Biology subjects

Grenci, G.

Publications and source records attributed to Grenci, G..

12 recordsLinked to original sources

bioRxiv2026

Microdomes: Micro-engineered Microdome arrays enable standardised and shear-free 3D biology with full-spectrum optical imaging compatibility

Three-dimensional cellular models such as organoids and spheroids hold major promise for developmental biology, disease modelling and precision medicine, yet their large-scale production and analysis remain constrained by handling-induced shear stress, sample fragility, positional instability and limited compatibility with advanced imaging workflows. Here, we introduce Microdomes, dense arrays of open-top, dome-shaped microcavities. Each cavity holds a single specimen in its own miniature aquarium, accessed through a narrow apical opening that admits cells, medium, matrix and staining reagents while shielding it from the shear generated during routine pipetting. Each Microdomes chip accommodates more than 100 spheroids or organoids, supporting diverse cell types, co-culture formats and both matrix-free and matrix-embedded culture. All specimens are retained through prolonged culture, repeated medium exchange, fixation and immunostaining. Because every specimen occupies a fixed, addressable position against a thin transparent film, the same spheroid can be relocated and re-imaged over weeks of culture and across microscopes, from array-wide overviews to subcellular details, without transfer, embedding or other perturbation. Microdomes also support AI-based automated segmentation, from whole-spheroid outlines to individual nuclei in 3D, using common image analysis software. Microdomes thereby turn each array into a self-contained quality control unit, providing specimen-level traceability that organoid production pipelines currently lack.

cell biology↗

bioRxiv2026

Curvature-guided chiral collective organization of myoblast tissues

Surface curvature is a fundamental geometric cue in tissue morphogenesis, yet its role in guiding collective cell organization has remained elusive. Here, we show that curvature acts as a geometric control parameter that shapes supracellular alignment and chirality while modulating myogenic differentiation in myoblast tissues. Cells cultured on curved substrates self-organize into robust helical assemblies whose handedness is set, and can be reversed, by the sign of curvature: convex fibers produce right-handed helices, whereas concave channels invert the chirality. We identify a previously hidden clockwise bias in single-cell motion associated with the helical actin cytoskeleton. A minimal continuum theory coupling an effective chiral drive to curvature quantitatively captures the emergence and reversal of tissue-scale chiral alignment. On substrates with spatially varying curvature, local curvature gradients organize patterned multicellular architectures while preserving a global handedness. Curvature is also associated with myogenic state, with higher curvature linked to reduced or delayed differentiation. Together, these findings reveal how complex geometries shape the alignment, symmetry, and cellular state of living tissues.

biophysics↗

bioRxiv2025

Fibrillar adhesions are the primary integrin complexes shaped by matrix topography

Mechanisms of matrix topography recognition are poorly understood. Here, we show that 5{beta}1-integrin mediated fibrillar adhesions serve this function. While on planar substrates, their formation requires fibronectin secretion, tensins, and actomyosin contractility, these requirements are bypassed on nanotopographical features. While focal adhesions avoid these features, fibrillar adhesions rapidly align along pre-existing fibrous cell-derived matrix or electrospun nanofibers where they can then template fibronectin fibrils. Topography-induced fibrillar adhesions depend primarily on 5{beta}1-integrin clustering and disassemble upon cortical stiffening driven by myosin-II overactivation or increased membrane tension. We propose a generic theoretical model where the adhesion receptor favours the membrane and substrate planes to be tilted relative to each other. This model matches experimental observations of preferential 5{beta}1-integrin clustering along nanofibers and concave edges of large negative curvature along micro-ridges. These findings establish fibrillar adhesions as primary adhesion complexes that form independently of focal adhesions in response to matrix topography.

cell biology↗

bioRxiv2025

Extracellular matrix deposition controls early differentiation patterns in human cardiac gastruloids

Amongst the various factors that affect differentiation and tissue organization, the autonomous deposition of extracellular matrix (ECM) has hardly been considered in the context of gastruloids. Using biofunctionalized colloidal particles as artificial organizing centers (aOCs), we patterned differentiation loci within 3D embryonic bodies. Our findings reveal that the spatial distribution of the aOCs induces various pattern organizations, mediated by endogenous deposition of ECM. We investigated how the interplay between meso-endoderm cell epithelial-to-mesenchymal transition, migration into germ layers, and the prolonged maintenance of local stem-cell niches orchestrates gastruloid structure and composition. These factors collectively regulate the complexity of emergent cardiac structures observed at later time points. This work uncovers a critical feedback loop between cell differentiation rates and endogenous ECM deposition patterns, which governs the differentiation paths in scaffolded cardiac gastruloids.

cell biology↗

bioRxiv2024

Ca2+-dependent vesicular and non-vesicular lipid transfer controls hypoosmotic plasma membrane expansion

Robust coordination of surface and volume changes is critical for cell integrity. Few studies have elucidated the plasma membrane (PM) remodeling events during cell surface and volume alteration, especially regarding PM sensing and its subsequent rearrangements. Here, using fission yeast protoplasts, we reveal a Ca2+-dependent mechanism for membrane addition that ensures PM integrity and allows its expansion during acute hypoosmotic cell swelling. We show that MscS-like mechanosensitive channels activated by PM tension control extracellular Ca2+ influx, which triggers direct lipid transfer at endoplasmic reticulum (ER)-PM contact sites by conserved extended-synaptotagmins and accelerates exocytosis, enabling PM expansion necessary for osmotic equilibrium. Defects in any of these key events result in rapid protoplast rupture upon severe hypotonic shock. Our numerical simulations of hypoosmotic expansion further propose a cellular strategy that combines instantaneous non-vesicular lipid transfer with bulk exocytic membrane delivery to maintain PM integrity for dramatic cell surface/volume adaptation.

cell biology↗

bioRxiv2024

Fast volumetric fluorescence lifetime imaging of multicellular systems using single-objective light-sheet microscopy

Fluorescence lifetime imaging (FLIM) is widely used for functional and multiplexed bioimaging. The lifetime of autofluorescence or fluorescent sensors encodes physiologically relevant parameters. Thus, FLIM is especially relevant for the investigation of living systems. However, application of FLIM to live specimen is hampered by its slow speed and high phototoxicity. To enable faster and gentler FLIM, we integrated single-objective light-sheet microscopy with pulsed excitation and time-resolved detection on a novel SPAD array detector. We achieved 10-100-fold acceleration compared to confocal FLIM, down to 100 ms acquisition time per image, with excellent quantitative agreement. The massively enhanced speed enables volumetric FLIM acquisitions on live multicellular specimens, which we demonstrate with lifetime-based multiplexing in 3D and time-lapse FLIM of tension probes on living embryonic organoids. We benchmark both scanned and static light-sheet modalities to facilitate adding FLIM capability to a large variety of light-sheet microscopes.

biophysics↗

bioRxiv2023

Cortical ductility governs cell-cell adhesion mechanics

This paper challenges our understanding of cell-cell adhesion by emphasising the role of mechanical dissipation at the cellular level. We have developed new microdevices to measure the energy dissipated during the rupture of junctions between cell-cell doublets. Using a synthetic cadherin approach, we decoupled the role of cadherin binding energy, signalling and downstream regulation of cytoskeletal architecture. This yielded a phase diagram in which cell junctions transition from a ductile to a brittle fracture mode based on their ratio of cortical tension and shape relaxation time. We recapitulated our results using a descriptive mechanical simulation approach. Our results shift our understanding of cell-cell adhesion from the current focus on bond energy and tension to the key role played by energy dissipation in the cytoskeleton during junction deformation and its active mechanosensitive regulation.

cell biology↗

bioRxiv2023

Digitalized organoids: integrated pipeline for 3D high-speed analysis of organoid structures using multilevel segmentation and cellular topology

Analysing the tissue morphogenesis and function is crucial for unravelling the underlying mechanisms of tissue development and disease. Organoids, 3D in vitro models that mimic the architecture and function of human tissues, offer a unique opportunity to study effects of external perturbators that are difficult to replicate in vivo. However, large-scale screening procedures for studying the effects of different stress on cellular morphology and topology of these 3D tissue-like system face significant challenges, including limitations in high-resolution 3D imaging, and accessible 3D analysis platforms. These limitations impede the scale and throughput necessary to accurately quantify the effects of mechanical and chemical cues. Here, we present a novel, fine-tuned pipeline for screening morphology and topology modifications in 3D cell culture using multilevel segmentations and cellular topology, based on confocal microscopy and validated across different image qualities. Our pipeline incorporates advanced image analysis algorithms and artificial intelligence (AI) for multiscale 3D segmentation, enabling quantification of morphology changes at both the nuclear and cytoplasmic levels, as well as at the organoid scale. Additionally, we investigate cell relative position and employ neighbouring topology analysis to identify tissue patterning and their correlations with organoid microniches. Eventually, we have organized all the extracted features, 3D segmented masks and raw images into a single database to allow statistical and data mining approaches to facilitate data analysis, in a biologist-friendly way. We validate our approach through proof-of-concept experiments, including well-characterized conditions and poorly explored mechanical stressors such as microgravity, showcasing the versatility of our pipeline. By providing a powerful tool for discovery-like assays in screening 3D organoid models, our pipeline has wide-ranging interests from biomedical applications in development and aging-related pathologies to tissue engineering and regenerative medicine.

cell biology↗

bioRxiv2023

Migrating neurons adapt motility modes to brain microenvironments via a mechanosensor, PIEZO1

Migration of newborn neurons is essential for brain morphogenesis and circuit formation, yet controversy exists regarding how neurons generate the driving force against strong mechanical stresses in crowded neural tissues. We found that cerebellar granule neurons adopt differential motility modes in distinct extracellular environments. In 3-dimensional (3D) confinement, actomyosin produces contractile forces at the posterior cell membrane, in addition to the traction force in the leading process that is exclusively observed in 2D cultures. The 3D migration is initiated by activation of a mechanosensitive channel PIEZO1. PIEZO1-induced calcium influx in the soma triggers the PKC-ezrin cascade, which recruits actomyosin to the posterior plasma membrane. Thus, migrating neurons use a mechano-sensing mechanism to activate multiple driving forces to maneuver in irregular brain tissue. One-Sentence SummaryCerebellar granule neurons use a mechanosensor PIEZO1 to switch migratory modes in confined spaces.

cell biology↗

bioRxiv2021

Bioengineering a Miniaturized In Vitro 3D Myotube Contraction Monitoring Chip For Modelization of Muscular Dystrophies

Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for muscular disorders. Muscle three-dimensional "Organ-on-chip" models usually require a substantial amount of biological material, which is problematic in the context of limited patient sample. Here we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments. Spontaneous contractions in myotubes derived from primary human myoblasts were observed. Analysis of nuclear morphology confirmed a similar organization between obtained myotubes and in vivo myofibers. LMNA-related Congenital Muscular Dystrophy (L-CMD) was modelled with successful development of mutant 3D myotubes displaying contractile dysfunction. This technology can thus be used to study contraction characteristics and evaluate how diseases affect muscle organization and force generation. Importantly, it requires significantly fewer starting materials than current systems, which should allow to substantially improve drug screening capability.

bioengineering↗

bioRxiv2021

High content 3D imaging method for quantitative characterization of organoid development and phenotype.

Quantitative analysis on a large number of organoids can provide meaningful information from the morphological variability observed in 3D organotypic cultures, called organoids. Yet, gathering statistics of growing organoids is currently limited by existing imaging methods and subsequent image analysis workflows that are either restricted to 2D, limited in resolution, or with a low throughput. Here, we present an automated high content imaging platform synergizing high density organoid cultures with 3D live light-sheet imaging. The platform is an add-on to a standard inverted microscope. We demonstrate our capacity to collect libraries of 3D images at a rate of 300 organoids per hour, enabling training of artificial intelligence-based algorithms to quantify the organoid morphogenetic organization at multiple scales with subcellular resolution. We validate our approach on different organotypic cell cultures (stem, primary, and cancer), and quantify the development of hundreds of neuroectoderm organoids (from human Embryonic Stem Cells) at cellular, multicellular and whole organoid scales.

biophysics↗

bioRxiv2018

Forces and constraints controlling podosome assembly and disassembly

Podosomes are a singular category of integrin-mediated adhesions important in the processes of cell migration, matrix degradation, and cancer cell invasion. Despite a wealth of biochemical studies, the effects of mechanical forces on podosome integrity and dynamics are poorly understood. Here, we show that podosomes are highly sensitive to two groups of physical factors. First, we describe the process of podosome disassembly induced by activation of myosin-IIA filament assembly. Next, we find that podosome integrity and dynamics depends upon membrane tension and can be experimentally perturbed by osmotic swelling and deoxycholate treatment. We have also found that podosomes can be disrupted in a reversible manner by single or cyclic radial stretching of the substratum. We show that disruption of podosomes induced by osmotic swelling is independent of myosin-II filaments. Inhibition of the membrane sculpting protein, dynamin-II, but not clathrin, resulted in activation of myosin-IIA filament formation and disruption of podosomes. The effect of dynamin-II inhibition on podosomes was however independent of myosin-II filaments. Moreover, formation of organized arrays of podosomes in response to microtopographic cues (the ridges with triangular profile) was not accompanied by reorganization of myosin-II filaments. Thus, mechanical elements such as myosin-II filaments and factors affecting membrane tension/sculpting independently modulate podosome formation and dynamics, underlying a versatile response of these adhesion structures to intracellular and extracellular cues.

cell biology↗