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Mazza, D.

Publications and source records attributed to Mazza, D..

14 recordsLinked to original sources

Compressive stress couples calcium signalling to translational control and proteostasis

Compressive stress is a pervasive feature of solid tumour growth, and mechanical loading is known to activate calcium signalling through the mechanosensitive channel Piezo1. How this signal is converted into changes in cellular biosynthetic capacity, however, remains unclear. Here we show that compressive stress engages a Piezo1-dependent calcium checkpoint that reduces translational output with increasing mechanical load, independently of mTORC1 and the integrated stress response. Compression triggers Piezo1-dependent release of endoplasmic reticulum Ca2, activating eEF2 kinase to slow translational elongation. This same Ca2 signal drives mitochondrial remodelling and reactive oxygen species production, which reinforce translational repression through redox-dependent oxidation of the translation initiation factor eIF2{beta}. In parallel, Ca2-dependent AMPK activation triggers autophagic ribosome degradation, alleviating compression-induced macromolecular crowding and restoring cytoplasmic rheology despite sustained mechanical load. These mechanisms act at distinct levels of translational control: initiation, elongation and ribosome abundance; yet converge to limit biosynthetic output under compression. Consistent with a role for this pathway in tumour adaptation to mechanical constraint, eEF2K expression correlates with poor overall survival in a public PDAC patient dataset. Together, these findings define a mechano-metabolic checkpoint through which cells actively tune biosynthetic capacity to their physical environment.

cell biology↗

A Cooperative Mechanism of Eukaryotic Transcription Factor Target Search

Rapid gene activation requires transcription factors (TFs) to locate their target motifs within vast genomes. In bacteria, TF target search is accelerated by combining 3D diffusion with 1D sliding, called facilitated diffusion, yet whether eukaryotic TFs rely on similar strategies has remained unresolved due to the lack of direct measurements in living cells. Here, we directly visualize eukaryotic TF target search in living cells by labeling a single TF per nucleus and visualizing its binding to its endogenous locus. Using the budding yeast TF Gal4, we find that efficient target localization occurs near the diffusion limit and does not require facilitated diffusion. Instead, rapid association requires cooperative self-interactions mediated by an intrinsically-disordered central region (IDR), independent of the activation domain. Replacing the Gal4 IDR with human self-interacting IDRs (EWS or FUS) restores efficient search, demonstrating that self-interactions are a general and portable feature for search. A second structured dimerization domain further cooperatives with the IDR to stabilize binding at neighboring motifs, revealing two mechanistically separable forms of cooperativity that together govern TF function. These findings highlight that facilitated diffusion is not strictly required and establish cooperative IDR-driven self-interactions as a key mechanism to enable rapid target recognition in eukaryotic cells.

molecular biology↗

NF-κB transcriptionally enhances p53 accumulation dynamics hampering DNA repair

Cells integrate multiple, often concurrent signals through intertwined genetic circuits whose dynamics shape transcriptional programs and cell fate decisions. Among these, the tumor suppressor p53 and the inflammatory transcription factor NF-{kappa}B are central regulators of stress responses in normal and cancer cells, yet their dynamic crosstalk under co-activation remains poorly characterized. Here, we combine genetic approaches, live cell imaging, transcriptomic analysis and mathematical modeling to dissect their dynamic interplay. We find that co-activation of NF-{kappa}B by the inflammatory cytokines TNF- and IL-1{beta} significantly enhance p53 nuclear accumulation upon genotoxic stress or Nutlin3a, and this effect is absent in NF-{kappa}B-deficient cells. Mechanistically, we show that cytokines induce an NF-{kappa}B-mediated increase of TP53 transcription, and mathematical modeling indicates that it is sufficient to account for the observed increased p53 accumulation. Functionally, NF-{kappa}B co-activation rewires p53-dependent transcriptional programs and impairs p53-mediated DNA repair following genotoxic stress, due to a shift of p53 dynamics from oscillatory to more sustained accumulation; p53 oscillatory dynamics and DNA repair remain largely unaltered in absence of NF-{kappa}B. Our results uncover an amplification of p53 response in presence of inflammatory cues that is transcriptionally mediated by NF-{kappa}B and that results, counterintuitively, in functional antagonism. SIGNIFICANCE STATEMENTp53 dynamics have been shown to correlate with the cellular responses of cancer cells to genotoxic insults such as those delivered by chemo- and radiotherapies. However, these dynamics have been mostly studied in settings that do not account for the pro-inflammatory cues that cancer cells might receive from the microenvironment. By addressing this gap, our study uncovers a previously unappreciated mechanism by which inflammatory signals induce an increased p53 accumulation, leading to reduced DNA repair capacity. Our results provide mechanistic understanding on the origin of p53-NF-{kappa}B antagonism and show how dynamically interconnected signalling pathways can produce counterintuitive functional outcomes. Translationally, the contribution of inflammation to the sensitivity of normal and cancer cells to DNA damage might be exploited in the management of chemo- and radiotherapies.

systems biology↗

QuantiTrack: A unified software to study protein dynamics in living cells

Linking the spatiotemporal dynamics of proteins in live cells to biological function is a fundamental challenge in biology. Single molecule tracking (SMT) has emerged as a powerful technique to investigate protein dynamics at the single molecule level. However, SMT analysis often requires expertise in biophysical modeling and programming, and integrating results from different analyses can be challenging. To address these barriers, we developed QuantiTrack: a MATLAB-based SMT analysis software with a simple graphical user interface. This provides a much-needed end-to-end solution where a user can load a movie, detect and track single molecules, and perform complementary downstream analyses within a standardized workflow. QuantiTrack includes quantitative metrics for selecting detection and tracking parameters and troubleshooting experimental design, and includes a detailed step-by-step User Guide. We used simulations to demonstrate how signal intensity, labeling density, and motion blur affect detection and tracking fidelity. Using multi-state simulations, we further benchmarked complementary methods to identify distinct mobility states from heterogeneous trajectory populations. Finally, we applied QuantiTrack to real experimental data where we address how the glucocorticoid receptor (GR), a hormone-regulated transcription factor, responds to treatment and washout of its cognate hormone. Hormone washout results in rapid (in minutes) downregulation of GR target genes to basal levels. By integrating complementary analyses within QuantiTrack, we showed that hormone washout substantially reduced the bound fraction of GR, its occupancy in the mobility state associated with GR activation, and dwell times. Together, these analyses showcase QuantiTrack as an integrated platform for extracting biologically meaningful measurements from single molecule trajectories.

biophysics↗

Mycobacterium tuberculosis diversity and macrophage heterogeneity dictate phagosomal acidification

Therapies counteracting pathogen-induced immune modulation of the host responses represent a promising improvement for tuberculosis treatments. Despite the documented role of host immune heterogeneity and bacterial genotypic background in determining infection outcomes, their interplay remains largely uncharacterized. We investigate the Mycobacterium tuberculosis (MTB)-human macrophage interaction considering both macrophage phenotypic variability and MTB genetic diversity. Using single-cell techniques, we show how diverse MTB lineages fine-tune phagosome acidification differently based on macrophage phenotype (M1, M2), revealing very heterogeneous host-pathogen interactions. Our findings underscore the multiplicity of outcomes due to the combinatorial interplay between MTB lineages and macrophage phenotypes, which may have implications in proper design for host-directed therapies. IMPORTANCEThe intricate interplay among host, pathogen, and environmental factors significantly contributes to susceptibility and clinical presentation of tuberculosis. These multifaceted mechanisms are often overlooked in tuberculosis studies, hindering our comprehensive understanding of infection progression and impeding the development of effective host-directed therapies which offer benefits such as reduced drug resistance emergence and heightened host compatibility. Contrary to the prevailing hypothesis, the results observed in this study demonstrate the nuanced response of different M. tuberculosis lineages within distinct macrophage phenotypes in terms of phagosome acidification. The findings of our investigation delineate a crucial yet under-characterized aspect of tuberculosis pathogenesis, emphasizing how the interactions between different M. tuberculosis lineages and macrophage phenotypes could significantly influence the efficacy of host-directed therapies, particularly those targeting phagolysosomal maturation. More broadly, recognizing the impact of both M. tuberculosis and macrophage heterogeneity is paramount in the development of effective strategies to combat tuberculosis.

microbiology↗

MYC modulates TOP2A diffusion to promote substrate detection and activity

Topoisomerases alleviate DNA supercoiling by cleaving and resealing DNA strands. Previously, we showed that the oncoprotein MYC recruits and stimulates topoisomerases to remove DNA entanglements generated by oncogenic transcription. Understanding this mechanism may suggest methods to inhibit MYC-driven topoisomerase activation, targeting tumor-specific transcription. Here, we demonstrate that the essential topoisomerase TOP2A in human cells exists in a dynamic equilibrium between sequestration in the nucleolus, substrate searching in transcription hubs, and active engagement on chromatin. This equilibrium is highly responsive to changes in DNA topology, allowing cells to regulate TOP2A levels. Using single molecule tracking, we show that MYC accelerates TOP2A diffusion in cells. We explain this phenotype by demonstrating that MYC limits TOP2A self-interaction in vitro, while decreasing the size of TOP2A complexes in cells. By increasing TOP2A diffusion, MYC promotes substrate binding and increases TOP2A engagement on chromatin genome-wide, revealing the mechanism underlying MYC stimulation of TOP2A activity.

cell biology↗

Vaginal microbiota changes during a natural menstrual cycle in the Women4Health cohort highlights specific features of the Italian population.

Large sex hormonal fluctuations are thought to influence vaginal microbiota, but little is known about the impact of small, physiological variations. Here we tracked changes in vaginal microbiota during four key menstrual cycle phases in 61 healthy, naturally menstruating Italian women from the Women4Health cohort. The microbiota, characterized using a high depth 16S rRNA amplicon sequencing approach covering four hypervariable regions, was primarily composed of Lactobacillus species, with Lactobacillus iners being the most abundant (average relative abundance 40%) and the most prevalent (prevalence 98%). Individual microbiota were generally stable, but beta diversity was higher during the follicular phase (p=0.007). Only 11 women exhibited compositional shifts, mostly occurring between follicular and ovulatory phases. Finally, using linear mixed models we assessed the association between taxa relative abundance and five sex-hormones along the menstrual cycle. Among these, 17-beta estradiol showed the largest number of significant associations, linking its increase to a decrease in relative abundance of taxa that are more common after menopause. Our study highlights specific features of the Italian population and points to the resilience of the vaginal microbiota to physiological hormonal changes. Noteworthy, the observed high abundance of L. iners contrasts with previous studies in European populations, challenging its proposed pathogenic role and suggesting distinct microbiota profiles within Europe. IMPORTANCEThe vaginal microbiota plays an important role in womens health, yet we know little about how it responds to normal hormonal fluctuations. In this study, we followed 61 healthy Italian women over a natural menstrual cycle to explore microbiota changes across different hormonal phases. We found that Lactobacillus iners was the most common species, unlike previous findings in Northern Europe, suggesting population-specific patterns. The common hypothesis that L. iners is invariably linked with poor health is called into question by our findings. They emphasize the importance of considering population context and hormonal status when assessing vaginal health. The vaginal microbiota was generally stable, with only a few changes observed between the follicular and ovulatory phases. When evaluating association between five sex hormones and taxa abundances, we found that 17-beta estradiol levels had the largest number of significant associations. These highlight an association between increase levels of 17-beta estradiol and increased relative abundance of rare bacterial taxa rather than dominant species like Lactobacillus. Our findings help define what constitutes a "healthy microbiota" in generally healthy Italian women of reproductive age and may inform future strategies for diagnosing or preventing womens health conditions.

microbiology↗

Development of a high-throughput 3D culture microfluidic platform for multi-parameter phenotypic and omics profiling of patient-derived organoids

Patient-derived organoids (PDOs) are poised to become central tools both in clinical practice, to preemptively identify patient optimal treatments, and in drug discovery, overcoming the limitations of cancer cell lines. However, the use of PDOs in these settings has been hampered by several bottlenecks, including sample requirements, assay time, and handling in the context of high-throughput assays. We developed a Microfluidic Platform for Organoids culture (MPO) that miniaturises and simplifies PDOs cultures in a 384-plate format. Both retrospective and prospective clinical studies demonstrate MPO predictive value and the straightforward implementation in the clinical setting. MPO allows subcellular phenotypic screenings, as imaging-based applications like Cell Painting, target engagement analyses, alongside the comprehensive definition of PDOs genomic, transcriptomic, proteomic, lipidomic, and metabolomic landscapes. Harnessing the pleiotropic capabilities of MPO, we uncovered the role of EZH2 inhibitors to prevent the long-term emergence of resistance to RAS inhibitors in metastatic colon cancer. In all, we demonstrate the potential of MPO to impact clinical practice, alongside exploration of the mechanisms underlying compound response and resistance.

cancer biology↗

Chromatin organization drives the exploration strategy of nuclear factors

Nuclear Factors (NFs) rapidly scan the genome for their targets, but the role of nuclear organization in such search is uncharted. Here we analyzed how multiple NFs explore chromatin, by combining live-cell single-molecule tracking with multifocal structured illumination of DNA density. We find that NFs displaying higher bound fractions sample DNA dense regions more exhaustively. Focusing on the tumor-suppressor p53, we demonstrated that this NF search for its targets by alternating between rapid diffusion in the interchromatin compartment and compact sampling of chromatin dense regions. Efficient p53 targeting requires balanced IDR/chromatin interactions: adding an exogenous IDR potentiates p53-mediated target gene activation, but excessive IDR/IDR interactions lead to p53 condensates, derailing its search and downregulating transcription. Our findings highlight the role of NF IDRs on their search and showcase a powerful method to generate traffic maps of the eukaryotic nucleus and dissect how nuclear organization guides NFs action.

biophysics↗

First responders shape a prompt and sharp NF-Bκ-mediated transcriptional response to TNF-α

SummaryNF-κB acts as the master regulator of the transcriptional response to inflammatory signals by translocating into the nucleus upon stimuli, but we lack a single-cell characterization of the resulting transcription dynamics. Here we show that transcription of NF-κB target genes is strongly heterogeneous in individual cells but dynamically coordinated at the population level, since the average nascent transcription is prompt (i.e. occurs almost immediately) and sharp (i.e. increases and decreases rapidly) compared to NF-κB nuclear localization. Using an NF-κB-controlled MS2 reporter we confirm that the population-level transcriptional activity emerges from a strongly heterogeneous response in single cells as compared to NF-κB translocation dynamics, including the presence of a fraction of “first responders”. Mathematical models show that a combination of NF-κB mediated gene activation and a gene activity module including a gene refractory state is enough to produce sharp and prompt transcriptional responses. Our data and models show how the expression of the target genes of a paradigmatic inducible transcription activator upon stimuli can be time-resolved at population level and yet heterogeneous across single cells.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

Integrated requirement of non-specific and sequence-specific DNA binding in MYC-driven transcription

Eukaryotic transcription factors recognize specific DNA sequence motifs, but are also endowed with generic, non-specific DNA-binding activity: how these binding modes are integrated to determine select transcriptional outputs remains unresolved. We designed mutants of the MYC transcription factor bearing substitutions in residues that contact either the DNA backbone or specific bases within the consensus binding motif (E-box), and profiled their DNA-binding and gene-regulatory activities in murine cells. Our data reveal that non-specific DNA binding is required for MYC to engage onto active regulatory elements in the genome, preceding sequence recognition; beyond merely stabilizing MYC onto select target loci, sequence-specific binding contributes to its precise positioning and - most unexpectedly - to transcriptional activation per se. In particular, at any given binding intensity, promoters targeted via the cognate DNA motif were more frequently activated by MYC. Hence, seemingly promiscuous chromatin interaction profiles actually encompass diverse DNA-binding modalities, driving defined, sequence-dependent transcriptional responses.

molecular biology↗

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↗

Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-{kappa}B (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an \"enhanceosome,\" nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.\n\nAuthor SummaryTo control transcription of a certain gene or a group of genes, both eukaryotes and prokaryotes express specialized proteins, transcription factors (TFs). During gene activation, TFs bind gene promotor sequences to recruit the transcriptional machinery including DNA polymerase II. TFs are often multi-subunit proteins containing a DNA-binding domain (DBD) as well as a protein-protein interaction interface. It was suggested that the duration of a TF-DNA binding event 1) depends on these two subunits and 2) dictates the outcome, i.e. the amount of mRNA produced from an activated gene. We set out to address these hypotheses using the transcriptional activator NF-{kappa}B (p65) as well as a number of mutants affecting different functional subunits. Using a combination of live-cell microscopy and RNA sequencing, we show that p65 DNA-binding time indeed correlates with the transcriptional output, but that this relationship depends on, and hence can be uncoupled by altering, the protein-protein interaction capacity. Our results suggest that, while p65 DNA binding times are dominated by the DBD, a transcriptional output can only be achieved with a functional protein-protein interaction subunit.

genetics↗

Scc2/Nipbl Hops Between Chromosomal Cohesin Rings After Loading

The cohesin complex mediates DNA-DNA interactions both between (sister chromatid cohesion) and within chromosomes (DNA looping) via a process thought to involve entrapment of DNAs within its tripartite ring. It has been suggested that intra- chromosome loops are generated through processive extrusion of DNAs through the lumen of cohesins ring. Scc2 (Nipbl) is essential for loading cohesin onto chromosomes but not for maintaining sister chromatid cohesion following DNA replication. It has therefore been assumed that Scc2 is involved exclusively in the cohesin loading process. However, it is possible that the stimulation of cohesins ABC-like ATPase by Scc2 also has a post-loading function, for example driving loop extrusion. Using fluorescence recovery after photobleaching (FRAP) and single-molecule tracking, we show that Scc2 binds dynamically to chromatin, principally through an association with cohesin. Scc2s movement within chromatin is consistent with a \"stop-and-go\" or \"hopping\" motion. We suggest that a low diffusion coefficient, a low stoichiometry relative to cohesin, and a high affinity for chromosomal cohesin enables Scc2 to move rapidly from one chromosomal cohesin complex to another, performing a function distinct from loading.

cell biology↗