per cluster, as well as areas and density of clusters. We show how the cluster features correlate with the breast cell type and how the chromatin decompaction influences the DEK clusters in both cell lines. Our results suggest that the DEK density and nano-organization are preserved and are not influenced by protein overexpression itself or by chromatin compaction changes.

biophysics↗

bioRxiv2022

Strategies and experimental tips for optimized quantitative single-molecule studies of membrane and membrane-associated proteins

The study of stoichiometry and supra-molecular organization of membrane (and membrane-associated) proteins plays a key role in understanding membrane structure and function. Single-molecule localization techniques (SML), besides providing imaging at unprecedented resolution, also offer quantitative tools such as stepwise photobleaching (SP) experiments and quantitative single-molecule localization (qSMLM). SML is becoming widely present in imaging core facilities but addressing biological problems by molecular counting experiments still remains not straightforward since experimental approaches for sample preparation require particular attention. We will focus on the experimental aspects that may prevent successful quantitative SML experiments of membrane-associated proteins. Depending on the specific experiment, to avoid artifacts and to miscount, fine-tuning of the expression levels and proper staining procedures are required, as well as optimized protocols and controls for counting. The work aims to highlight the crucial aspects that must be faced when quantitative single-molecule experiments are performed, helping to match the gap between sample preparation and the application of quantitative fluorescence microscopy techniques.

biophysics↗

bioRxiv2022

Optical-quality assessment of a miniaturized intraocular telescope

PurposeEvaluating the optical transmission and geometrical aberrations of an intraocular device, namely, the Small-Incision New Generation Implantable Miniature Telescope (SING IMT, Samsara Vision), designed to correct age-related macular degeneration. MethodsOptical transmission in the spectral range 350-750 nm of the implantable optics was recorded with a fiber-optic spectrometer. Geometrical aberrations were studied by measuring the wavefront of a laser beam after passing through the implantable optics and performing an expansion of the measured wavefront into a Zernike polynomial basis. The study was conducted under in-vitro experimental conditions. A second monofocal intraocular lens (SY60WF, Alcon) was tested and used as reference for assessing the optical quality of the SING IMT device. ResultsSpectroscopy measurements revealed that the SING IMT and monofocal IOL element feature UV-rejection and blue-rejection capabilities, respectively. Wavefront concavity indicated that the SING IMT behaves as a diverging lens with a focal length of approximately -100 mm; Zernike analysis showed that SING IMT has negligible coma, trefoil, astigmatism, and spherical aberrations of any order and along any direction. ConclusionsThe SING IMT exhibited even optical transmission in the whole visible spectrum and curvature capable of magnifying the retinal images without introducing geometrical aberrations, which proves the feasibility of this device as high-quality optical element for imaging. The rigidity of the compound lens of the SING IMT prevents mechanically-induced distortions, an issue encountered with polymeric lenses. Translational RelevanceSpectrometry and in vitro wavefront analysis provide evidence supporting the new generation miniaturized telescopic intraocular lens as a favorable option to intraocular implant in age-related macular degeneration.

biophysics↗

bioRxiv2022

Nanoscale engagement of immune checkpoint inhibitor PD-L1 in membrane lipid rafts

Programmed death ligand 1 (PD-L1) plays a key role in several human tumors, and it has recently become a crucial target for cancer therapy. Yet, its subtle regulatory roles beside the well characterized immunosuppression in PD-1/PD-L1 immune checkpoint are still obscure and should be strictly related to its molecular properties in different cell settings. In this study we targeted the plasma membrane organization of PD-L1 in a Non-Small Cell Lung Cancer (NSCLC) cell line by a multiscale fluorescence imaging toolbox that included super-resolution microscopy approaches to reach the nanoscale. Our findings revealed for the first time that PD-L1 is prevalently engaged in the cholesterol-enriched "raft" regions of the cell membrane. Clusterization and engagement in membrane rafts may afford novel targets for the immuno-oncology strategies in the framework of NSCLC and, possibly, other tumor types.

biophysics↗

bioRxiv2022

Focus-ISM for Sharp and Gentle Super-Resolved Microscopy

Super-resolution microscopy is routinely used for fixed and thin samples, while its feasibility for imaging live and thick samples is still limited. In the case of stimulated emission depletion (STED) microscopy, the high-intensity illumination required to achieve effective sub-diffraction resolution can introduce photo-damage, thus reducing the compatibility of the technique with live-cell imaging. Moreover, the out-of-focus fluorescence background may overcome the often faint signal stemming from the focal point, thus constraining imaging to thin samples. Here, we combined STED microscopy with image-scanning microscopy (ISM) to mitigate these limitations without any practical disadvantages. We first enhanced a laser scanning microscope (LSM) by introducing a detector array, hence providing access to a set of additional spatial information that is not available with a typical single-element detector. Then, we exploited this extended dataset to implement focus-ISM, a novel method that relaxes the high-intensity requirement of STED microscopy and removes the out-of-focus background. Additionally, we generalized the focus-ISM method to conventional LSM, namely without a STED beam. The proposed approach requires minimal architectural changes compared with conventional STED microscopes but provides substantial advantages for live and thick sample imaging while maintaining all compatibility with all recent advances in STED and confocal microscopy. As such, focus-ISM represents an essential step towards a universal super-resolved LSM technique for subcellular imaging.

biophysics↗

bioRxiv2021

EVALUATION OF STED SUPER-RESOLUTION IMAGE QUALITY BY IMAGE CORRELATION SPECTROSCOPY (QuICS)

Quantifying the imaging performances in an unbiased way is of outmost importance in super-resolution microscopy. Here, we describe an algorithm based on image correlation spectroscopy (ICS) that can be used to assess the quality of super-resolution images. The algorithm is based on the calculation of an autocorrelation function and provides three different parameters: the width of the autocorrelation function, related to the spatial resolution; the brightness, related to the image contrast; the relative noise variance, related to the signal-to-noise ratio of the image. We use this algorithm to evaluate the quality of stimulated emission depletion (STED) images of DNA replication foci in U937 cells acquired under different imaging conditions. Increasing the STED power improves the resolution but may reduce the image contrast. Increasing the number of line averages improves the signal-to-noise ratio but facilitates the onset of photobleaching and subsequent reduction of the image contrast. Finally, we evaluate the performances of two different separation of photons by lifetime tuning (SPLIT) approaches: the method of tunable STED power and the commercially available Leica Tau-STED. We find that SPLIT provides an efficient way to improve the resolution and contrast in STED microscopy.

biophysics↗

bioRxiv2021

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells

We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. We deployed this toolbox to characterize subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results suggest that in these cells the variant of concern B.1.1.7, (aka Alpha variant), became the predominant circulating variant in several countries by a clear transmission advantage. In fact, in these cells B.1.1.7 outcompetes its ancestor B.1.177 in terms of a much faster kinetics of entry. Given the cell-entry scenario dominated by the endosomal "late pathway", the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the main role of clathrin as mediator of late-entry endocytosis, reconciling it with the membrane localization of the ACE2 receptor previously attributed to caveolin-enriched rafts. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells.

biophysics↗

bioRxiv2020

The piRNA pathway sustains adult neurogenesis by repressing protein synthesis

Adult Neural progenitor cells (aNPCs) ensure lifelong neurogenesis in the mammalian hippocampus. Proper regulation of aNPC fate entails important implications for brain plasticity and healthy aging. Piwi proteins and the small noncoding RNAs interacting with them (piRNAs) are best known in gonads as repressors of transposons. Here, we show that Piwil2 (Mili) and piRNAs are abundant in aNPCs of the postnatal mouse hippocampus and demonstrate that this pathway is essential for proper neurogenesis. Particularly, depleting the piRNA pathway in aNPCs impaired neurogenesis, increased senescence and accordingly the generation of reactive glia. Moreover, this manipulation primarily elevated 5S ribosomal RNA, SINEB1 and mRNAs encoding ribosomal proteins and regulators of translation, resulting in higher polysome density and protein synthesis upon differentiation. Our results provide evidence of an essential role for the piRNA pathway in maintaining homeostasis to sustain neural stem cell fate, underpinning its possible involvement in brain plasticity and successful aging.

neuroscience↗

bioRxiv2019

Electrostatics and Solvation: Essential Determinants of Chromatin Compaction

Chromatin compaction is a process of fundamental importance in Biology, as it greatly influences cellular function and gene expression. The dynamics of compaction is determined by the interactions between DNA and histones, which are mainly mechanical and electrostatic. The high charge of DNA makes electrostatics extremely important for chromatin topology and dynamics. Besides their mechanical and steric role in the chromatin fibre, linker DNA length and linker histone presence and binding position also bear great electrostatic consequences. Electrostatics in chromatin is also indirectly linked to the DNA sequence: the presence of high-curvature AT-rich segments in DNA can cause conformational variations with electrostatic repercussions, attesting to the fact that the role of DNA is both structural and electrostatic. Electrostatics in this system has been analysed by extensively examining at the computational level the repercussions of varying ionic concentration, using all-atom, coarse-grained, and continuum models. There have been some tentative attempts to describe the force fields governing chromatin conformational changes and the energy landscapes of these transitions, but the intricacy of the system has hampered reaching a consensus. Chromatin compaction is a very complex issue, depending on many factors and spanning orders of magnitude in space and time in its dynamics. Therefore, comparison and complementation of theoretical models with experimental results is fundamental. Here, we present existing approaches to analyse electrostatics in chromatin and the different points of view from which this issue is treated. We pay particular attention to solvation, often overlooked in chromatin studies. We also present some numerical results on the solvation of nucleosome core particles. We discuss experimental techniques that have been combined with computational approaches and present some related experimental data such as the Z-potential of nucleosomes at varying ionic concentrations. Finally, we discuss how these observations support the importance of electrostatics and solvation in chromatin models.\n\nSIGNIFICANCEThis work explores the determinants of chromatin compaction, focusing on the importance of electrostatic interactions and solvation. Chromatin compaction is an intrinsically multiscale issue, since processes concerning chromatin occur on a wide range of spatial and temporal scales. Since DNA is a highly charged macromolecule, electrostatic interactions are extremely significant for chromatin compaction, an effect examined in this work from many angles, such as the importance of ionic concentration and different ionic types, DNA-protein interactions, and solvation. Solvation is often overlooked in chromatin studies, especially in coarse-grained models, where the nucleosome core, the building block of the chromatin fibre, is represented as a rigid body, even though it has been observed that solvation influences chromatin even at the base-pair level.

biophysics↗

bioRxiv2019

Nanoscale distribution of nuclear sites analyzed by superresolution STED-ICCS

Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, superresolution microscopy has gained considerable interest as it can be used to probe the spatial organization of functional sites in intact single cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a pre-segmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet.\n\nHere we combine dual color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that STED-ICCS provides not only a value of colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the Proliferating cell nuclear antigen (PCNA) protein have a high level of proximity and are correlated at a nanometer distance which is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of nanoscale distribution of functional sites in the nucleus.\n\nStatement of significanceSeveral methods are available to quantify the proximity of two labeled molecules from dual color images. Among them, image cross-correlation spectroscopy (ICCS) is attractive as it does not require a pre-segmentation of the image into objects and can be used to detect dynamic interactions. Here, we combine for the first time ICCS with superresolution stimulated emission depletion (STED) microscopy (STED-ICCS) to quantify the spatial distribution of functional sites in the nucleus. Our results show that STED-ICCS, in addition to quantifying the colocalized fraction, detects characteristic nanometer distances associated to correlated nuclear sites. This work shows that STED-ICCS can be a powerful tool to quantify the nanoscale distribution of functional sites in the nucleus.

biophysics↗

bioRxiv2019

Volumetric Lissajous Confocal Microscopy

Dynamic biological systems present challenges to existing three-dimensional (3D) optical microscopes because of their continuous temporal and spatial changes. Most techniques are based on rigid architectures, as in confocal microscopy, where a laser beam is sequentially scanned at a predefined spatial sampling rate and pixel dwell time. Here, we developed volumetric Lissajous confocal microscopy to achieve unsurpassed 3D scanning speed with a tunable sampling rate. The system combines an acoustic liquid lens for continuous axial focus translation with a resonant scanning mirror. Accordingly, the excitation beam follows a dynamic Lissajous trajectory enabling sub-millisecond acquisitions of image series containing 3D information at a sub-Nyquist sampling rate. By temporal accumulation and/or advanced interpolation algorithms, volumetric imaging rate is selectable using a post-processing step at the desired spatiotemporal resolution for events of interest. We demonstrate multicolor and calcium imaging over volumes of tens of cubic microns with acquisition speeds up to 5 kHz.

biophysics↗

bioRxiv2019

Chromatin nanoscale compaction in live cells visualized by acceptor-donor ratio corrected FRET between DNA dyes

Chromatin nanoscale architecture in live cells can be studied by Forster Resonance Energy Transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor-donor ratio rather than distance. Here we show that a FRET value independent of the acceptor-donor ratio can be obtained by Fluorescence Lifetime Imaging (FLIM) detection of FRET combined with a normalization of the FRET level to a pixel-wise estimation of the acceptor-donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that acceptor-donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser-induced micro-irradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.

biophysics↗

bioRxiv2019

Easy Two-Photon Image Scanning Microscopy with SPAD Array and Blind Image Reconstruction

Two-photon excitation (2PE) microscopy is the imaging modality of choice, when one desires to work with thick biological samples, possibly in-vivo. However, the resolution in two-photon microscopy is poor, below confocal microscopy, and the lack of an optical pinhole becomes apparent in complex samples as reduced quality of optical sectioning. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced ISM platform - based on a new single-photon avalanche diode array detector - coupled with a novel blind image reconstruction method, is shown to improve the optical resolution, as well as the overall image quality in various test samples. Most importantly, our 2PE-ISM implementation requires no calibration or other input from the user - it works like any old and familiar two-photon system, but simply produces higher resolution images (in real-time). Making the complexity disappear, in our view, is the biggest novelty here, and the key for making 2PE-ISM mainstream.

bioengineering↗

bioRxiv2019

The oncoprotein DEK affects the outcome of PARP1/2 inhibition during replication stress

DNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.

cancer biology↗

bioRxiv2019

Fourier ring correlation simplifies image restoration in fluorescence microscopy

Fourier ring correlation (FRC) has recently gained some popularity among (super-resolution) fluorescence microscopists as a straightforward and objective method to measure the effective resolution of a microscopy image. While the knowledge of the numeric resolution value is helpful in e.g. interpreting imaging results, much more practical use can be made of FRC analysis - in this article we propose novel blind image restoration methods enabled by it. We apply FRC to perform image de-noising by frequency domain filtering. We propose novel blind linear and non-linear image deconvolution methods that use FRC to estimate the effective point-spread-function, directly from the images, with no need for prior knowledge of the instrument or sample characteristics. The deconvolution is shown to work exquisitely with both two- and three-dimensional images. We also show how FRC can be used as a powerful metric to observe the progress of iterative deconvolution. While developing the image restoration methods, we also addressed two important limitations in FRC that are of more general interest: how to make FRC work with single images and with three-dimensional images with anisotropic resolution.

bioinformatics↗

bioRxiv2018

Measuring mobility in chromatin by intensity sorted FCS

The architectural organization of chromatin can play an important role in genome regulation by affecting the mobility of molecules within its surroundings via binding interactions and molecular crowding. The diffusion of molecules at specific locations in the nucleus can be studied by Fluorescence Correlation Spectroscopy (FCS), a well-established technique based on the analysis of fluorescence intensity fluctuations detected in a confocal observation volume. However, detecting subtle variations of mobility between different chromatin regions remains challenging with currently-available FCS methods.\n\nHere we introduce a method that samples multiple positions by slowly scanning the FCS observation volume across the nucleus. Analyzing the data in short time segments, we preserve the high temporal resolution of single-point FCS while probing different nuclear regions in the same cell. Using the intensity level of the probe (or a DNA marker) as a reference, we efficiently sort the FCS segments into different populations and obtain average correlation functions that are associated to different chromatin regions. This sorting and averaging strategy renders the method statistically robust while preserving the observation of intranuclear variations of mobility.\n\nUsing this approach, we quantified diffusion of monomeric GFP in high versus low chromatin density regions. We found that GFP mobility was reduced in heterochromatin, especially within perinucleolar heterochromatin. Moreover, we found that modulation of chromatin compaction by ATP depletion, or treatment with solution of different osmolarity, differentially-affected the ratio of diffusion in both regions. Then, we used the approach to probe the mobility of estrogen receptor- (ER) in the vicinity of an integrated multicopy prolactin gene array. Finally, we discussed the coupling of this method with stimulated emission depletion (STED)-FCS, for performing FCS at sub-diffraction spatial scales.

biophysics↗