Transcription inhibition suppresses nuclear blebbing and rupture independently of nuclear rigidity.

Chromatin plays an essential role in the nuclear mechanical response and determining nuclear shape, which maintain nuclear compartmentalization and function. However, major genomic functions, such as transcription activity, might also impact cell nuclear shape via blebbing and rupture through their effects on chromatin structure and dynamics. To test this idea, we inhibited transcription with several RNA polymerase II inhibitors in wild-type cells and perturbed cells that presented increased nuclear blebbing. Transcription inhibition suppressed nuclear blebbing for several cell types, nuclear perturbations and transcription inhibitors. Furthermore, transcription inhibition suppressed nuclear bleb formation, bleb stabilization and bleb-based nuclear ruptures. Interestingly, transcription inhibition did not alter the histone H3 lysine 9 (H3K9) modification state, nuclear rigidity, and actin compression and contraction, which typically control nuclear blebbing. Polymer simulations suggested that RNA polymerase II motor activity within chromatin could drive chromatin motions that deform the nuclear periphery. Our data provide evidence that transcription inhibition suppresses nuclear blebbing and rupture, in a manner separate and distinct from chromatin rigidity.

Frequency Propagation: Multimechanism Learning in Nonlinear Physical Networks.

We introduce frequency propagation, a learning algorithm for nonlinear physical networks. In a resistive electrical circuit with variable resistors, an activation current is applied at a set of input nodes at one frequency and an error current is applied at a set of output nodes at another frequency. The voltage response of the circuit to these boundary currents is the superposition of an activation signal and an error signal whose coefficients can be read in different frequencies of the frequency domain. Each conductance is updated proportionally to the product of the two coefficients. The learning rule is local and proved to perform gradient descent on a loss function. We argue that frequency propagation is an instance of a multimechanism learning strategy for physical networks, be it resistive, elastic, or flow networks. Multimechanism learning strategies incorporate at least two physical quantities, potentially governed by independent physical mechanisms, to act as activation and error signals in the training process. Locally available information about these two signals is then used to update the trainable parameters to perform gradient descent. We demonstrate how earlier work implementing learning via chemical signaling in flow networks (Anisetti, Scellier, et al., 2023) also falls under the rubric of multimechanism learning.

Extracellular Vimentin as a Target Against SARS-CoV-2 Host Cell Invasion.

Infection of human cells by pathogens, including SARS-CoV-2, typically proceeds by cell surface binding to a crucial receptor. The primary receptor for SARS-CoV-2 is the angiotensin-converting enzyme 2 (ACE2), yet new studies reveal the importance of additional extracellular co-receptors that mediate binding and host cell invasion by SARS-CoV-2. Vimentin is an intermediate filament protein that is increasingly recognized as being present on the extracellular surface of a subset of cell types, where it can bind to and facilitate pathogens' cellular uptake. Biophysical and cell infection studies are done to determine whether vimentin might bind SARS-CoV-2 and facilitate its uptake. Dynamic light scattering shows that vimentin binds to pseudovirus coated with the SARS-CoV-2 spike protein, and antibodies against vimentin block in vitro SARS-CoV-2 pseudovirus infection of ACE2-expressing cells. The results are consistent with a model in which extracellular vimentin acts as a co-receptor for SARS-CoV-2 spike protein with a binding affinity less than that of the spike protein with ACE2. Extracellular vimentin may thus serve as a critical component of the SARS-CoV-2 spike protein-ACE2 complex in mediating SARS-CoV-2 cell entry, and vimentin-targeting agents may yield new therapeutic strategies for preventing and slowing SARS-CoV-2 infection.

Thermal Fluctuations of Singular Bar-Joint Mechanisms.

A bar-joint mechanism is a deformable assembly of freely rotating joints connected by stiff bars. Here we develop a formalism to study the equilibration of common bar-joint mechanisms with a thermal bath. When the constraints in a mechanism cease to be linearly independent, singularities can appear in its shape space, which is the part of its configuration space after discarding rigid motions. We show that the free-energy landscape of a mechanism at low temperatures is dominated by the neighborhoods of points that correspond to these singularities. We consider two example mechanisms with shape-space singularities and find that they are more likely to be found in configurations near the singularities than others. These findings are expected to help improve the design of nanomechanisms for various applications.

Dynamic Nuclear Structure Emerges from Chromatin Cross-Links and Motors.

The cell nucleus houses the chromosomes, which are linked to a soft shell of lamin protein filaments. Experiments indicate that correlated chromosome dynamics and nuclear shape fluctuations arise from motor activity. To identify the physical mechanisms, we develop a model of an active, cross-linked Rouse chain bound to a polymeric shell. System-sized correlated motions occur but require both motor activity and cross-links. Contractile motors, in particular, enhance chromosome dynamics by driving anomalous density fluctuations. Nuclear shape fluctuations depend on motor strength, cross-linking, and chromosome-lamina binding. Therefore, complex chromosome dynamics and nuclear shape emerge from a minimal, active chromosome-lamina system.

Spongelike Rigid Structures in Frictional Granular Packings.

We show how rigidity emerges in experiments on sheared two-dimensional frictional granular materials by using generalizations of two methods for identifying rigid structures. Both approaches, the force-based dynamical matrix and the topology-based rigidity percolation, agree with each other and identify similar rigid structures. As the system becomes jammed, at a critical contact number z_{c}=2.4±0.1, a rigid backbone interspersed with floppy, particle-filled holes of a broad range of sizes emerges, creating a spongelike morphology. While the pressure within rigid structures always exceeds the pressure outside the rigid structures, they are not identified with the force chains of shear jamming. These findings highlight the need to focus on mechanical stability arising through arch structures and hinges at the mesoscale.

The role of vimentin-nuclear interactions in persistent cell motility through confined spaces.

The ability of cells to move through small spaces depends on the mechanical properties of the cellular cytoskeleton and on nuclear deformability. In mammalian cells, the cytoskeleton is composed of three interacting, semi-flexible polymer networks: actin, microtubules, and intermediate filaments (IF). Recent experiments of mouse embryonic fibroblasts with and without vimentin have shown that the IF vimentin plays a role in confined cell motility. Here, we develop a minimal model of a cell moving through a microchannel that incorporates explicit effects of actin and vimentin and implicit effects of microtubules. Specifically, the model consists of a cell with an actomyosin cortex and a deformable cell nucleus and mechanical linkages between the two. By decreasing the amount of vimentin, we find that the cell speed increases for vimentin-null cells compared to cells with vimentin. The loss of vimentin increases nuclear deformation and alters nuclear positioning in the cell. Assuming nuclear positioning is a read-out for cell polarity, we propose a new polarity mechanism which couples cell directional motion with cytoskeletal strength and nuclear positioning and captures the abnormally persistent motion of vimentin-null cells, as observed in experiments. The enhanced persistence indicates that the vimentin-null cells are more controlled by the confinement and so less autonomous, relying more heavily on external cues than their wild-type counterparts. Our modeling results present a quantitative interpretation for recent experiments and have implications for understanding the role of vimentin in the epithelial-mesenchymal transition.

A spontaneous missense mutation in the chromodomain helicase DNA-binding protein 8 (CHD8) gene: a novel association with congenital myasthenic syndrome.

AIMS: Congenital myasthenic syndromes (CMS) are characterized by muscle weakness, ptosis and episodic apnoea. Mutations affect integral protein components of the neuromuscular junction (NMJ). Here we searched for the genetic basis of CMS in female monozygotic twins. METHODS: We employed whole-exome sequencing for mutation detection and Sanger sequencing for segregation analysis. Immunohistology was done with antibodies against CHD8, rapsyn, ß-catenin (ßCAT) and golgin on fi-bro-blasts, human and mouse muscle. We recorded superresolution images of the NMJ using 3D-structured illumination microscopy. RESULTS: We discovered a spontaneous missense mutation in CHD8 [chr14:g.21,884,051G>A, GRCh37.p11 | c.1732C>T, NM_00117062 | p.(R578C)], the gene encoding chromodomain helicase DNA-binding protein 8. This is the first missense mutation affecting Duplin, the short 110 kDa isoform of CHD8. It is known that CHD8/Duplin negatively regulates ßCAT signalling in the WNT pathway and plays a role in chromatin remodelling. Inactivating CHD8 mutations are associated with autism spectrum disorder and intellectual disability in combination with facial dysmorphism, overgrowth and macrocephalus. No muscle-specific phenotype has been reported to date. Co-immunostaining with rapsyn on human and mouse muscle revealed a strong presence of CHD8 at the NMJ being located towards the sarcoplasmic side of the rapsyn cluster, where it co-localizes with ßCAT. CONCLUSION: We hypothesize CHD8 to have a role in the maintenance of the structural integrity and function of the NMJ. Both patients benefited from treatment with 3,4-diaminopyridine, a reversible blocker of voltage-gated potassium channels at the nerve terminal that prolongs the action potential and increases acetylcholine release.

Loops versus lines and the compression stiffening of cells.

Both animal and plant tissue exhibit a nonlinear rheological phenomenon known as compression stiffening, or an increase in moduli with increasing uniaxial compressive strain. Does such a phenomenon exist in single cells, which are the building blocks of tissues? One expects an individual cell to compression soften since the semiflexible biopolymer-based cytoskeletal network maintains the mechanical integrity of the cell and in vitro semiflexible biopolymer networks typically compression soften. To the contrary, we find that mouse embryonic fibroblasts (mEFs) compression stiffen under uniaxial compression via atomic force microscopy studies. To understand this finding, we uncover several potential mechanisms for compression stiffening. First, we study a single semiflexible polymer loop modeling the actomyosin cortex enclosing a viscous medium modeled as an incompressible fluid. Second, we study a two-dimensional semiflexible polymer/fiber network interspersed with area-conserving loops, which are a proxy for vesicles and fluid-based organelles. Third, we study two-dimensional fiber networks with angular-constraining crosslinks, i.e. semiflexible loops on the mesh scale. In the latter two cases, the loops act as geometric constraints on the fiber network to help stiffen it via increased angular interactions. We find that the single semiflexible polymer loop model agrees well with the experimental cell compression stiffening finding until approximately 35% compressive strain after which bulk fiber network effects may contribute. We also find for the fiber network with area-conserving loops model that the stress-strain curves are sensitive to the packing fraction and size distribution of the area-conserving loops, thereby creating a mechanical fingerprint across different cell types. Finally, we make comparisons between this model and experiments on fibrin networks interlaced with beads as well as discuss implications for single cell compression stiffening at the tissue scale.

Small-scale demixing in confluent biological tissues.

Surface tension governed by differential adhesion can drive fluid particle mixtures to sort into separate regions, i.e., demix. Does the same phenomenon occur in confluent biological tissues? We begin to answer this question for epithelial monolayers with a combination of theory via a vertex model and experiments on keratinocyte monolayers. Vertex models are distinct from particle models in that the interactions between the cells are shape-based, as opposed to distance-dependent. We investigate whether a disparity in cell shape or size alone is sufficient to drive demixing in bidisperse vertex model fluid mixtures. Surprisingly, we observe that both types of bidisperse systems robustly mix on large lengthscales. On the other hand, shape disparity generates slight demixing over a few cell diameters, a phenomenon we term micro-demixing. This result can be understood by examining the differential energy barriers for neighbor exchanges (T1 transitions). Experiments with mixtures of wild-type and E-cadherin-deficient keratinocytes on a substrate are consistent with the predicted phenomenon of micro-demixing, which biology may exploit to create subtle patterning. The robustness of mixing at large scales, however, suggests that despite some differences in cell shape and size, progenitor cells can readily mix throughout a developing tissue until acquiring means of recognizing cells of different types.

Soft yet Sharp Interfaces in a Vertex Model of Confluent Tissue.

How can dense biological tissue maintain sharp boundaries between coexisting cell populations? We explore this question within a simple vertex model for cells, focusing on the role of topology and tissue surface tension. We show that the ability of cells to independently regulate adhesivity and tension, together with neighbor-based interaction rules, lets them support strikingly unusual interfaces. In particular, we show that mechanical- and fluctuation-based measurements of the effective surface tension of a cellular aggregate yield different results, leading to mechanically soft interfaces that are nevertheless extremely sharp.

Shape-Shifting Droplet Networks.

We consider a three-dimensional network of aqueous droplets joined by single lipid bilayers to form a cohesive, tissuelike material. The droplets in these networks can be programed to have distinct osmolarities so that osmotic gradients generate internal stresses via local fluid flows to cause the network to change shape. We discover, using molecular dynamics simulations, a reversible folding-unfolding process by adding an osmotic interaction with the surrounding environment which necessarily evolves dynamically as the shape of the network changes. This discovery is the next important step towards osmotic robotics in this system. We also explore analytically and numerically how the networks become faceted via buckling and how quasi-one-dimensional networks become three dimensional.

Rigid Cluster Decomposition Reveals Criticality in Frictional Jamming.

We study the nature of the frictional jamming transition within the framework of rigidity percolation theory. Slowly sheared frictional packings are decomposed into rigid clusters and floppy regions with a generalization of the pebble game including frictional contacts. Our method suggests a second-order transition controlled by the emergence of a system-spanning rigid cluster accompanied by a critical cluster size distribution. Rigid clusters also correlate with common measures of rigidity. We contrast this result with frictionless jamming, where the rigid cluster size distribution is noncritical.

On the modeling of endocytosis in yeast.

The cell membrane deforms during endocytosis to surround extracellular material and draw it into the cell. Results of experiments on endocytosis in yeast show general agreement that 1) actin polymerizes into a network of filaments exerting active forces on the membrane to deform it, and 2) the large-scale membrane deformation is tubular in shape. In contrast, there are three competing proposals for precisely how the actin filament network organizes itself to drive the deformation. We use variational approaches and numerical simulations to address this competition by analyzing a meso-scale model of actin-mediated endocytosis in yeast. The meso-scale model breaks up the invagination process into three stages: 1) initiation, where clathrin interacts with the membrane via adaptor proteins; 2) elongation, where the membrane is then further deformed by polymerizing actin filaments; and 3) pinch-off. Our results suggest that the pinch-off mechanism may be assisted by a pearling-like instability. We rule out two of the three competing proposals for the organization of the actin filament network during the elongation stage. These two proposals could be important in the pinch-off stage, however, where additional actin polymerization helps break off the vesicle. Implications and comparisons with earlier modeling of endocytosis in yeast are discussed.

Energy barriers and cell migration in densely packed tissues.

Recent observations demonstrate that confluent tissues exhibit features of glassy dynamics, such as caging behavior and dynamical heterogeneities, although it has remained unclear how single-cell properties control this behavior. Here we develop numerical and theoretical models to calculate energy barriers to cell rearrangements, which help govern cell migration in cell monolayers. In contrast to work on sheared foams, we find that energy barrier heights are exponentially distributed and depend systematically on the cell's number of neighbors. Based on these results, we predict glassy two-time correlation functions for cell motion, with a timescale that increases rapidly as cell activity decreases. These correlation functions are used to construct simple random walks that reproduce the caging behavior observed for cell trajectories in experiments. This work provides a theoretical framework for predicting collective motion of cells in wound-healing, embryogenesis and cancer tumorogenesis.

Cocaine diminishes functional network robustness and destabilizes the energy landscape of neuronal activity in the medial prefrontal cortex.

We present analysis of neuronal activity recordings from a subset of neurons in the medial prefrontal cortex of rats before and after the administration of cocaine. Using an underlying modern Hopfield model as a description for the neuronal network, combined with a machine learning approach, we compute the underlying functional connectivity of the neuronal network. We find that the functional connectivity changes after the administration of cocaine with both functional-excitatory and functional-inhibitory neurons being affected. Using conventional network analysis, we find that the diameter of the graph, or the shortest length between the two most distant nodes, increases with cocaine, suggesting that the neuronal network is less robust. We also find that the betweenness centrality scores for several of the functional-excitatory and functional-inhibitory neurons decrease significantly, while other scores remain essentially unchanged, to also suggest that the neuronal network is less robust. Finally, we study the distribution of neuronal activity and relate it to energy to find that cocaine drives the neuronal network towards destabilization in the energy landscape of neuronal activation. While this destabilization is presumably temporary given one administration of cocaine, perhaps this initial destabilization indicates a transition towards a new stable state with repeated cocaine administration. However, such analyses are useful more generally to understand how neuronal networks respond to perturbations.

Vimentin promotes collective cell migration through collagen networks via increased matrix remodeling and spheroid fluidity.

The intermediate filament (IF) protein vimentin is associated with many diseases with phenotypes of enhanced cellular migration and aggressive invasion through the extracellular matrix (ECM) of tissues, but vimentin's role in in-vivo cell migration is still largely unclear. Vimentin is important for proper cellular adhesion and force generation, which are critical to cell migration; yet the vimentin cytoskeleton also hinders the ability of cells to squeeze through small pores in ECM, resisting migration. To identify the role of vimentin in collective cell migration, we generate spheroids of wide-type and vimentin-null mouse embryonic fibroblasts (mEFs) and embed them in a 3D collagen matrix. We find that loss of vimentin significantly impairs the ability of the spheroid to collectively expand through collagen networks and remodel the collagen network. Traction force analysis reveals that vimentin null spheroids exert less contractile force than their wild-type counterparts. In addition, spheroids made of mEFs with only vimentin unit length filaments (ULFs) exhibit similar behavior as vimentin-null spheroids, suggesting filamentous vimentin is required to promote 3D collective cell migration. We find the vimentin-mediated collective cell expansion is dependent on matrix metalloproteinase (MMP) degradation of the collagen matrix. Further, 3D vertex model simulation of spheroid and embedded ECM indicates that wild-type spheroids behave more fluid-like, enabling more active pulling and reconstructing the surrounding collagen network. Altogether, these results signify that VIF plays a critical role in enhancing migratory persistence in 3D matrix environments through MMP transportation and tissue fluidity.

Enhanced extracellular matrix remodeling due to embedded spheroid fluidization.

Tumor spheroids are in vitro three-dimensional, cellular collectives consisting of cancerous cells. Embedding these spheroids in an in vitro fibrous environment, such as a collagen network, to mimic the extracellular matrix (ECM) provides an essential platform to quantitatively investigate the biophysical mechanisms leading to tumor invasion of the ECM. To understand the mechanical interplay between tumor spheroids and the ECM, we computationally construct and study a three-dimensional vertex model for a tumor spheroid that is mechanically coupled to a cross-linked network of fibers. In such a vertex model, cells are represented as deformable polyhedrons that share faces. Some fraction of the boundary faces of the tumor spheroid contain linker springs connecting the center of the boundary face to the nearest node in the fiber network. As these linker springs actively contract, the fiber network remodels. By toggling between fluid-like and solid-like spheroids via changing the dimensionless cell shape index, we find that the spheroid rheology affects the remodeling of the fiber network. More precisely, fluid-like spheroids displace the fiber network more on average near the vicinity of the spheroid than solid-like spheroids. We also find more densification of the fiber network near the spheroid for the fluid-like spheroids. These spheroid rheology-dependent effects are the result of cellular motility due to active cellular rearrangements that emerge over time in the fluid-like spheroids to generate spheroid shape fluctuations. Our results uncover intricate morphological-mechanical interplay between an embedded spheroid and its surrounding fiber network with both spheroid contractile strength and spheroid shape fluctuations playing important roles in the pre-invasion stages of tumor invasion.

How cells wrap around virus-like particles using extracellular filamentous protein structures.

Nanoparticles, such as viruses, can enter cells via endocytosis. During endocytosis, the cell surface wraps around the nanoparticle to effectively eat it. Prior focus has been on how nanoparticle size and shape impacts endocytosis. However, inspired by the noted presence of extracellular vimentin affecting viral and bacteria uptake, as well as the structure of coronaviruses, we construct a computational model in which both the cell-like construct and the virus-like construct contain filamentous protein structures protruding from their surfaces. We then study the impact of these additional degrees of freedom on viral wrapping. We find that cells with an optimal density of filamentous extracellular components (ECCs) are more likely to be infected as they uptake the virus faster and use relatively less cell surface area per individual virus. At the optimal density, the cell surface folds around the virus, and folds are faster and more efficient at wrapping the virus than crumple-like wrapping. We also find that cell surface bending rigidity helps generate folds, as bending rigidity enhances force transmission across the surface. However, changing other mechanical parameters, such as the stretching stiffness of filamentous ECCs or virus spikes, can drive crumple-like formation of the cell surface. We conclude with the implications of our study on the evolutionary pressures of virus-like particles, with a particular focus on the cellular microenvironment that may include filamentous ECCs.