Biology subjects

Cosma, M. P.

Publications and source records attributed to Cosma, M. P..

9 recordsLinked to original sources

bioRxiv2026

Retinal resuscitation in post-mortem eyes

Vision loss compromises the quality of life of millions of people worldwide. Currently, vision-restoring therapies are lacking. Post-mortem preservation of human eyes may enable whole eye transplantation (WET) or therapeutic development. We developed an approach to cannulate the ophthalmic artery and perfuse post-mortem pig and human eyes using a custom-built device, Eye-in-Care-Box (ECaBox). Retinal vasculature imaging and reconstruction were used to assess perfusion efficacy using a supervised deep learning model. Without intervention, the retina degrades post-mortem, whereas perfusion preserves retinal structure and cell viability for up to 24-hours. Eyes were perfused within 30 minutes post-extraction, and regained light-responses persisted for up to 10 hours after death, challenging the notion that response to light ends at death. Our findings show that intact eyes can be resuscitated and preserved outside the body, supporting the ECaBox platform for WET preservation and pre-clinical therapeutic testing. Ultimately, ECaBox may bring vision-restoring treatments to patients.

neuroscience↗

bioRxiv2023

coRIC map: a framework for the exploration of multifaceted subcellular RNA-binding proteins

The ability of RNA-binding proteins (RBPs) to form complexes with other biomolecules underpins a broad range of functions and remarkable structural properties. Understanding the precise spatiotemporal dynamics of RBPs and their interacting partners in the steady state and upon perturbation is key to deciphering these aspects. Here, we present the coRIC (compartmentalized RNA Interactome Capture) map, an experimental resource and analytical pipeline to study the subcellular dynamics of RBPs through multimodal dataset integration and machine learning. Using this approach, we have generated an atlas comprising 1,768 RBPs distributed in a broad panel of subcellular compartments and delineated their intermolecular and intercompartmental relationships. We have also defined the hierarchy of RBP-containing complexes at multiple scales across the cell, uncovering previously unknown functions of multiple RBPs. Furthermore, we have investigated the changes in RBP complex composition and subcellular distribution in response to C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia dipeptide repeats and DNA damage stress. The coRIC map provides a valuable new approach for defining the roles of RBPs in homeostasis and disease.

cell biology↗

bioRxiv2023

Active Transcription and Epigenetic Reactions Synergistically RegulateMeso-Scale Genomic Organization

In interphase nuclei, chromatin is organized into interspersed dense domains with characteristic sizes, both in the nuclear interior and periphery. However, the quantitative impact of transcription and histone modifications on the size and distribution of these domains remains unclear. Here, we introduce a mesoscale theoretical model that investigates the relationship between heterochromatic domain sizes and loop extrusion rates from these domains. The model considers chromatin-chromatin and chromatin-lamina interactions, methylation and acetylation kinetics, and diffusion of epigenetic marks and nucleoplasm. Our model generates testable predictions that help reveal the biophysics underlying chromatin organization in the presence of transcription-driven loop extrusion. This process is kinetically captured through the conversion of heterochromatin to euchromatin in response to RNAPII activity. We discovered that a balance between diffusive and reactive fluxes governs the steady-state sizes of heterochromatin domains. Using theory and simulations, we predicted that a loss of transcription results in increased chromatin compaction and larger heterochromatin domain sizes. To validate our predictions, we employed complementary super-resolution and nano-imaging techniques on five different cell lines with impaired transcription. We quantitatively assessed how domain sizes scale with loop extrusion rates at the hetero-euchromatin interfaces. Our analysis of previously obtained super-resolution images of nuclei revealed that excessive loop extrusion leads to smaller heterochromatin domains. The model successfully recapitulated these observations, explaining how transcription loss can counteract the effects of cohesin overloading. As the general biophysical mechanisms regulating heterochromatin domain sizes are independent of cell type, our findings have significant implications for understanding the role of transcription in global genome organization.

biophysics↗

bioRxiv2023

SMARCAD1 and TOPBP1 contribute to heterochromatin maintenance at the transition from the 2C-like to the pluripotent state

Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells. This conversion is marked by a global reorganization and dispersion of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. By profiling the chromatin-bound proteome (chromatome) through genome capture of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation. We identified TOPBP1 and investigated its binding partner SMARCAD1. SMARCAD1 and TOPBP1 associate with H3K9me3-heterochromatin in ESCs. Interestingly, the nuclear localization of SMARCAD1 is lost in 2C-like cells. SMARCAD1 or TOPBP1 depletion in mouse embryos leads to developmental arrest, reduction of H3K9me3, and remodeling of heterochromatin foci. Collectively, our findings contribute to comprehending the maintenance of chromocenters during early development.

developmental biology↗

bioRxiv2022

The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency

Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage segregation in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

developmental biology↗

bioRxiv2019

Senolytic treatment targets aberrant p21-expression to restore liver regeneration in adult mice

Young mammals possess a limited regenerative capacity in tissues such as the liver, heart and limbs, but which is quickly lost upon maturation or transition to adulthood. Chronic cellular senescence is a known mediator of decreased tissue function in aging and disease. Here we investigated whether senescence plays a role in the progressive loss of liver regenerative capacity that develops in young adult mice. We find that following partial hepatectomy, the senescence markers p21, p16Ink4a and p19Arf become dynamically expressed at an age when regenerative capacity decreases. In addition, we demonstrate that treatment with a senescence-inhibiting drug improves regenerative capacity, through targeting of aberrant p21 expression. Surprisingly, we also find that the senescence marker p16Ink4a is expressed in a different cell-population to p21, and is unaffected by senescence targeting. This work suggests that senescence may initially develop as a heterogeneous cellular response, and that treatment with senolytic drugs may aid in promoting organ regeneration.

cell biology↗

bioRxiv2019

Canonical Wnt pathway controls mESCs self-renewal through inhibition of spontaneous differentiation via β-catenin/TCF/LEF functions

The Wnt/{beta}-catenin signalling pathway is a key regulator of embryonic stem cell self-renewal and differentiation. Constitutive activation of this pathway has been shown to significantly increase mouse embryonic stem cell (mESC) self-renewal and pluripotency marker expression. In this study, we generated a novel {beta}-catenin knock-out model in mESCs by using CRISPR/Cas9 technology to delete putatively functional N-terminally truncated isoforms observed in previous knock-out models. While we showed that aberrant N-terminally truncated isoforms are not functional in mESCS, we observed that canonical Wnt signalling is not active in mESCs, as {beta}-catenin ablation does not alter mESC transcriptional profile in LIF-enriched culture conditions; on the other hand, Wnt signalling activation represses mESC spontaneous differentiation. We also showed that transcriptionally silent {beta}-catenin ({Delta}C) isoforms can rescue {beta}-catenin knock-out self-renewal defects in mESCs, cooperating with TCF1 and LEF1 in the inhibition of mESC spontaneous differentiation in a Gsk3 dependent manner.

cell biology↗

bioRxiv2018

Nanoscale decompaction of nucleosomal DNA revealed through multi-color super-resolution microscopy

Chromatin organization plays an important role in regulating gene expression. Previously, we showed that chromatin is organized in the form of nucleosome groups or clutches. The size and nucleosome packing density of clutches decreased in hyperacetylated cells having more open chromatin. While hyperacetylation is thought to disrupt histone-DNA and inter-nucleosome interactions, its impact on higher order DNA compaction by groups of nucleosomes in vivo is not clear. To elucidate this question, we carried out two-color super-resolution imaging of histones and DNA in cells treated with the Histone Deacetylase (HDAC) inhibitor Trichostatin A (TSA). We showed that a lower percentage of DNA was associated to clutches in hyperacetylated cells, suggesting a decrease in nucleosome occupancy. We further identified the presence of \"clutch\" DNA within a nanoscale distance around the clutches. Upon histone hyperacetylation, the radius of the clutch DNA decreased leading to DNA release from the clutches, consistent with disruption of DNA-histone interactions. Finally, the most dramatic decompaction was observed for groups of clutches in close spatial proximity, suggesting that neighboring clutches influence each others DNA compaction.\n\nSummarySuper-resolution imaging of histones and DNA reveals that DNA is compacted by groups of nucleosomes - clutches - at the nanoscale level and clutch compaction of DNA is affected by histone tail acetylation especially in highly folded regions containing several nearby clutches.

cell biology↗

bioRxiv2018

A tunable dual-input system for on-demand dynamic geneexpression regulation.

Cellular systems have evolved numerous mechanisms to finely control signalling pathway activation and properly respond to changing environmental stimuli. This is underpinned by dynamic spatiotemporal patterns of gene expression. Indeed, in addition to gene transcription and translation regulation, modulation of protein levels, dynamics and localization are also essential checkpoints that govern cell functions. The introduction of tetracycline-inducible promoters has allowed gene expression control using orthogonal small molecules, facilitating rapid and reversible manipulation to study gene function in biological systems. However, differing protein stabilities means this solely transcriptional regulation is insufficient to allow precise ON-OFF dynamics, thus hindering generation of temporal profiles of protein levels seen in vivo. We developed an improved Tet-On based system augmented with conditional destabilising elements at the post-translational level that permits simultaneous control of gene expression and protein stability. Integrating these properties to control expression of a fluorescent protein in mouse Embryonic Stem Cells (mESCs), we found that adding protein stability control allows faster response times to changes in small molecules, fully tunable and enhanced dynamic range, and vastly improved microfluidic-based in-silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to finely modulate levels of signalling pathway components in stem cells.

synthetic biology↗