Biology subjects

Stone, H. A.

Publications and source records attributed to Stone, H. A..

At least 19 recordsLinked to original sources

bioRxiv2026

An implantable sensor network for large-scale cellular force mapping in living embryos

Mechanical forces generated by cells are fundamental regulators of embryonic development, yet how locally generated forces are integrated across tissues to drive large-scale morphogenesis remains poorly understood. Addressing this question requires direct quantitative mapping of intercellular forces across tissue scale with cellular resolution while preserving native tissue architectures and dynamics, a capability not yet achieved. Here, we introduce FORCENET, an implantable force-sensing network that enables quantitative tissue-scale force mapping at cellular resolution within living embryos. FORCENET consists of a microfabricated network of ultrathin elastic sensors whose three-dimensional deformations are tracked over time and converted into intercellular force maps through physics-based reconstruction. FORCENET covers hundreds of cells while occupying a total volume 6- to 20-fold smaller than that of a single cell. Anatomical measurements, three-dimensional fluorescent imaging, and cell density and speed tracking confirmed that FORCENET seamlessly integrates within developing embryos without perturbing cell motion and proliferation, tissue architecture, and embryonic development. Applied to zebrafish gastrulation, FORCENET revealed that intercellular forces are dynamically coordinated across supracellular length scales of ~30-210 m. Inhibition of fibroblast growth factor signaling accelerated force accumulation and shortened the force-coordination length scales despite only modest changes in cell density and kinematics, demonstrating that FORCENET uncovers information not readily inferred from cell tracking alone. FORCENET establishes a scalable platform for in vivo force mapping and reveals that cell-generated forces are coordinated into supracellular mechanical patterns that are regulated by developmental signaling.

developmental biology↗

bioRxiv2026

Emergence of large-scale polar microtubule swarms for dense molecular transport

Microtubules (MTs) and their motor proteins collectively harness chemical energy to generate mechanical work, driving some of the most coordinated self-organized dynamics in living cells. The unique properties of these molecules also make them versatile building blocks of cytoskeletal active matter and biomimetic nanomachines that recapitulate cellular motility, emergent pattern formation, and motor-driven transport. However, these canonical systems use MTs of fixed length and do not incorporate the natural ability of MTs to grow and regenerate. Here, we go beyond these limits by using dynamic self-amplifying branched MT networks. Driven by kinesin-1 and cytoplasmic dynein activity, surface-gliding branched MT bundles undergo swarming that yields large-scale collective MT architectures with several sought-after features. They are polar and orientationally aligned, dense, span millimeter scales, and persist over hours. We then show that these features enable molecular transport along the swarm at unprecedented capacities, with up to six million motor complexes walking in parallel across millimeter-scale distances over hours. Our results introduce a new regime in cytoskeletal active matter in which the interplay between motor-driven activity and filament generation via branching leads to emergent polar order in proliferating swarms. Such emergent polarity makes these swarms suitable for engineering scalable transport nanotechnologies and programmable soft materials.

biophysics↗

bioRxiv2026

Capillary bundling of microtubules by condensates

The cytoskeleton organizes the cellular interior using cytoskeletal filaments that rely on bundling, usually executed by stable and ordered crosslinking proteins. Bundling often requires protein complexes with at least two defined microtubule binding regions, as present in many molecular motors. Here, we establish a mechanism of microtubule bundling based on capillary forces, analogous to how wet hair sticks together. We show using in vitro experiments and theory that condensates can bundle microtubules through capillary forces, wherein liquid-like capillary bridges form between microtubules and adhere them together through interfacial and wetting forces. We quantify the structure and dynamics of these capillary bundles using total internal reflection fluorescence microscopy, and directly measure the charge-dependent interfacial tensions of condensates on microtubules using atomic force microscopy. Lastly, we show that these capillary bridges provide viscous resistance to motor-driven microtubule sliding that is insensitive to the bulk protein concentration. Taken together, we provide a novel mechanism for how cytoskeletal filaments bundle: through condensate-mediated capillary forces.

biophysics↗

bioRxiv2025

A handheld microfluidic manifold for massively multiplexed CRISPR-based nucleic acid detection

Multiplexed methods for nucleic acid detection are immensely challenging to deploy outside of laboratory settings. Conversely, field-deployable methods are limited to low levels of multiplexing. Here, we introduce Scalable On-site Nucleic Acid Testing Architecture (SONATA), enabling >100-chamber reaction partitioning for multiplexed nucleic acid amplification and detection with a portable microfluidic manifold. The manifold directs a diluted sample into individual reaction chambers, each of which contains lyophilized reagents and a small stir bar or bead for mixing. Samples can be loaded using a syringe by hand, greatly simplifying the testing process. We demonstrate the integration of the platform with Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a sensitive and deployable CRISPR-based detection technology. We show that deployed with SONATA, SHINE retains its sensitivity, enabling highly multiplexed pathogen detection in [≤] 1 hour. In addition, we demonstrate the detection of single-nucleotide variants, including mutations associated with drug susceptibility.

bioengineering↗

bioRxiv2025

Sperm hyperactivation drives a circling-and-wandering migration strategy

During migration through the female reproductive tract, sperm undergo physiological changes known as capacitation, including a motility transition termed hyperactivation. Hyperactivation is essential for various aspects of fertilization, particularly effective migration within the tract. However, how hyperactivation facilitates this migration remains elusive. Here, we profiled bull sperm hyperactivation in Newtonian and complex fluids near microfluidic surfaces, mimicking generic swimming conditions in the tract. We identified three swim gaits: wandering (persistent random walks), circling, and an intriguing circling-and- wandering mode marked by stochastic transitions between the two. All gaits exhibit diffusive behavior over long time scales, with wandering showing a tenfold higher diffusivity than circling, and the diffusivity of circling-and-wandering falling in between. We found that while wandering sperm scatter from convex and concave surfaces, circling sperm become trapped around pillars, highlighting the distinctive nature of each phase. Additionally, stochastic simulations of swimming in porous media showed that as the geometrical complexity of the environment increases, circling-and-wandering outperforms either strategy alone in spreading through the media. Our findings suggest that while wandering promotes exploration and circling supports local exploitation, circling-and-wandering combines the strengths of both strategies by balancing exploration and exploitation to adapt motility, enhance migration, and potentially improve target search.

biophysics↗

bioRxiv2025

Suspension physics govern the multiscale dynamics of blood flow in sickle cell disease

In diseases from diabetes to malaria, blood dynamics are significantly altered, resulting in poor clinical outcomes. However, the multiscale mechanisms that determine blood flow in the microcirculation in health and disease are undefined, largely owing to the difficulty in directly linking cell properties to whole-blood rheology. Here, we overcome these difficulties by developing a microfluidic platform to measure red blood cell properties and flow dynamics in the same blood samples from donors. We focus on sickle cell disease (SCD), a genetic disorder that causes red blood cells to stiffen in deoxygenated conditions, with disease pathology driven by oxygen-dependent blood rheology. Our linked cell and whole-blood measurements establish that increases in effective resistances in heterogeneous suspensions are driven by increases in the proportion of stiff cells, similar macroscopically to the behavior of rigid-particle suspensions. Furthermore, by combining simulations with spatially resolved measurements of cell dynamics, we show how the spatio-temporal organization of stiff and deformable cells determines blood rheology and drives disease pathophysiology. In the presence of deformable cells, the stiffened cells marginate towards channel walls, increasing effective wall friction. In fully deoxygenated conditions in which all cells are stiffened, significant heterogeneity in cell volume fraction along the direction of flow causes localized jamming, drastically increasing effective viscous flow resistance. Our work defines the relevant suspension physics required to understand pathological blood rheology in SCD and other diseases affecting red blood cell properties. More broadly, we reveal the multiscale processes that determine emergent rheology in heterogeneous particle suspensions.

physiology↗

bioRxiv2024

Boundary-Sensing Mechanism in Branched Microtubule Networks

The self-organization of cytoskeletal networks in confined geometries requires sensing and responding to mechanical cues at nanometer to micron scales that allow for dynamic adaptation. Here, we show that the branching of microtubules (MTs) via branching MT nucleation combined with dynamic instability constitutes a boundary-sensing mechanism within confined spaces. Using a nanotechnology platform, we observe the self-organization of a branched MT network in a channel featuring a narrow junction and a closed end. Our observations reveal that branching MT nucleation occurs in the post-narrowing region only if that region exceeds a certain length before it terminates at the channels closed end. The length-dependent occurrence of branching MT nucleation arises from the dynamic instability of existing MTs when they interact with the channels closed end, combined with the specific timescale required for new MTs to nucleate at a point distant from the closed end, creating a mechanical feedback. Increasing the concentration of the base branching factor TPX2 accelerates nucleation kinetics and thus tunes the minimum length scale needed for occurrence of branching MT nucleation. As such, this feedback not only allows for adaptation to the local geometry, but also allows for tunable formation of MT networks in narrow (micron and submicron scale) channels. However, while a high concentration of TPX2 increases the kinetic rate of branching MT nucleation, it also stabilizes MTs at the channels closed end leading to MT growth and nucleation in the reversed direction, and thus hinders boundary sensing. After experimental characterization of boundary-sensing feedback, we propose a minimal model and execute numerical simulations. We investigate how this feedback, wherein growing MTs dynamically sense their physical environment and provide nucleation sites for new MTs, sets a length/time scale that steers the architecture of MT networks in confined spaces. This "search- and-branch" mechanism has implications for the formation of MT networks during neuronal morphogenesis, including axonal growth and the formation of highly branched dendritic networks, as well as for plant development and MT-driven guidance in fungi, and engineering nanotechnologies.

bioengineering↗

bioRxiv2024

Electric charge controls plasmodesma conductivity

While plant cells are enclosed by rigid cell walls that counteract intracellular hydrostatic pressure1, their plasma membrane, cytosol, and endoplasmic reticulum (ER) remain connected through plasmodesmata, nanoscopic cell wall pores2. Plasmodesmal cell-to-cell transport occurs in the cytosolic sleeve between the plasma membrane and the ER membrane3-5, and is generally thought to be limited by the size of the moving particle alone6. Given that biological membranes carry negative electric surface charges7-9, this steric notion conflicts with physical theory of ion diffusion in nanometer-sized pores with charged walls10. Quantifying the movements of differently sized and charged fluorescent dyes in Tradescantia stamen hairs, we found that anionic fluorophores of up to 1 kDa traversed plasmodesmata whereas much smaller cationic ones did not. While this agrees with theoretical expectations of different size exclusion limits for cations and anions, it questions current dogma concerning plasmodesma function and also structure, as it implies positively rather than negatively charged surfaces within plasmodesmal pores. Our findings call for re-evaluations of current models of symplasmic transport, especially of charged molecules like the phytohormone auxin (indole-acetic acid) and certain amino acids.

cell biology↗

bioRxiv2023

Free-floating Bacteria Transcriptionally Respond to Shear Flow

Planktonic (free-floating) cells are typically assumed to be oblivious to any flow that carries them. Here we discover that planktonic bacteria can sense flow to induce gene expression changes that are beneficial in flow. Specifically, planktonic P. aeruginosa induce shear-rate-dependent genes that promote growth in low oxygen environments. Untangling this mechanism revealed that in flow, motile P. aeruginosa spatially redistribute, leading to cell density changes that activate quorum sensing, which in turn enhances oxygen uptake rate. In diffusion-limited environments, including those commonly encountered by bacteria, flow-induced cell density gradients also independently generate oxygen gradients that alter gene expression. Mutants deficient in this newly-discovered flow sensing mechanism exhibit decreased fitness in flow, suggesting that this dynamic coupling of biological and mechanical processes can be physiologically significant. One-Sentence SummaryFree-floating bacteria integrate biological and mechanical responses to adaptively alter gene expression in flow environments

microbiology↗

bioRxiv2023

Community behaviors of bacteria in flow: Divergent roles for transport and collective morphology

Fluid flows can impact bacterial behaviors in unexpected ways (1-3). The high shear rate in heart valves should reduce colonization, but in endocarditis, valves are often counter-intuitively colonized by Staphylococcus aureus and Enterococcus faecalis (4, 5). Here we discover bacteria-specific mechanisms for preferential surface colonization in higher shear rate environments. This behavior enables bacteria that are outcompeted in low flow to dominate in high flow. Flow-dependent colonization by S. aureus and E. faecalis are mediated by distinct mechanisms that depend on each species microcolony morphologies: transport of a dispersal signaling molecule for clustered S. aureus and mechanical forces for linear chains of E. faecalis. These results suggest that microcolony morphologies have previously unappreciated costs and benefits in different environments, like those introduced by flow. One-Sentence SummaryBacterial surface colonization in high fluid flow depends upon the species clustered or chained microcolony morphologies.

microbiology↗

bioRxiv2023

Building on-chip cytoskeletal circuits via branched microtubule networks

Controllable platforms to engineer robust cytoskeletal scaffolds have the potential to create novel on-chip nanotechnologies. Inspired by axons, we combined the branching microtubule (MT) nucleation pathway with microfabrication to develop "cytoskeletal circuits". This active matter platform allows control over the adaptive self-organization of uniformly polarized MT arrays via geometric features of microstructures designed within a microfluidic confinement. We build and characterize basic elements, including turns and divisions, as well as complex regulatory elements, such as biased division and MT diodes, to construct various MT architectures on a chip. Our platform could be used in diverse applications, ranging from efficient on-chip molecular transport to mechanical nano-actuators. Further, cytoskeletal circuits can serve as a tool to study how the physical environment contributes to MT architecture in living cells. SignificanceMicrotubules have essential functions within the cell, including providing a robust railroad for motor-driven cargo transport. The unique properties of microtubules have stimulated attempts to harness these characteristics for targeted delivery of molecular complexes, novel material design, and developing nanotechnologies with precision comparable to living organisms. However, Previous efforts mainly focused on microtubules with fixed length and layout and no controlled MT generation, setting a limit on designing MT architecture. In this study, we integrated nanofabrication with microtubule branching reactions borrowed directly from the cells toolkit to construct cytoskeletal circuits and generate microtubule architectures from scratch. That is, our system enables control over microtubule growth and autocatalytic nucleation on a microfluidic chip with micro/nanostructures patterned within.

bioengineering↗

bioRxiv2022

Interface resistance of biomolecular condensates

A hallmark of biomolecular condensates formed via liquid-liquid phase separation is that they dynamically exchange material with their surroundings, and this process can be crucial to condensate function. Intuitively, the rate of exchange can be limited by the flux from the dilute phase or by the mixing speed in the dense phase. Surprisingly, a recent experiment suggests that exchange can also be limited by the dynamics at the droplet interface, implying the existence of an "interface resistance". Here, we first derive an analytical expression for the timescale of condensate material exchange, which clearly conveys the physical factors controlling exchange dynamics. We then utilize sticker-spacer polymer models to show that interface resistance can arise when incident molecules transiently touch the interface without entering the dense phase, i.e., the molecules "bounce" from the interface. Our work provides insight into condensate exchange dynamics, with implications for both natural and synthetic systems.

biophysics↗

bioRxiv2022

Acentrosomal spindles assemble from branching microtubule nucleation near chromosomes

Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstituted microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and found that chromosomes alone can form spindles. We visualized microtubule nucleation at chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. The initial branches nucleate near and towards kinetochores, helping explain how kinetochores might be efficiently captured. By depleting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient around them that spatially biases branching nucleation. In the presence of motors, these branched networks are organized into multipolar spindles.

biophysics↗

bioRxiv2020

Confinement size determines the architecture of Ran-induced microtubule networks

The organization of microtubules (MTs) within cells is critical for its internal organization during interphase and mitosis. During mitotic spindle assembly, MTs are made and organized around chromosomes in a process regulated by RanGTP. The role of RanGTP has been explored in Xenopus egg extracts, which are not limited by a cell membrane. Here, we investigated whether cell-sized confinements affect the assembly of RanGTP-induced MT networks in Xenopus egg extracts. We used microfluidics to encapsulate extract within monodisperse extract-in-oil droplets. Importantly, we find that the architecture of Ran-induced MT networks depends on the droplet diameter and the Ran concentration, and differs from structures formed in bulk extract. Our results highlight that both MT nucleation and physical confinement play critical roles in determining the spatial organization of the MT cytoskeleton. STATEMENT OF SIGNIFICANCEDuring cell division, chromosomes are segregated by the mitotic spindle, whose framework consists of up to hundreds of thousands of microtubules (MTs). Spindle MTs are generated via several pathways, one of which is regulated by RanGTP. Yet, how Ran-induced MTs self-organize within cell-sized confinement remains unclear. This work reports unexpected architectures of Ran-induced MT networks confined in cell-sized droplets, which depend on the droplet diameter and the RanGTP concentration. Thus, MT nucleation and confinement together give rise to specific MT network architectures, which are otherwise not observed in unconfined assays. The findings provide a simple strategy to engineer the architectures of MT networks and could have direct implications in nucleation-controlled soft material processing.

biophysics↗

bioRxiv2019

Molecular mechanism of microtubule nucleation from gamma-tubulin ring complex

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, {gamma}-tubulin ring complex ({gamma}-TuRC) has been identified, precisely how {gamma}-TuRC nucleates a MT remains poorly understood. Here we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis {gamma}-TuRC. We reveal a high {gamma}-/{beta}-tubulin affinity, which facilitates assembly of a MT from {gamma}-TuRC. Whereas spontaneous nucleation requires assembly of 8 {beta}-tubulins, nucleation from {gamma}-TuRC occurs efficiently with a cooperativity of 4 {beta}-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally-associated {beta}-tubulins drive {gamma}-TuRC into a closed conformation. Putative activation domain of CDK5RAP2, NME7 and TPX2 do not enhance {gamma}-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal {gamma}-/{beta}-tubulin interaction.

cell biology↗

bioRxiv2019

Spatiotemporal organization of branched microtubule networks

To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use Xenopus egg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and {gamma}-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/{gamma}-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks.\n\nImpact StatementA sequential reaction pathway involving TPX2, augmin and {gamma}-TuRC governs the assembly and architecture of branched microtubule networks.

biophysics↗

bioRxiv2018

Cell membranes resist flow

The fluid-mosaic model posits a liquid-like plasma membrane, which can flow in response to tension gradients. It is widely assumed that membrane flow transmits local changes in membrane tension across the cell in milliseconds. This conjectured signaling mechanism has been invoked to explain how cells coordinate changes in shape, motility, and vesicle fusion, but the underlying propagation has never been observed. Here we show that propagation of membrane tension occurs quickly in cell-attached blebs, but is largely suppressed in intact cells. The failure of tension to propagate in cells is explained by a fluid dynamical model that incorporates the flow resistance from cytoskeleton-bound transmembrane proteins. In primary endothelial cells, local increases in membrane tension lead only to local activation of mechanosensitive ion channels and to local vesicle fusion. Thus membrane tension is not a mediator of long-range intra-cellular signaling, but local variations in tension mediate distinct processes in sub-cellular domains.

biophysics↗

bioRxiv2017

Spatial gene drives and pushed genetic waves

Gene drives have the potential to rapidly replace a harmful wild-type allele with a gene drive allele engineered to have desired functionalities. However, an accidental or premature release of a gene drive construct to the natural environment could damage an ecosystem irreversibly. Thus, it is important to understand the spatiotemporal consequences of the super-Mendelian population genetics prior to potential applications. Here, we employ a reaction-diffusion model for sexually reproducing diploid organisms to study how a locally introduced gene drive allele spreads to replace the wild-type allele, even though it posses a selective disadvantage s > 0. Using methods developed by N. Barton and collaborators, we show that socially responsible gene drives require 0.5 < s < 0.697, a rather narrow range. In this \"pushed wave\" regime, the spatial spreading of gene drives will be initiated only when the initial frequency distribution is above a threshold profile called \"critical propagule\", which acts as a safeguard against accidental release. We also study how the spatial spread of the pushed wave can be stopped by making gene drives uniquely vulnerable (\"sensitizing drive\") in a way that is harmless for a wild-type allele. Finally, we show that appropriately sensitized drives in two dimensions can be stopped even by imperfect barriers perforated by a series of gaps.

synthetic biology↗