Biology subjects

Lanzano, L.

Publications and source records attributed to Lanzano, L..

7 recordsLinked to original sources

bioRxiv2025

Tracking DNA damage localization and chromatin remodeling in live cells using time-resolved quantitative analysis of DNA counterstains

DNA damage profoundly impacts genome stability and cellular homeostasis, and its repair is tightly coordinated with local chromatin remodeling. However, monitoring these rapid chromatin changes in living cells remains challenging. We recently developed QUANDO, an imaging-based method that exploits a simple DNA counterstain to investigate the subnuclear localization of DNA damage in fixed cells. Here, we adapt this approach to track chromatin remodeling at laser-induced DNA damage sites in live cells using Hoechst-based staining. Specifically, PARP1-expressing HeLa cells are exposed to UV laser micro-irradiation in a defined nuclear region, and PARP1 and chromatin dynamics are monitored in real time. We observe that PARP1 rapidly accumulates in the irradiated region but with a heterogeneous pattern: PARP1 initially localizes to high-density chromatin regions (where the concentration of the sensitizer is higher) and gradually redistributes over the whole irradiated region. At the same time, we observe rapid chromatin relaxation, as indicated by decreasing Hoechst intensity and coefficient of variation (CV). In this framework, the PARP inhibitor Talazoparib has the following effects: it slows down PARP1 accumulation, it freezes the PARP1 heterogeneous pattern and blocks chromatin relaxation. Finally, we show that Hoechst-only imaging is sufficient to observe chromatin remodeling: measured chromatin relaxation kinetics are similar in transfected and non-transfected cells, confirming that staining with Hoechst is sufficient for studying chromatin dynamics bypassing the complexities of transfection. These findings underscore the dynamic interplay between DNA damage and chromatin remodeling, demonstrating how conventional nuclear counterstaining can reveal rapid chromatin changes at damage sites, offering new perspectives for investigating genome stability in live cells.

biophysics↗

bioRxiv2025

Nuclear Pore Transport: Toward an Integrated Perspective

Nuclear pore complexes (NPCs) mediate the selective exchange of proteins and RNAs between nucleus and cytoplasm. Despite decades of study, the molecular mechanism of transport remains debated, particularly the role of FG-nucleoporin dynamics in facilitating translocation. Here, we compare high-resolution single-molecule trajectories obtained with MINFLUX microscopy with earlier correlation-based measurements, revealing that actual pore-crossing events are typically completed within 2-4 localization steps, corresponding to 2-6 ms depending on localization rates. These values closely match transit times obtained from fluorescence fluctuation analysis and align with models in which cargo binding induces transient FG-nucleoporin collapse. Together with recent independent observations, this synthesis supports a converging view of NPC transport as a fast, directional, FG-assisted process. We propose that integrating fluctuation-based and trajectory-based approaches provides a robust framework for reconciling mechanistic hypotheses and refining our understanding of selective gating.

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

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↗

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↗

bioRxiv2018

Enigma at the nanoscale: can the NPC act as an intrinsic reporter for isotropic expansion microscopy?

Expansion microscopy is a super-resolution method that allows expanding uniformly biological samples, by increasing the relative distances among fluorescent molecules labeling specific components. The main \"enigma\" regarding this approach is given by the isotropic behavior at the nanoscale. The present study aims to determine the robustness of such a technique, quantifying the expansion parameters i.e. scale factor, isotropy, uniformity. Our focus is on the nuclear pore complex (NPC), as well-known nanoscale component endowed of a preserved and symmetrical structure localized on the nuclear envelope. Here, we show that Nup153 is a good reporter to quantitatively address the isotropy of the expansion process. The quantitative analysis carried out on NPCs, at different spatial scales, allows concluding that expansion microscopy can be used at the nanoscale with a uniform accuracy in the range of 20 nm. In addition, it is an excellent method for structural studies of macromolecular complexes.

biophysics↗

bioRxiv2018

Photon-separation to enhance the spatial resolution in pulsed STED microscopy

Stimulated emission depletion microscopy (STED) is one of the pivotal super-resolution techniques. It overcomes the spatial resolution limit imposed by the diffraction by using an additional laser beam, the STED beam, whose intensity is directly related to the achievable resolution. Despite achieving nanometer resolution, much effort in recent years has been devoted to reduce the STED beam intensity because it may lead to photo-damaging effects. Exploring the temporal dynamics of the detected fluorescence photons and accessing the encoded spatial information has proven to be a powerful strategy, and has contributed to the separation by lifetime tuning (SPLIT) technique. The SPLIT technique uses the phasor analysis to efficiently distinguish photons emitted from the center and the periphery of the excitation spot. It thus improves the resolution without increasing the STED beam intensity. This method was proposed for architectures based on STED beam running in continuous wave (CW-STED microscopy). Here, we extend it to architectures based on pulsed STED beam (pSTED microscopy). We show, through simulated and experimental data, that the SPLIT-pSTED method reduces the detection volume of the pSTED microscope without significantly reducing the signal-to-noise ratio of the final image, thus effectively improving the resolution without increasing the STED beam intensity.

biophysics↗