Cell cycle-dependent centrosome clustering precedes proplatelet formation.

Platelet-producing megakaryocytes (MKs) primarily reside in the bone marrow, where they duplicate their DNA content with each cell cycle resulting in polyploid cells with an intricate demarcation membrane system. While key elements of the cytoskeletal reorganizations during proplatelet formation have been identified, what initiates the release of platelets into vessel sinusoids remains largely elusive. Using a cell cycle indicator, we observed a unique phenomenon, during which amplified centrosomes in MKs underwent clustering following mitosis, closely followed by proplatelet formation, which exclusively occurred in G1 of interphase. Forced cell cycle arrest in G1 increased proplatelet formation not only in vitro but also in vivo following short-term starvation of mice. We identified that inhibition of the centrosomal protein kinesin family member C1 (KIFC1) impaired clustering and subsequent proplatelet formation, while KIFC1-deficient mice exhibited reduced platelet counts. In summary, we identified KIFC1- and cell cycle-mediated centrosome clustering as an important initiator of proplatelet formation from MKs.

Focal adhesions are controlled by microtubules through local contractility regulation.

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

Helical motors and formins synergize to compact chiral filopodial bundles: A theoretical perspective.

Chiral actin bundles have been shown to play an important role in cell dynamics, but our understanding of the molecular mechanisms which combine to generate chirality remains incomplete. To address this, we numerically simulate a crosslinked filopodial bundle under the actions of helical myosin motors and/or formins and examine the collective buckling and twisting of the actin bundle. We first show that a number of proposed mechanisms to buckle polymerizing actin bundles without motor activity fail under biologically-realistic parameters. We then demonstrate that a simplified model of myosin spinning action at the bundle base effectively "braids" the bundle, but cannot control compaction at the fiber tips. Finally, we show that formin-mediated polymerization and motor activity can act synergitically to compact filopodium bundles, as motor activity bends filaments into shapes that activate twist forces induced by formins. Stochastic fluctuations of actin polymerization rates and slower cross linking dynamics both increase buckling and decrease compaction. We discuss implications of our findings for mechanisms of cytoskeletal chirality.

Friction patterns guide actin network contraction.

The shape of cells is the outcome of the balance of inner forces produced by the actomyosin network and the resistive forces produced by cell adhesion to their environment. The specific contributions of contractile, anchoring and friction forces to network deformation rate and orientation are difficult to disentangle in living cells where they influence each other. Here, we reconstituted contractile actomyosin networks in vitro to study specifically the role of the friction forces between the network and its anchoring substrate. To modulate the magnitude and spatial distribution of friction forces, we used glass or lipids surface micropatterning to control the initial shape of the network. We adapted the concentration of Nucleating Promoting Factor on each surface to induce the assembly of actin networks of similar densities and compare the deformation of the network toward the centroid of the pattern shape upon myosin-induced contraction. We found that actin network deformation was faster and more coordinated on lipid bilayers than on glass, showing the resistance of friction to network contraction. To further study the role of the spatial distribution of these friction forces, we designed heterogeneous micropatterns made of glass and lipids. The deformation upon contraction was no longer symmetric but biased toward the region of higher friction. Furthermore, we showed that the pattern of friction could robustly drive network contraction and dominate the contribution of asymmetric distributions of myosins. Therefore, we demonstrate that during contraction, both the active and resistive forces are essential to direct the actin network deformation.

Recycling of the actin monomer pool limits the lifetime of network turnover.

Intracellular organization is largely mediated by actin turnover. Cellular actin networks continuously assemble and disassemble, while maintaining their overall appearance. This behavior, called "dynamic steady state," allows cells to sense and adapt to their environment. However, how structural stability can be maintained during the constant turnover of a limited actin monomer pool is poorly understood. To answer this question, we developed an experimental system where polystyrene beads are propelled by an actin comet in a microwell containing a limited amount of components. We used the speed and the size of the actin comet tails to evaluate the system's monomer consumption and its lifetime. We established the relative contribution of actin assembly, disassembly, and recycling for a bead movement over tens of hours. Recycling mediated by cyclase-associated protein (CAP) is the key step in allowing the reuse of monomers for multiple assembly cycles. ATP supply and protein aging are also factors that limit the lifetime of actin turnover. This work reveals the balancing mechanism for long-term network assembly with a limited amount of building blocks.

Crawling, waving, inch worming, dilating, and pivoting mechanics of migrating cells: Lessons from Ken Jacobson.

Research on the locomotion of single cells on hard, flat surfaces brought insight into the mechanisms of leading-edge protrusion, spatially graded adhesion, front-rear coordination, and how intracellular and traction forces are harnessed to execute various maneuvers. Here, we highlight how, by studying a variety of cell types, shapes, and movements, Ken Jacobson and his collaborators made several discoveries that triggered the mechanistic understanding of cell motility. We then review the recent advancements and current perspectives in this field.

Electric field-guided collective motility initiation of large epidermal cell groups.

Recent research has elucidated mechanochemical pathways of single cell polarization, but much less is known about collective motility initiation in adhesive cell groups. We used galvanotactic assays of zebrafish keratocyte cell groups, pharmacological perturbations, electric field switches, particle imaging velocimetry, and cell tracking to show that large cell groups initiate motility in minutes toward the cathode. Interestingly, while PI3K-inhibited single cells are biased toward the anode, inhibiting PI3K does not affect the cathode-directed cell group migration. We observed that control groups had the fastest cathode-migrating cell at the front, while the front cells in PI3K-inhibited groups were the slowest. Both control and PI3K-inhibited groups rapidly repolarized when the electric field direction was reversed, and the group migration continued after the electric field was switched off. Inhibiting myosin disrupted the cohesiveness of keratocyte groups and abolished the collective directionality and ability to switch direction when the electric field is reversed. Our data are consistent with a model according to which cells in the group sense the electric field individually and mechanical integration of the cells results in coherent group motility.

Actin polymerisation and crosslinking drive left-right asymmetry in single cell and cell collectives.

Deviations from mirror symmetry in the development of bilateral organisms are common but the mechanisms of initial symmetry breaking are insufficiently understood. The actin cytoskeleton of individual cells self-organises in a chiral manner, but the molecular players involved remain essentially unidentified and the relationship between chirality of an individual cell and cell collectives is unclear. Here, we analysed self-organisation of the chiral actin cytoskeleton in individual cells on circular or elliptical patterns, and collective cell alignment in confined microcultures. Screening based on deep-learning analysis of actin patterns identified actin polymerisation regulators, depletion of which suppresses chirality (mDia1) or reverses chirality direction (profilin1 and CapZß). The reversed chirality  is mDia1-independent but requires the function of actin-crosslinker α-actinin1. A robust correlation between the effects of a variety of actin assembly regulators on chirality of individual cells and cell collectives is revealed. Thus, actin-driven cell chirality may underlie tissue and organ asymmetry.

Mechanosensation: Capping actin filaments for robustness.

Actin networks adapt to resistance by becoming denser. A recent investigation shows that this mechanosensation relies on a force-sensitive mechanical ratchet of capping actin filaments to reorganize the network. This and other mechanical feedback mechanisms make actin-based protrusion amazingly robust.

F-actin bending facilitates net actomyosin contraction By inhibiting expansion with plus-end-located myosin motors.

Contraction of actomyosin networks underpins important cellular processes including motility and division. The mechanical origin of actomyosin contraction is not fully-understood. We investigate whether contraction arises on the scale of individual filaments, without needing to invoke network-scale interactions. We derive discrete force-balance and continuum partial differential equations for two symmetric, semi-flexible actin filaments with an attached myosin motor. Assuming the system exists within a homogeneous background material, our method enables computation of the stress tensor, providing a measure of contractility. After deriving the model, we use a combination of asymptotic analysis and numerical solutions to show how F-actin bending facilitates contraction on the scale of two filaments. Rigid filaments exhibit polarity-reversal symmetry as the motor travels from the minus to plus-ends, such that contractile and expansive components cancel. Filament bending induces a geometric asymmetry that brings the filaments closer to parallel as a myosin motor approaches their plus-ends, decreasing the effective spring force opposing motor motion. The reduced spring force enables the motor to move faster close to filament plus-ends, which reduces expansive stress and gives rise to net contraction. Bending-induced geometric asymmetry provides both new understanding of actomyosin contraction mechanics, and a hypothesis that can be tested in experiments.

CellDynaMo-stochastic reaction-diffusion-dynamics model: Application to search-and-capture process of mitotic spindle assembly.

We introduce a Stochastic Reaction-Diffusion-Dynamics Model (SRDDM) for simulations of cellular mechanochemical processes with high spatial and temporal resolution. The SRDDM is mapped into the CellDynaMo package, which couples the spatially inhomogeneous reaction-diffusion master equation to account for biochemical reactions and molecular transport within the Langevin Dynamics (LD) framework to describe dynamic mechanical processes. This computational infrastructure allows the simulation of hours of molecular machine dynamics in reasonable wall-clock time. We apply SRDDM to test performance of the Search-and-Capture of mitotic spindle assembly by simulating, in three spatial dimensions, dynamic instability of elastic microtubules anchored in two centrosomes, movement and deformations of geometrically realistic centromeres with flexible kinetochores and chromosome arms. Furthermore, the SRDDM describes the mechanics and kinetics of Ndc80 linkers mediating transient attachments of microtubules to the chromosomal kinetochores. The rates of these attachments and detachments depend upon phosphorylation states of the Ndc80 linkers, which are regulated in the model by explicitly accounting for the reactions of Aurora A and B kinase enzymes undergoing restricted diffusion. We find that there is an optimal rate of microtubule-kinetochore detachments which maximizes the accuracy of the chromosome connections, that adding chromosome arms to kinetochores improve the accuracy by slowing down chromosome movements, that Aurora A and kinetochore deformations have a small positive effect on the attachment accuracy, and that thermal fluctuations of the microtubules increase the rates of kinetochore capture and also improve the accuracy of spindle assembly.

Non-centrosomal microtubules at kinetochores promote rapid chromosome biorientation during mitosis in human cells.

Proper segregation of chromosomes during mitosis depends on "amphitelic attachments"-load-bearing connections of sister kinetochores to the opposite spindle poles via bundles of microtubules, termed as the "K-fibers." Current models of spindle assembly assume that K-fibers arise largely from stochastic capture of microtubules, which occurs at random times and locations and independently at sister kinetochores. We test this assumption by following the movements of all kinetochores in human cells and determine that most amphitelic attachments form synchronously at a specific stage of spindle assembly and within a spatially distinct domain. This biorientation domain is enriched in bundles of antiparallel microtubules, and perturbation of microtubule bundling changes the temporal and spatial dynamics of amphitelic attachment formation. Structural analyses indicate that interactions of kinetochores with microtubule bundles are mediated by non-centrosomal short microtubules that emanate from most kinetochores during early prometaphase. Computational analyses suggest that momentous molecular motor-driven interactions with antiparallel bundles rapidly convert these short microtubules into nascent K-fibers. Thus, load-bearing connections to the opposite spindle poles form simultaneously on sister kinetochores. In contrast to the uncoordinated sequential attachments of sister kinetochores expected in stochastic models of spindle assembly, our model envisions the formation of amphitelic attachments as a deterministic process in which the chromosomes connect with the spindle poles synchronously at a specific stage of spindle assembly and at a defined location determined by the spindle architecture. Experimental analyses of changes in the kinetochore behavior in cells with perturbed activity of molecular motors CenpE and dynein confirm the predictive power of the model.

Interplay between Brownian motion and cross-linking controls bundling dynamics in actin networks.

Morphology changes in cross-linked actin networks are important in cell motility, division, and cargo transport. Here, we study the transition from a weakly cross-linked network of actin filaments to a heavily cross-linked network of actin bundles through microscopic Brownian dynamics simulations. We show that this transition occurs in two stages: first, a composite bundle network of small and highly aligned bundles evolves from cross-linking of individual filaments and, second, small bundles coalesce into the clustered bundle state. We demonstrate that Brownian motion speeds up the first stage of this process at a faster rate than the second. We quantify the time to reach the composite bundle state and show that it strongly increases as the mesh size increases only when the concentration of cross-links is small and that it remains roughly constant if we decrease the relative ratio of cross-linkers as we increase the actin concentration. Finally, we examine the dependence of the bundling timescale on filament length, finding that shorter filaments bundle faster because they diffuse faster.

Mechanical Torque Promotes Bipolarity of the Mitotic Spindle Through Multi-centrosomal Clustering.

Intracellular forces shape cellular organization and function. One example is the mitotic spindle, a cellular machine consisting of multiple chromosomes and centrosomes which interact via dynamic microtubule filaments and motor proteins, resulting in complicated spatially dependent forces. For a cell to divide properly, it is important for the spindle to be bipolar, with chromosomes at the center and multiple centrosomes clustered into two 'poles' at opposite sides of the chromosomes. Experimental observations show that in unhealthy cells, the spindle can take on a variety of patterns. What forces drive each of these patterns? It is known that attraction between centrosomes is key to bipolarity, but what prevents the centrosomes from collapsing into a monopolar configuration? Here, we explore the hypothesis that torque rotating chromosome arms into orientations perpendicular to the centrosome-centromere vector promotes spindle bipolarity. To test this hypothesis, we construct a pairwise-interaction model of the spindle. On a continuum version of the model, an integro-PDE system, we perform linear stability analysis and construct numerical solutions which display a variety of spatial patterns. We also simulate a discrete particle model resulting in a phase diagram that confirms that the spindle bipolarity emerges most robustly with torque. Altogether, our results suggest that rotational forces may play an important role in dictating spindle patterning.

Simulations of dynamically cross-linked actin networks: Morphology, rheology, and hydrodynamic interactions.

Cross-linked actin networks are the primary component of the cell cytoskeleton and have been the subject of numerous experimental and modeling studies. While these studies have demonstrated that the networks are viscoelastic materials, evolving from elastic solids on short timescales to viscous fluids on long ones, questions remain about the duration of each asymptotic regime, the role of the surrounding fluid, and the behavior of the networks on intermediate timescales. Here we perform detailed simulations of passively cross-linked non-Brownian actin networks to quantify the principal timescales involved in the elastoviscous behavior, study the role of nonlocal hydrodynamic interactions, and parameterize continuum models from discrete stochastic simulations. To do this, we extend our recent computational framework for semiflexible filament suspensions, which is based on nonlocal slender body theory, to actin networks with dynamic cross linkers and finite filament lifetime. We introduce a model where the cross linkers are elastic springs with sticky ends stochastically binding to and unbinding from the elastic filaments, which randomly turn over at a characteristic rate. We show that, depending on the parameters, the network evolves to a steady state morphology that is either an isotropic actin mesh or a mesh with embedded actin bundles. For different degrees of bundling, we numerically apply small-amplitude oscillatory shear deformation to extract three timescales from networks of hundreds of filaments and cross linkers. We analyze the dependence of these timescales, which range from the order of hundredths of a second to the actin turnover time of several seconds, on the dynamic nature of the links, solvent viscosity, and filament bending stiffness. We show that the network is mostly elastic on the short time scale, with the elasticity coming mainly from the cross links, and viscous on the long time scale, with the effective viscosity originating primarily from stretching and breaking of the cross links. We show that the influence of nonlocal hydrodynamic interactions depends on the network morphology: for homogeneous meshworks, nonlocal hydrodynamics gives only a small correction to the viscous behavior, but for bundled networks it both hinders the formation of bundles and significantly lowers the resistance to shear once bundles are formed. We use our results to construct three-timescale generalized Maxwell models of the networks.

Supracellular organization confers directionality and mechanical potency to migrating pairs of cardiopharyngeal progenitor cells.

Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that multicellular arrangements confer biochemical and biomechanical properties contributing to tissue-scale organization. The Ciona cardiopharyngeal progenitors provide the simplest model of collective cell migration, with cohesive bilateral cell pairs polarized along the leader-trailer migration path while moving between the ventral epidermis and trunk endoderm. We use the Cellular Potts Model to computationally probe the distributions of forces consistent with shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we identify cardiopharyngeal progenitors as the simplest cell collective maintaining supracellular polarity with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy to align collective polarity with the direction of migration, as observed with three or more cells in silico and in vivo. Our approach reveals emerging properties of the migrating collective: cell pairs are more persistent, migrating longer distances, and presumably with higher accuracy. Simulations suggest that cell pairs can overcome mechanical resistance of the trunk endoderm more effectively when they are polarized collectively. We propose that polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.

Protein friction and filament bending facilitate contraction of disordered actomyosin networks.

We use mathematical modeling and computation to investigate how protein friction facilitates contraction of disordered actomyosin networks. We simulate two-dimensional networks using an agent-based model, consisting of a system of force-balance equations for myosin motor proteins and semiflexible actin filaments. A major advantage of our approach is that it enables direct calculation of the network stress tensor, which provides a quantitative measure of contractility. Exploiting this, we use repeated simulations of disordered networks to confirm that both protein friction and actin filament bending are required for contraction. We then use simulations of elementary two-filament systems to show that filament bending flexibility can facilitate contraction on the microscopic scale. Finally, we show that actin filament turnover is necessary to sustain contraction and prevent filament aggregation. Simulations with and without turnover also exhibit contractile pulses. However, these pulses are aperiodic, suggesting that periodic pulsation can only arise because of additional regulatory mechanisms or more complex mechanical behavior.

Stress fibres are embedded in a contractile cortical network.

Contractile actomyosin networks are responsible for the production of intracellular forces. There is increasing evidence that bundles of actin filaments form interconnected and interconvertible structures with the rest of the network. In this study, we explored the mechanical impact of these interconnections on the production and distribution of traction forces throughout the cell. By using a combination of hydrogel micropatterning, traction force microscopy and laser photoablation, we measured the relaxation of traction forces in response to local photoablations. Our experimental results and modelling of the mechanical response of the network revealed that bundles were fully embedded along their entire length in a continuous and contractile network of cortical filaments. Moreover, the propagation of the contraction of these bundles throughout the entire cell was dependent on this embedding. In addition, these bundles appeared to originate from the alignment and coalescence of thin and unattached cortical actin filaments from the surrounding mesh.