Biology subjects

Barone, V.

Publications and source records attributed to Barone, V..

8 recordsLinked to original sources

bioRxiv2025

Characterization of cellular wound resistance in the giant ciliate Stentor coeruleus

Resistance to mechanical stress is essential for cells to prevent wounding and maintain structural integrity. This capability is especially critical for free-living single-celled organisms, which routinely encounter mechanical stress from their natural habitats. We investigated Stentor coeruleus, a single-celled ciliate known for its remarkable wound repair capacity, as a model for studying mechanical wound resistance. While previous work focused on wound repair in Stentor, the structures that enable it to resist wounding remain poorly understood. We characterized how Stentor resisted mechanical stress during transit through a microfluidic constriction. Using high- speed imaging, we tracked the transit dynamics of the cells and linked them to wounding outcomes. Larger cells experienced longer transit times in the constriction and were more prone to rupture, often failing to recover shape due to membrane rupture and loss of cytoplasm. To elucidate the role of the Stentor cytoskeleton, we performed drug-mediated disruption of KM fibers, which are microtubule bundles in the Stentor cytoskeleton. Drug-treated cells exhibited an increased likelihood of membrane rupture at the constriction, implicating KM fibers in wound resistance. To investigate the resistance of Stentor cells to hydrodynamic stress, we injected the cells at increasing flow rates through the constriction. Interestingly, cells were more resistant to larger hydrodynamic stresses up to a threshold, potentially due to shear-thinning of the cytoplasm. Together, these results suggest that Stentor relies on both cytoskeletal architecture and cytoplasmic rheology to withstand mechanical stress, offering insights into cellular strategies for wound resistance in the absence of rigid extracellular structures.

cell biology↗

bioRxiv2024

MAPK15 Protects Against The Development Of Metabolic Dysfunction-Associated Steatotic Liver Disease

Accumulation of lipids in the liver characterizes metabolic dysfunction-associated steatotic liver disease (MASLD), the most prevalent chronic liver disease worldwide. As liver injury progresses to metabolic dysfunction-associated steatohepatitis (MASH), MASLD can predispose individuals to cirrhosis and hepatocellular carcinoma. Here, we characterized the first knockout mouse model for mitogen-activated protein kinase 15 (MAPK15) and revealed its critical role in controlling lipid homeostasis in the liver. Indeed, Mapk15-/- mice exhibited a MASLD-like phenotype, and hepatocellular models allowed us to demonstrate that dysregulated accumulation of lipids was due to increased expression and membrane localization of the CD36 fatty acid translocase. Consistently, Mapk15-/- mice exhibited elevated hepatic levels of CD36 and feeding them with a western-type diet significantly accelerated their progression to a MASH-like phenotype. Ultimately, transcriptomic analysis of human cohorts revealed increased liver expression of MAPK15 in MASLD patients, compared to unaffected individuals, ultimately supporting a protective role for MAPK15 against this disease. Overall, our data highlight a critical role for MAPK15 in liver physiopathology, by contributing to maintain physiological intracellular levels of lipids in this tissue.

cell biology↗

bioRxiv2024

Advances in culturing of the sea star Patiria miniata

The use of the sea star Patiria miniata as a model system has produced groundbreaking advances in a disparate set of biomedical research fields, including embryology, immunology, regeneration, cell biology and evolution of development. Nonetheless, the life cycle of P. miniata has not yet been closed in the laboratory, precluding the generation of stable transgenic and mutant lines, which would greatly expand the toolset for experimentation with this model system. Rearing P. miniata in the laboratory has been challenging due to limited knowledge about metamorphosis cues, feeding habits of juveniles and their relatively long generation time. Here we report protocols to rear P. miniata embryos through sexual maturity in a laboratory setting. We provide detailed staging of early embryonic development at different temperatures, and show that larvae can be raised to competence in as little as 15 days. We find that retinoic acid induces metamorphosis effectively and present methods to rear juveniles on commercially available foods. We show that in a flow-through system, juveniles double in size every 2 months and reach sexual maturity in approximately 2 years. We report the first example of P. miniata raised through sexual maturity in a laboratory setting, paving the way for the generation of stable mutant sea star lines.

developmental biology↗

bioRxiv2024

Failure to Resolve Inflammation Contributes to Juvenile-Onset Cardiomyopathy in a Mouse Model of Duchenne Muscular Dystrophy

Absence of dystrophin protein causes cardiac dysfunction in patients with Duchenne muscular dystrophy (DMD). Unlike boys with DMD, the common mouse model of DMD (B10-mdx) does not manifest cardiac deficits until late adulthood. This has limited our understanding of the mechanism and therapeutic approaches to target the pediatric onset of cardiac pathology in DMD. Here we show that the mdx mouse model on the DBA/2J genetic background (D2-mdx) displays juvenile-onset cardiac degeneration. Molecular and histological analysis revealed that cardiac damage in this model is linked to increased leukocyte chemotactic signaling and an inability to resolve inflammation. These deficiencies result in chronic inflammation and fibrotic conversion of the extracellular matrix (ECM) in the juvenile D2-mdx heart. To address these pathologies, we tested the utility of pro-resolution therapy to clear chronic cardiac inflammation. Use of an N-formyl peptide receptor (FPR) agonist helped physiologically resolve inflammation and mitigate the downstream events that lead to fibrotic degeneration of cardiomyocytes, preventing juvenile onset cardiac muscle loss. These results establish the utility of D2-mdx model to study events associated with pediatric-onset cardiac damage and demonstrates pro-resolution therapy as an alternate to anti-inflammatory therapy for treating cardiac degenerative pathology responsible for cardiomyopathy in DMD patients.

cell biology↗

bioRxiv2024

Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium.

Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes adopting a shape named "scutoid" that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here we use live-imaging of the sea star embryo coupled with deep learning-based segmentation, to dissect the relative contributions of cell density, tissue compaction, and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing just after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development. Summary statementThe study uses sea star embryogenesis as a model of a proliferating epithelium to highlight how cell division induces 3D cell rearrangements during development.

developmental biology↗

bioRxiv2022

Live imaging of echinoderm embryos to illuminate evo-devo

Echinoderm embryos have been model systems for cell and developmental biology for over 150 years, in good part because of their optical clarity. Discoveries that shaped our understanding of fertilization, cell division and cell differentiation were only possible because of the transparency of sea urchin eggs and embryos, which allowed direct observations of intracellular structures. More recently, live imaging of sea urchin embryos, coupled with fluorescence microscopy, has proven pivotal to uncovering mechanisms of epithelial to mesenchymal transition, cell migration and gastrulation. However, live imaging has mainly been performed on sea urchin embryos, while echinoderms include numerous experimentally tractable species that present interesting variation in key aspects of morphogenesis, including differences in embryo compaction and mechanisms of blastula formation. The study of such variation would allow us not only to understand how tissues are formed in echinoderms, but also to identify which changes in cell shape, cell-matrix and cell-cell contact formation are more likely to result in evolution of new embryonic shapes. Here we argue that adapting live imaging techniques to more echinoderm species will be fundamental to exploit such an evolutionary approach to the study of morphogenesis, as it will allow measuring differences in dynamic cellular behaviors - such as changes in cell shape and cell adhesion - between species. We briefly review existing methods for live imaging of echinoderm embryos and describe in detail how we adapted those methods to allow long-term live imaging of several species, namely the sea urchin Lytechinus pictus and the sea stars Patiria miniata and Patiriella regularis. We outline procedures to successfully label, mount and image early embryos for 10-16 hours, from cleavage stages to early blastula. We show that data obtained with these methods allows 3D segmentation and tracking of individual cells over time, the first step to analyze how cell shape and cell contact differ among species. The methods presented here can be easily adopted by most cell and developmental biology laboratories and adapted to successfully image early embryos of additional species, therefore broadening our understanding of the evolution of morphogenesis.

developmental biology↗

bioRxiv2022

Cell size asymmetries in the sea star embryo

Cell size asymmetries are often linked to cell fate decisions, due to cell volumes and cell fate determinants being unequally partitioned during asymmetric cell divisions. A clear example is found in the sea urchin embryo, where a characteristic and obvious unequal 4th cleavage generates micromeres, which are necessary for mesendoderm cell fate specification. Unlike sea urchin development, sea star development is generally thought to have only equal cleavage. However, subtle cell size asymmetries can be observed in sea star embryos; whether those cell size asymmetries are consistently produced during sea star development and if they are involved in cell fate decisions remains unknown. Using confocal live imaging of early embryos we quantified cell size asymmetries in 16-cell stage embryos of two sea star species, Patiria miniata and Patiriella regularis. Using photoconversion to perform lineage tracing, we find that the position of the smallest cells of P. miniata embryos is biased toward anterior ventral tissues. However, both blastomere dissociation and mechanical removal of one small cell do not prevent dorsoventral (DV) axis formation, suggesting that embryos compensate for the loss of those cells and asymmetric partitioning of maternal determinants is not strictly necessary for DV patterning. Finally, we show that manipulating cell size to introduce artificial cell size asymmetries is not sufficient to direct the positioning of the future DV axis in P. miniata embryos. Our results show that although cell size asymmetries are consistently produced during sea star early cleavage and may be predictive of the DV axis, they are not necessary to instruct DV axis formation.

developmental biology↗

bioRxiv2017

Live Tracking Of Moving Samples In Confocal Microscopy For Vertically Grown Plant Roots

Roots navigate through soil integrating environmental signals to orient their growth. The Arabidopsis root is a widely used model for developmental, physiological and cell biological studies. Live imaging greatly aids these efforts, but the horizontal sample position and continuous root tip displacement present significant difficulties. Here, we develop a confocal microscope setup for vertical sample mounting and integrated directional illumination. We present TipTracker - a custom software for automatic tracking of diverse moving objects usable on various microscope setups. Combined, this enables observation of root tips growing along the natural gravity vector over prolonged periods of time, as well as the ability to induce rapid gravity or light stimulation. We also track migrating cells in the developing zebrafish embryo, demonstrating the utility of this system in the acquisition of high resolution data sets of dynamic samples. We provide detailed descriptions of the tools enabling the easy implementation on other microscopes.

plant biology↗