Biology subjects

Tacchetti, C.

Publications and source records attributed to Tacchetti, C..

4 recordsLinked to original sources

bioRxiv2024

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↗

bioRxiv2024

A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism

One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.

cell biology↗

bioRxiv2022

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↗

bioRxiv2020

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↗