Hiraiwa, T. | bioRxiv Science

bioRxiv2025

Force Scaling in Active Swarm of Microtubules via Magnetic Manipulation

Scalability refers to the ability of a system to enhance performance with increasing size. This ability is a defining feature of natural swarms such as ant and bee colonies. Extending this principle to artificial active matter has motivated the creation of molecular swarms composed of myosin-driven actin filaments and kinesin-driven microtubules (MTs). While these swarms exhibit cooperative transport behaviors that surpass the abilities of individual filaments, their collective force generation has remained unquantified. Here, we introduce a straightforward electromagnetic tweezer approach to directly measure the forces generated by MT swarms propelled by surface-bound kinesin motors. We find that force output changes with swarm size, demonstrating quantitative scalability and linking collective organization to mechanical performance. These results establish MT swarms as a model system for scalable, force-producing active matter and provide a foundation for designing biomolecular devices that exploit collective dynamics for microscale actuation and transport. Significance StatementThe cooperative activity of multiple motile agents is observed in nature, known as swarming. In artificial systems, swarming has been demonstrated using active agents like kinesin and MTs, successfully enabling efficient cargo transportation. However, the force generated by the swarm remains unexplored, which is one of the keys to understanding the principles underlying swarm systems. This study introduces a simple method employing electromagnetic tweezers and magnetic beads to measure the force of the MT swarm accurately. The measured forces within the microtubule swarms of different sizes unveil the scaling effect, the additive nature of force generation as the kinesin unit increases. The force study sheds light on the fundamental properties, work performance, efficiency, and execution of the MT swarm.

biophysics↗

bioRxiv2025

Critical phenomenon underlies de novo luminogenesis during mammalian follicle development

A key process during mammalian folliculogenesis is the formation of a fluid-filled antrum around the oocyte. While it is known that the antrum is critical for oocyte maturation and the eventual ovulation, the detailed process underlying de novo luminogenesis remains poorly understood. In this study, we investigated the spatiotemporal dynamics of lumen growth and the cellular mechanism driving this process, using advanced microscopy, molecular perturbations and computational modelling. We found that in secondary follicles, the interstitial gaps exist in a near-critical regime, characterised by a highly dynamic and interconnected fluid network with gap size obeying a power-law distribution. Above a critical size of 180 m, we observed an onset of cell death and the emergence of a stably growing dominant fluid cavity resembling phase separation. By modelling the granulosa cells and the interstitial fluid as a binary fluid, we reproduced the near-critical and phase-separated regimes and found that a spatial gradient of cell-fluid interfacial tension, as observed experimentally, is sufficient to robustly maintain the secondary follicles in a near-critical state. Reducing cell-fluid interfacial tension globally by weakening the cell-cell adhesion between granulosa cells leads to fragmentation of fluid and growth arrest of the dominant cavity, as predicted by the model. Importantly, perturbing the critical state in secondary follicles leads to impaired follicle growth. Altogether, our study reveals how collective spatiotemporal regulation of cell junction mechanics and cell death can contribute to the effective transition between distinct regimes of fluids, which are indispensable for functional maturation of follicles.

developmental biology↗

bioRxiv2025

Condensation of Sidekick at tricellular junctions organizes mechanical forces for cell-cell adhesion remodeling

Epithelial morphogenesis relies on coordinated multicellular movements regulated by adhesion and force-generating molecules. It remains elusive how cells ensure organization of these molecules in space and time. Combining in vitro reconstitution and fly genetics, we demonstrate that condensation of an adhesive transmembrane protein shapes molecular dynamics at tricellular junctions to facilitate cell-cell junction remodeling. Specifically, we identify that the intracellular domain of Sidekick, known to localize to tricellular junctions and recruit mechanical force-generating myosin II, undergoes phase separation into condensates. This condensation controls the physiological localization and concentration of Sidekick on lipid membranes. Altering the molecular dynamics of these condensates disrupts Sidekicks localization against cellular movements, misdirecting myosin II and leading to delayed junction formation. These findings suggest that cells exploit the mechano-resistant properties of tricellular junction-anchored condensates to spatially allocate force-regulating molecules. This study underscores the importance of condensation to organize tricellular junctions for robust multicellular movement.

cell biology↗

bioRxiv2025

Stress anisotropy in 3D active curved structures

Layers composed of lateral connections prevail in biological systems from subcellular membranes to epithelial sheets. This work presents a continuum framework to describe the effects of mechanical forces within a 3D curved layer with supporting lateral mesh. We provide detailed discussion on the emergence of stress anisotropy as a function of depth in different curvature settings, building on Lou et al. Phys. Rev. Lett. 130, 108401 (2023). We principally consider an epithelial monolayer to explain how the interplay between layer curvature and cell mechanics determines the stress anisotropy. We show that this can lead to irregular cellular shapes in 3D, including scutoid-like geometries. Our framework is general, and can be extended to a diverse set of biologically relevant systems.

biophysics↗

bioRxiv2025

The cellular and behavioral blueprints of chordate rheotaxis.

The oceans are filled with life-forms exhibiting complex adaptations to help navigate fluid environments and take advantage of fluid motion to locomote and disperse. However, the neuronal and behavioral underpinnings of navigating in fluid environments outside vertebrates are poorly understood. We present behavioral and computational modelling-based evidence that the pre-vertebrate chordate Ciona intestinalis actively modulates its heading and angular velocities to counter oncoming flows and perform positive rheotaxis. We demonstrate that a distributed network of ciliated peripheral sensory neurons is responsible for sensing hydrodynamic information such as flow velocity and direction. Pharmacological removal of sensory cilia impedes rheotactic behavior and reduces stimulus evoked neuronal activity. Whole-brain calcium imaging experiments reveal that the central nervous system of Ciona can encode the onset and offset of flow, flow direction, and flow velocity, suggesting that miniature chordate brain is capable of surprisingly sophisticated computations.

neuroscience↗

bioRxiv2024

Epithelium Stratifies via Nucleation and Growth Induced by Foam-Geometric Instability

The epithelium undergoes stratification, transitioning from a monolayer to a multilayer structure, across broad phenomena. Recent studies have identified several cell behaviors as triggers, including junctional tension, cell density and geometry, and topological defects. However, how these factors drive stratification throughout the entire epithelium remains poorly understood. Here, we report a mechanism underlying epithelial stratification that mirrors the physics of phase transition. Combining cell culture with three-dimensional vertex modeling, we demonstrate that epithelial stratification is analogous to a structural phase transition driven by the nucleation-growth process, i.e., multilayer origins dispersedly appear and expand across the epithelium via unordered intermediate states. This transition is induced by a mechanical instability inherent in the foam-like geometry of the epithelium. Moreover, the nucleation-growth concept applies to embryonic skin development and intestinal cancer transformation. These findings conceptualize epithelial stratification as a form of a phase transition governed by foam mechanics, offering a physical perspective on various epithelial developments.

biophysics↗

bioRxiv2024

Strain softening and hysteresis arising from 3D multicellular dynamics during long-term large deformation

Living tissues exhibit complex mechanical properties, including viscoelastic and elastoplastic responses, that are crucial for regulating cell behaviors and tissue deformations. Despite their significance, the intricate properties of three-dimensional (3D) multicellular tissues are not well understood and are inadequately implemented in biomaterial engineering. To address this gap, we developed a numerical method to analyze the dynamic properties of multicellular tissues using a 3D vertex model framework. By focusing on 3D tissues composed of confluent homogeneous cells, we characterized their properties in response to various deformation magnitudes and time scales. Stress relaxation tests revealed that large deformations initially induced relaxation in the shapes of individual cells. This process is amplified by subsequent transient cell rearrangements, homogenizing cell shapes and leading to tissue fluidization. Additionally, dynamic viscoelastic analyses showed that tissues exhibited strain softening and hysteresis during large deformations. Interestingly, this strain softening originates from multicellular structures independent of cell rearrangement, while hysteresis arises from cell rearrangement. Moreover, tissues exhibit elastoplastic responses over the long term, which are well represented by the Ramberg-Osgood model. These findings highlight the characteristic properties of multicellular tissues emerging from their structures and rearrangements, especially during long-term large deformations. The developed method offers a new approach to uncover the dynamic nature of 3D tissue mechanics and could serve as a technical foundation for exploring tissue mechanics and advancing biomaterial engineering.

bioengineering↗

bioRxiv2024

Asymmetry of acto-myosin cortices as active fluids shape cells in organoids

Cell cortex is a thin sheet of actin cytoskeleton spanning cell boundaries with rich out-of-equilibrium dynamics. A theoretical description of the cortex as an active fluid enables to capture cell shapes dynamics in 3D epithelial tissues. However models integrated with calibration of parameters and quantitative experiments are lacking so far. Here we report that cells in organoids and in cysts have conserved apico-basal-lateral asymmetric compositions in actin and in myosin, and we quantify their densities and mechanical properties. This allows to calibrate a new model coupling active fluids with a phase field which reproduces the main features of cell shapes. To test our approach, we successfully predict changes in cell shapes by modulating actin polymerisation, myosin activity, and adhesion in experiments and in simulations. Our study shows how active fluid theory integrated with experiments can determine cell shapes in epithelial tissues.

biophysics↗

bioRxiv2024

Control of lumen geometry and topology by cell proliferation rate and pressure

Many internal organs in multicellular organisms comprise epithelia which enclose fluid-filled cavities. These are referred to as lumens and their formation is regulated by a wide range of processes, including epithelial polarization, secretion, exocytosis and actomyosin contractility [1, 2]. While these mechanisms have shed light on lumen growth, what controls lumen morphology remains enigmatic. Here we use pancreas organoids to explore how lumens acquire either a spherical shape or a branched topology [3]. Combining computational simulations based on a phase field model with experimental measurements we reveal that lumen morphology arises from the balance between the cell cycle duration and lumen pressure, with more complex lumen at low pressure and fast proliferation rates. Moreover, the manipulation of proliferation and lumen pressure in silico and in vitro is sufficient to alter and reverse the morphological trajectories of the lumens. Increasing epithelial permeability of spherical lumens lead to lower lumen pressure and converts their morphology to complex lumen shapes, highlighting its crucial role. In summary, the study underscores the importance of balancing cell proliferation, lumen pressure, and epithelial permeability in determining lumen morphology, providing insights relevant to other organs, for tissue engineering and cystic disease understanding and treatment [4].

developmental biology↗

bioRxiv2024

Predicting Organoid Morphology Through a Phase Field Model: Insights into Cell Division and Lumenal Pressure

Organoids are ideal systems to predict the phenotypes of organs. However, there is currently a lack of understanding regarding the generalized rules that enable use of simple cellular principles to make morphological predictions of entire organoids. Therefore, we employed a phase field model with the following basic components: the minimum conditions for the timing and volume of cell division, lumen nucleation rules, and lumenal pressure. Through our model, we could compute and generate a myriad of organoid phenotypes observed till date. We propose morphological indices necessary to characterize the shapes and construct phase diagrams and show their dependencies on proliferation time and lumen pressure. Additionally, we introduced the lumen-index parameter, which helped in examining the criteria to maintain organoids as spherical structures comprising a single layer of cells and enclosing an intact lumen. Finally, we predict a star-like organoid phenotype that did not undergo differentiation, suggesting that the volume constraint during cell division may determine the final phenotype. In summary, our approach provides researchers with guidelines to test the mechanisms of self-organization and predict the shape of organoid. Author summaryIn nature, a wide variety of organ morphologies are observed. Owing to the complexity of the process underlying the acquisition of organs morphology, it is challenging to investigate the mechanisms that lead to such variations. A promising approach to study these variations is the use of "computational organoid" study, which is the computational-based study of self-organizing shapes in multicellular assemblies and fluid-filled cavities called lumens that develop from a few proliferating cells. This study explores general mechanisms that dictate how various mechanical factors affect the growing self-organized multicellular assembly. We relied on computer simulations of the mathematical model called multicellular phase-field model with lumens and explored the mechanical factor effects, such as the lumen pressure while considering the time and volume conditions required for cell division. These simulations generated and categorized a wide range of organoid phenotypes based on the varying lumen pressure and cell division conditions. These phenotypes were characterized into seven distinct classes, based on the morphological index sets, including a cellular monolayer/multilayer surrounding single or multiple lumens and branch formation. These phenotypes were obtained without the assumption of differentiation. Our study elucidates the mechanisms underlying the organoid and organ formation with different shapes, thereby highlighting the significance of mechanical forces in shaping these complex biological structures.

cell biology↗

bioRxiv2024

Generic rules of lumen nucleation and fusion in epithelial organoids

Many internal organs in the body harbor a fluid-filled lumen. The mechanisms of lumens initiation and fusion have been reported as dependent on organ-type during organogenesis. In contrast, the physics of lumen suggests that force balance between luminal pressure and cell mechanics could lead to conserved rules which may unify their self-organisation. However, this hypothesis lacks experimental evidence. Here we compare lumen dynamics for three different systems (MDCK cysts, pancreatic spheres, and epiblast cysts) by using quantitative cell biology, microfabrication and theory. We report that initial cell number determines the maximum number of lumens but does not impact the steady state which is a final single lumen. In addition, lumen numbers exhibit two phases over time, a nucleation phase followed by a fusion phase. In the nucleation phase, lumens form between two cells in pancreatic and MDCK cysts whereas they form at the rosette stage between ten cells in epiblasts. In the second phase, lumens fuse by an increase in lumen volume for pancreatic spheres and MDCK cysts, whereas cell convergent directional motion leads to lumens fusion in epiblasts. We show that these phenomena are associated to the luminal hydrostatic pressure. We support these results with theoretical arguments and numerical simulations. We finally use MDCK cysts to manipulate cell adhesion and lumen volume and we successfully reproduce the fusion dynamics of pancreatic spheres and epiblasts. Our results reveal self-organisation rules of lumens across systems with relevance for morphogenesis during development and for the design of synthetic organs.

biophysics↗

bioRxiv2023

Co-evolution of spheroid fluidization and mechanical fracture in a morphogenic cascade

Cells migrate and invade tissues during development, immune responses, and cancer. Collective invasion is generally understood to be driven by invading cells unjamming and pushing through barriers such as the extracellular matrix and surrounding tissues. Whether these barriers actively contribute to invasion remains unclear. Using ovarian adenocarcinoma spheroids invading mesothelium derived from benign pleural effusions as an experimental model, combined with modelling, we examine invasion across molecular to multicellular scales. We identify intercellular integrin adhesions linking invasive leader cells to the tissue barrier, triggering apical constrictions within the barrier. This constriction shrinks cell-cell contacts, leading to barrier rupture. Thus, the tissue barrier plays a mechanically active role in invasion. Rather than cells pushing through, we find that coordinated subcellular contractility between the invading leader cell and the barrier drives barrier tensile rupture and invasion, independent of a jamming transition. Together, our findings challenge prevailing paradigms of collective cell invasion.

cell biology↗

bioRxiv2023

Spatio-temporal Coordination of Active Deformation Forces and Wnt / Hippo-Yap Signaling in Hydra Regeneration

Ample evidence suggests that Wnt signaling and tissue deformation are key determinants for pattern formation in animals. The coordination of these biochemical and biomechanical spatio-temporal asymmetries is often unknown or controversial. We investigated this relationship by studying regeneration in the freshwater polyp Hydra. In both reaggregates of dissociated cells and tissue regenerates, we found significant tissue contraction waves and upregulation of Wnt signaling. Applying a simple mechanical model to the mode analysis of the active deformations, we quantitatively defined the phase reversal of size change and axial deformation in those oscillations as the time point of "biomechanical" symmetry breaking. Moreover, overexpression and inhibition of canonical Wnt signaling modulated the timing of this biomechanical symmetry breaking. A direct comparison with the RNAseq data indicates that the biomechanical symmetry breaking occurs only after the upregulation of canonical Wnt signaling. Further data suggest that biochemical signaling and biomechanical active deformation synergistically stabilize the body axis and hence the following head structure formation by Hippo-Yap signaling. The symmetry breaking mechanism identified here in Hydra most likely represents a patterning module that is evolutionary conserved from early metazoan to bilaterian animals.

biophysics↗

bioRxiv2023

The interplay between lumen pressure and cell proliferation determines organoid morphology in a multicellular phase field model

Organoids are ideal systems to predict the phenotypes of organs. However, there is currently a lack of understanding regarding the generalized rules that enable use of simple cellular principles to make morphological predictions of entire organoids. Therefore, we employed a phase field model with the following basic components: the minimum conditions for the timing and volume of cell division, lumen nucleation rules, and lumenal pressure. Through our model, we could compute and generate a myriad of organoid phenotypes observed till date. We propose morphological indices necessary to characterize the shapes and construct phase diagrams and show their dependencies on proliferation time and lumen pressure. Additionally, we introduced the lumen-index parameter, which helped in examining the criteria to maintain organoids as spherical structures comprising a single layer of cells and enclosing an intact lumen. Finally, we predict a star-like organoid phenotype that did not undergo differentiation, suggesting that the volume constraint during cell division may determine the final phenotype. In summary, our approach provides researchers with guidelines to test the mechanisms of self-organization and predict the shape of organoid. Author summaryIn nature, a wide variety of organ morphologies are observed. Owing to the complexity of the process underlying the acquisition of organs morphology, it is challenging to investigate the mechanisms that lead to such variations. A promising approach to study these variations is the use of "computational organoid" study, which is the computational-based study of self-organizing shapes in multicellular assemblies and fluid-filled cavities called lumens that develop from a few proliferating cells. This study explores general mechanisms that dictate how various mechanical factors affect the growing self-organized multicellular assembly. We relied on computer simulations of the mathematical model called multicellular phase-field model with lumens and explored the mechanical factor effects, such as the lumen pressure while considering the time and volume conditions required for cell division. These simulations generated and categorized a wide range of organoid phenotypes based on the varying lumen pressure and cell division conditions. These phenotypes were characterized into seven distinct classes, based on the morphological index sets, including a cellular monolayer/multilayer surrounding single or multiple lumens and branch formation. These phenotypes were obtained without the assumption of differentiation. Our study elucidates the mechanisms underlying the organoid and organ formation with different shapes, thereby highlighting the significance of mechanical forces in shaping these complex biological structures.

systems biology↗

bioRxiv2023

Tight junctions regulate lumen morphology via hydrostatic pressure and junctional tension

Formation of fluid filled lumen by epithelial tissues is a fundamental process for organ development. How epithelial cells regulate the hydraulic and cortical forces to control lumen morphology is not completely understood. Here, we quantified the mechanical role of tight junctions in lumen formation using genetically modified MDCKII cysts. We found that the paracellular ion barrier formed by claudin receptors is not required for hydraulic inflation of lumen. However, depletion of the zonula occludens scaffold resulted in lumen collapse and folding of apical membranes. Combining quantitative measurements and perturbations of hydrostatic lumen pressure and junctional tension with modelling, we were able to predict lumen morphologies from the pressure-tension force balance. We found that in MDCK tissue the tight junction promotes formation of spherical lumen by decreasing cortical tension via inhibition of myosin. In addition, we found that the apical surface area of cells is largely uncoupled from lumen volume changes, suggesting that excess apical area contributes to lumen opening in the low-pressure regime. Overall, our findings provide a mechanical understanding of how epithelial cells use tight junctions to modulate tissue and lumen shape.

cell biology↗

bioRxiv2023

Modelling contractile ring formation and division to daughter cells for simulating proliferative multicellular dynamics

Cell proliferation is a fundamental process underlying embryogenesis, homeostasis, wound healing, and cancer. The process involves multiple events during each cell cycle, such as cell growth, contractile ring formation, and division to daughter cells, which affect the surrounding cell population geometrically and mechanically. However, existing methods do not comprehensively describe the dynamics of multicellular structures involving cell proliferation at a subcellular resolution. In this study, we present a novel model for proliferative multicellular dynamics at the subcellular level by building upon the nonconservative fluid membrane (NCF) model that we developed in earlier research. The NCF model utilizes a dynamically-rearranging closed triangular mesh to depict the shape of each cell, enabling us to analyze cell dynamics over extended periods beyond each cell cycle, during which cell surface components undergo dynamic turnover. The proposed model represents the process of cell proliferation by incorporating cell volume growth and contractile ring formation through an energy function and topologically dividing each cell at the cleavage furrow formed by the ring. Numerical simulations demonstrated that the model recapitulated the process of cell proliferation at subcellular resolution, including cell volume growth, cleavage furrow formation, and division to daughter cells. Further analyses suggested that the orientation of actomyosin stress in the contractile ring plays a crucial role in the cleavage furrow formation, i.e., circumferential orientation can form a cleavage furrow but isotropic orientation cannot. Furthermore, the model replicated tissue-scale multicellular dynamics, where the successive proliferation of adhesive cells led to the formation of a cell sheet and stratification on the substrate. Overall, the proposed model provides a basis for analyzing proliferative multicellular dynamics at subcellular resolution.

biophysics↗

bioRxiv2022

Curvature-induced cell rearrangements in biological tissues

On a curved surface, epithelial cells can adapt to geometric constraints by tilting and by exchanging their neighbors from apical to basal sides, known as an apicobasal T1 (AB-T1) transition. The relationship between cell tilt, AB-T1 transitions, and tissue curvature still lacks a unified understanding. Here, we propose a general framework for cell packing in curved environments and explain the formation of AB-T1 transitions under different conditions. We find that steep curvature gradients can lead to cell tilting and induce AB-T1 transitions. Conversely, large curvature anisotropy can drive AB-T1 transitions by hydrostatic pressure. The two mechanisms compete to determine the impact of tissue geometry and mechanics on optimized cell rearrangements in 3D.

biophysics↗

bioRxiv2021

Mechanics defines the spatial pattern of compensatory proliferation

The number of cells in tissues is tightly controlled by cell division and cell death, and misregulation of cell numbers could lead to pathological conditions such as cancer. To maintain cell numbers in a tissue, a cell elimination process named programmed cell death or apoptosis, stimulates the proliferation of neighboring cells. This mechanism is called apoptosis-induced compensatory proliferation, which was originally reported more than 40 years ago. While only a limited number of the neigboring cells need to divide to compensate for apoptotic cell loss, the mechanisms that select cells for undergoing division remain an open question. Here we found that the spatial inhomogeneity in mechanotransduction through a growth-promoting transcription co-activator Yes-associated protein (YAP) in the neighboring tissue, accounts for the inhomogeneity of compensatory proliferation. Such inhomogeneous mechanotransduction arises from the combination of the non-uniform distribution of nuclear size, which is inherent in tissues, and the non-uniform pattern of mechanical force applied to the neighboring cells upon apoptosis. Our findings from a mechanical perspective complement the current biochemical understanding of compensatory growth and provide additional insights into cellular functions of how tissue precisely maintains its homeostasis.

cell biology↗