Biology subjects

Paterno, G.

Publications and source records attributed to Paterno, G..

3 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

SPROUTS_DB: an implemented database of contaminants for extracellular vesicle proteomics studies

BackgroundCurrent proteomics techniques allow rapid identification and quantification of proteins within any given biological source. In particular, nanoUHPLC/High-Resolution nanoESI-MS/MS enables the characterization of proteins in complex biological samples due to its high sensitivity, accuracy, and scalability. However, LC-MS/MS proteomics might still be susceptible to laboratory and sample-associated contaminants, which can significantly compromise the quality and reliability of data. Therefore, an accurate identification and annotation of such contaminants is crucial for the development of robust proteomics databases and spectral-libraries related search engines. This approach is of special interest in the field of secretome and extracellular vesicles (EVs), membrane-enclosed nanostructures that contain a variety of proteins crucial for cell-to-cell communication and translational applications. ResultsWhen working in ex vivo/in vitro settings, proteins from fetal bovine serum (FBS), commonly employed in standard cell culture media, may interfere with the proteome analysis. To address this issue, we conceived and designed SPROUTS_DB, Serum Protein Repository Of Unwanted Target(ed) Sequences DataBase, a dedicated resource to catalog serum-derived contaminants. Starting from media supplemented with EV-depleted FBS, we simulated cell growth conditions - in the absence of cells - followed by ultracentrifugation. LC-MS/MS analysis of these samples resulted in the identification of a novel set of 1,288 contaminant proteins, which has been deposited in the ProteomeXchange repository (identifier PXD044137). SPROUTS_DB contains primarily soluble proteins, mainly related to the Gene Ontology categories Extracellular Region and Extracellular Space, in line with the nature of the starting sample. In contrast, only a small fraction of the contaminants is classified as membrane-associated proteins, supporting the limited vesicle contamination in the complete medium, due to the use of EV-depleted FBS. Of note, we demonstrated that SPROUTS_DB outperforms existing contaminants databases, ensuring that only peptide spectra relevant to the examined sample are retained and identified as true positive data. ConclusionsConsidering that even proteins from phylogenetically distant organisms share extensive stretches of sequences, SPROUTS_DB is designed to discern contaminants from real sample proteins of interest, minimizing false positive identifications. To the best of our knowledge, SPROUTS_DB is the most updated database of contaminants useful for proteomics investigations of cellular secretomes and EV-containing samples.

cell biology↗

bioRxiv2023

Complement C1q-dependent engulfment of alpha-synuclein induces ENS-resident macrophage exhaustion and accelerates Parkinsons-like gut pathology

Deposition of misfolded -synuclein (syn) in the enteric nervous system (ENS) is found in multiple neurodegenerative diseases. It is hypothesized that ENS synucleinopathy contributes to both the pathogenesis and non-motor morbidity in Parkinsons Disease (PD), but the cellular and molecular mechanisms that shape enteric histopathology and dysfunction are poorly understood. Here, we demonstrate that ENS-resident macrophages, which play a critical role in maintaining ENS homeostasis, initially respond to enteric neuronal syn pathology by upregulating machinery for complement-mediated engulfment. Pharmacologic depletion of ENS-macrophages or genetic deletion of C1q enhanced enteric neuropathology. Conversely, C1q deletion ameliorated gut dysfunction, indicating that complement partially mediates syn-induced gut dysfunction. Internalization of syn led to increased endo-lysosomal stress that resulted in macrophage exhaustion and temporally correlated with the progression of ENS pathology. These novel findings highlight the importance of enteric neuron-macrophage interactions in removing toxic protein aggregates that putatively shape the earliest stages of PD in the periphery.

neuroscience↗