Disrupted Stiffness Ratio Alters Nuclear Mechanosensing.

The ability of endothelial cells to sense and respond to dynamic changes in blood flow is critical for vascular homeostasis and cardiovascular health. The mechanical and geometric properties of the nuclear and cytoplasmic compartments affect mechanotransduction. We hypothesized that alterations to these parameters have resulting mechanosensory consequences. Using atomic force microscopy and mathematical modeling, we assessed how the nuclear and cytoplasmic compartment stiffnesses modulate shear stress transfer to the nucleus within aging endothelial cells. Our computational studies revealed that the critical parameter controlling shear transfer is not the individual mechanics of these compartments, but the stiffness ratio between them. Replicatively aged cells had a reduced stiffness ratio, attenuating shear transfer, while the ratio was not altered in a genetic model of accelerated aging. We provide a theoretical framework suggesting that dysregulation of the shear stress response can be uniquely imparted by relative mechanical changes in subcellular compartments.

Force Production by a Bundle of Growing Actin Filaments Is Limited by Its Mechanical Properties.

Bundles of actin filaments are central to a large variety of cellular structures such as filopodia, stress fibers, cytokinetic rings, and focal adhesions. The mechanical properties of these bundles are critical for proper force transmission and force bearing. Previous mathematical modeling efforts have focused on bundles' rigidity and shape. However, it remains unknown how bundle length and buckling are controlled by external physical factors. In this work, we present a biophysical model for dynamic bundles of actin filaments submitted to an external load. In combination with in vitro motility assays of beads coated with formins, our model allowed us to characterize conditions for bead movement and bundle buckling. From the deformation profiles, we determined key biophysical properties of tethered actin bundles such as their rigidity and filament density.

Plastic Deformation and Fragmentation of Strained Actin Filaments.

The assembly of actin filaments and filament networks generate forces that drive cell and vesicle movement. These structures and the comprising actin filaments must be mechanically stable to sustain these forces and maintain their structural integrity. Filaments in these dynamic structures must also be disassembled to recycle and replenish the pool of actin monomers available for polymerization. Actin-severing proteins such as cofilin and contractile myosin motor proteins fragment these nominally stable structures. We developed a mesoscopic-length-scale actin filament model to investigate force-induced filament fragmentation. We show that fragmentation in our model occurs at curvatures similar to previous measurements of fragmentation within (cofil)actin and actin-cofilactin boundaries. Boundaries between bare and cofilin-decorated segments are brittle and fragment at small bending and twisting deformations. Extending filaments disperses strain uniformly over subunit interfaces, and filaments fragment with no detectable partial rupture or plastic deformation. In contrast, bending or twisting filaments imposes nonuniform interface strain and leads to partial interface rupture, accelerating filament fragmentation. As a result, the rupture force under compressive loads is an order of magnitude lower than under tensile loads. Partial interface rupture may be a primary mechanism of accelerating actin filament fragmentation by other actin-destabilizing proteins.

Intraluminal Ultrasonic Palpation Imaging Technique Revisited for Anisotropic Characterization of Healthy and Atherosclerotic Coronary Arteries: A Feasibility Study.

Accurate mechanical characterization of coronary atherosclerotic lesions remains essential for the in vivo detection of vulnerable plaques. Using intravascular ultrasound strain measurements and based on the mechanical response of a circular and concentric vascular model, E. I. Céspedes, C. L. de Korte and A. F. van der Steen developed an elasticity-palpography technique in 2000 to estimate the apparent stress-strain modulus palpogram of the thick subendoluminal arterial wall layer. More recently, this approach was improved by our group to consider the real anatomic shape of the vulnerable plaque. Even though these two studies highlighted original and promising approaches for improving the detection of vulnerable plaques, they did not overcome a main limitation related to the anisotropic mechanical behavior of the vascular tissue. The present study was therefore designed to extend these previous approaches by considering the orthotropic mechanical properties of the arterial wall and lesion constituents. Based on the continuum mechanics theory prescribing the strain field, an elastic anisotropy index was defined. This new anisotropic elasticity-palpography technique was successfully applied to characterize ten coronary plaque and one healthy vessel geometries of patients imaged in vivo with intravascular ultrasound. The results revealed that the anisotropy index-palpograms were estimated with a good accuracy (with a mean relative error of 26.8 ± 48.8%) compared with ground true solutions.

Adaptive Response of Actin Bundles under Mechanical Stress.

Actin is one of the main components of the architecture of cells. Actin filaments form different polymer networks with versatile mechanical properties that depend on their spatial organization and the presence of cross-linkers. Here, we investigate the mechanical properties of actin bundles in the absence of cross-linkers. Bundles are polymerized from the surface of mDia1-coated latex beads, and deformed by manipulating both ends through attached beads held by optical tweezers, allowing us to record the applied force. Bundle properties are strikingly different from the ones of a homogeneous isotropic beam. Successive compression and extension leads to a decrease in the buckling force that we attribute to the bundle remaining slightly curved after the first deformation. Furthermore, we find that the bundle is solid, and stiff to bending, along the long axis, whereas it has a liquid and viscous behavior in the transverse direction. Interpretation of the force curves using a Maxwell visco-elastic model allows us to extract the bundle mechanical parameters and confirms that the bundle is composed of weakly coupled filaments. At short times, the bundle behaves as an elastic material, whereas at long times, filaments flow in the longitudinal direction, leading to bundle restructuring. Deviations from the model reveal a complex adaptive rheological behavior of bundles. Indeed, when allowed to anneal between phases of compression and extension, the bundle reinforces. Moreover, we find that the characteristic visco-elastic time is inversely proportional to the compression speed. Actin bundles are therefore not simple force transmitters, but instead, complex mechano-transducers that adjust their mechanics to external stimulation. In cells, where actin bundles are mechanical sensors, this property could contribute to their adaptability.

Actin Filament Strain Promotes Severing and Cofilin Dissociation.

Computational and structural studies have been indispensable in investigating the molecular origins of actin filament mechanical properties and modulation by the regulatory severing protein cofilin. All-atom molecular dynamics simulations of cofilactin filament structures determined by electron cryomicroscopy reveal how cofilin enhances the bending and twisting compliance of actin filaments. Continuum mechanics models suggest that buckled cofilactin filaments localize elastic energy at boundaries between bare and cofilin-decorated segments because of their nonuniform elasticity, thereby accelerating filament severing. Here, we develop mesoscopic length-scale (cofil)actin filament models and evaluate the effects of compressive and twisting loads on strain energy distribution at specific interprotein interfaces. The models reliably capture the filament bending and torsional rigidities and intersubunit torsional flexibility measured experimentally with purified protein components. Buckling is predicted to enhance cofilactin filament severing with minimal effects on cofilin occupancy, whereas filament twisting enhances cofilin dissociation without compromising filament integrity. Preferential severing at actin-cofilactin boundaries of buckled filaments is more prominent than predicted by continuum models because of the enhanced spatial resolution. The models developed here will be valuable for evaluating the effects of filament shape deformations on filament stability and interactions with regulatory proteins, and analysis of single filament manipulation assays.

The size-speed-force relationship governs migratory cell response to tumorigenic factors.

Tumor development progresses through a complex path of biomechanical changes leading first to cell growth and contraction and then cell deadhesion, scattering, and invasion. Tumorigenic factors may act specifically on one of these steps or have a wider spectrum of actions, leading to a variety of effects and thus sometimes to apparent contradictory outcomes. Here we used micropatterned lines of collagen type I/fibronectin on deformable surfaces to standardize cell behavior and measure simultaneously cell size, speed of motion and magnitude of the associated traction forces at the level of a single cell. We analyzed and compared the normal human breast cell line MCF10A in control conditions and in response to various tumorigenic factors. In all conditions, a wide range of biomechanical properties was identified. Despite this heterogeneity, normal and transformed motile cells followed a common trend whereby size and contractile forces were negatively correlated with cell speed. Some tumorigenic factors, such as activation of ErbB2 or loss of the ßsubunit of casein kinase 2, shifted the whole population toward a faster speed and lower contractility state. Treatment with transforming growth factor ß induced some cells to adopt opposing behaviors such as extremely high versus extremely low contractility. Thus tumor transformation amplified preexisting population heterogeneity and led some cells to exhibit biomechanical properties that were more extreme than those observed with normal cells.

Architecture and Connectivity Govern Actin Network Contractility.

Actomyosin contractility plays a central role in a wide range of cellular processes, including the establishment of cell polarity, cell migration, tissue integrity, and morphogenesis during development. The contractile response is variable and depends on actomyosin network architecture and biochemical composition. To determine how this coupling regulates actomyosin-driven contraction, we used a micropatterning method that enables the spatial control of actin assembly. We generated a variety of actin templates and measured how defined actin structures respond to myosin-induced forces. We found that the same actin filament crosslinkers either enhance or inhibit the contractility of a network, depending on the organization of actin within the network. Numerical simulations unified the roles of actin filament branching and crosslinking during actomyosin contraction. Specifically, we introduce the concept of "network connectivity" and show that the contractions of distinct actin architectures are described by the same master curve when considering their degree of connectivity. This makes it possible to predict the dynamic response of defined actin structures to transient changes in connectivity. We propose that, depending on the connectivity and the architecture, network contraction is dominated by either sarcomeric-like or buckling mechanisms. More generally, this study reveals how actin network contractility depends on its architecture under a defined set of biochemical conditions.

Mechanical heterogeneity favors fragmentation of strained actin filaments.

We present a general model of actin filament deformation and fragmentation in response to compressive forces. The elastic free energy density along filaments is determined by their shape and mechanical properties, which were modeled in terms of bending, twisting, and twist-bend coupling elasticities. The elastic energy stored in filament deformation (i.e., strain) tilts the fragmentation-annealing reaction free-energy profile to favor fragmentation. The energy gradient introduces a local shear force that accelerates filament intersubunit bond rupture. The severing protein, cofilin, renders filaments more compliant in bending and twisting. As a result, filaments that are partially decorated with cofilin are mechanically heterogeneous (i.e., nonuniform) and display asymmetric shape deformations and energy profiles distinct from mechanically homogenous (i.e., uniform), bare actin, or saturated cofilactin filaments. The local buckling strain depends on the relative size of the compliant segment as well as the bending and twisting rigidities of flanking regions. Filaments with a single bare/cofilin-decorated boundary localize energy and force adjacent to the boundary, within the compliant cofilactin segment. Filaments with small cofilin clusters were predicted to fragment within the compliant cofilactin rather than at boundaries. Neglecting contributions from twist-bend coupling elasticity underestimates the energy density and gradients along filaments, and thus the net effects of filament strain to fragmentation. Spatial confinement causes compliant cofilactin segments and filaments to adopt higher deformation modes and store more elastic energy, thereby promoting fragmentation. The theory and simulations presented here establish a quantitative relationship between actin filament fragmentation thermodynamics and elasticity, and reveal how local discontinuities in filament mechanical properties introduced by regulatory proteins can modulate both the severing efficiency and location along filaments. The emergent behavior of mechanically heterogeneous filaments, particularly under confinement, emphasizes that severing in cells is likely to be influenced by multiple physical and chemical factors.

Measurement of cell traction forces with ImageJ.

The quantification of cell traction forces requires three key steps: cell plating on a deformable substrate, measurement of substrate deformation, and the numerical estimation of the corresponding cell traction forces. The computing steps to measure gel deformation and estimate the force field have somehow limited the adoption of this method in cell biology labs. Here we propose a set of ImageJ plug-ins so that every lab equipped with a fluorescent microscope can measure cell traction forces.

MAP65 coordinate microtubule growth during bundle formation.

Microtubules (MTs) are highly dynamical structures that play a crucial role in cell physiology. In cooperation with microtubule-associated proteins (MAPs), MTs form bundles endowing cells with specific mechanisms to control their shape or generate forces. Whether the dynamics of MTs is affected by the lateral connections that MAPs make between MTs during bundle formation is still under debate. Using in vitro reconstitution of MT bundling, we analyzed the dynamics of MT bundles generated by two plant MAP65 (MAP65-1/4), MAP65-1 being the plant ortholog of vertebrate PRC1 and yeast Ase1. MAP65-1/4 limit the amplitude of MT bundle depolymerization and increase the elongation phases. The subsequent sustained elongation of bundles is governed by the coordination of MT growth, so that MT ends come in close vicinity. We develop a model based on the assumption that both MAP65-1/4 block MT depolymerization. Model simulations reveal that rescue frequencies are higher between parallel than between anti-parallel MTs. In consequence the polarity of bundled MTs by MAP65 controls the amplitude of bundle's growth. Our results illustrate how MAP-induced MT-bundling, which is finely tuned by MT polarity, robustly coordinates MT elongation within bundles.

How actin network dynamics control the onset of actin-based motility.

Cells use their dynamic actin network to control their mechanics and motility. These networks are made of branched actin filaments generated by the Arp2/3 complex. Here we study under which conditions the microscopic organization of branched actin networks builds up a sufficient stress to trigger sustained motility. In our experimental setup, dynamic actin networks or "gels" are grown on a hard bead in a controlled minimal protein system containing actin monomers, profilin, the Arp2/3 complex and capping protein. We vary protein concentrations and follow experimentally and through simulations the shape and mechanical properties of the actin gel growing around beads. Actin gel morphology is controlled by elementary steps including "primer" contact, growth of the network, entanglement, mechanical interaction and force production. We show that varying the biochemical orchestration of these steps can lead to the loss of network cohesion and the lack of effective force production. We propose a predictive phase diagram of actin gel fate as a function of protein concentrations. This work unveils how, in growing actin networks, a tight biochemical and physical coupling smoothens initial primer-caused heterogeneities and governs force buildup and cell motility.

Actin network architecture can determine myosin motor activity.

The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

Cofilin-linked changes in actin filament flexibility promote severing.

The actin regulatory protein, cofilin, increases the bending and twisting elasticity of actin filaments and severs them. It has been proposed that filaments partially decorated with cofilin accumulate stress from thermally driven shape fluctuations at bare (stiff) and decorated (compliant) boundaries, thereby promoting severing. This mechanics-based severing model predicts that changes in actin filament compliance due to cofilin binding affect severing activity. Here, we test this prediction by evaluating how the severing activities of vertebrate and yeast cofilactin scale with the flexural rigidities determined from analysis of shape fluctuations. Yeast actin filaments are more compliant in bending than vertebrate actin filaments. Severing activities of cofilactin isoforms correlate with changes in filament flexibility. Vertebrate cofilin binds but does not increase the yeast actin filament flexibility, and does not sever them. Imaging of filament thermal fluctuations reveals that severing events are associated with local bending and fragmentation when deformations attain a critical angle. The critical severing angle at boundaries between bare and cofilin-decorated segments is smaller than in bare or fully decorated filaments. These measurements support a cofilin-severing mechanism in which mechanical asymmetry promotes local stress accumulation and fragmentation at boundaries of bare and cofilin-decorated segments, analogous to failure of some nonprotein materials.

Turnover of branched actin filament networks by stochastic fragmentation with ADF/cofilin.

Cell motility depends on the rapid assembly, aging, severing, and disassembly of actin filaments in spatially distinct zones. How a set of actin regulatory proteins that sustains actin-based force generation during motility work together in space and time remains poorly understood. We present our study of the distribution and dynamics of Arp2/3 complex, capping protein (CP), and actin-depolymerizing factor (ADF)/cofilin in actin "comet tails," using a minimal reconstituted system with nucleation-promoting factor (NPF)-coated beads. The Arp2/3 complex concentrates at nucleation sites near the beads as well as in the first actin shell. CP colocalizes with actin and is homogeneously distributed throughout the comet tail; it serves to constrain the spatial distribution of ATP/ADP-P(i) filament zones to areas near the bead. The association of ADF/cofilin with the actin network is therefore governed by kinetics of actin assembly, actin nucleotide state, and CP binding. A kinetic simulation accurately validates these observations. Following its binding to the actin networks, ADF/cofilin is able to break up the dense actin filament array of a comet tail. Stochastic severing by ADF/cofilin loosens the tight entanglement of actin filaments inside the comet tail and facilitates turnover through the macroscopic release of large portions of the aged actin network.

Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment boundaries.

Actin-based motility demands the spatial and temporal coordination of numerous regulatory actin-binding proteins (ABPs), many of which bind with affinities that depend on the nucleotide state of actin filament. Cofilin, one of three ABPs that precisely choreograph actin assembly and organization into comet tails that drive motility in vitro, binds and stochastically severs aged ADP actin filament segments of de novo growing actin filaments. Deficiencies in methodologies to track in real time the nucleotide state of actin filaments, as well as cofilin severing, limit the molecular understanding of coupling between actin filament chemical and mechanical states and severing. We engineered a fluorescently labeled cofilin that retains actin filament binding and severing activities. Because cofilin binding depends strongly on the actin-bound nucleotide, direct visualization of fluorescent cofilin binding serves as a marker of the actin filament nucleotide state during assembly. Bound cofilin allosterically accelerates P(i) release from unoccupied filament subunits, which shortens the filament ATP/ADP-P(i) cap length by nearly an order of magnitude. Real-time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and cofilin-decorated filament segments, thereby controlling the overall filament length, depending on cofilin binding density.

Origin of twist-bend coupling in actin filaments.

Actin filaments are semiflexible polymers that display large-scale conformational twisting and bending motions. Modulation of filament bending and twisting dynamics has been linked to regulatory actin-binding protein function, filament assembly and fragmentation, and overall cell motility. The relationship between actin filament bending and twisting dynamics has not been evaluated. The numerical and analytical experiments presented here reveal that actin filaments have a strong intrinsic twist-bend coupling that obligates the reciprocal interconversion of bending energy and twisting stress. We developed a mesoscopic model of actin filaments that captures key documented features, including the subunit dimensions, interaction energies, helicity, and geometrical constraints coming from the double-stranded structure. The filament bending and torsional rigidities predicted by the model are comparable to experimental values, demonstrating the capacity of the model to assess the mechanical properties of actin filaments, including the coupling between twisting and bending motions. The predicted actin filament twist-bend coupling is strong, with a persistence length of 0.15-0.4 µm depending on the actin-bound nucleotide. Twist-bend coupling is an emergent property that introduces local asymmetry to actin filaments and contributes to their overall elasticity. Up to 60% of the filament subunit elastic free energy originates from twist-bend coupling, with the largest contributions resulting under relatively small deformations. A comparison of filaments with different architectures indicates that twist-bend coupling in actin filaments originates from their double protofilament and helical structure.

Nucleation geometry governs ordered actin networks structures.

Actin filaments constitute one of the main components of cell cytoskeleton. Assembled into bundles in filopodia or in stress fibres, they play a pivotal role in eukaryotes during cell morphogenesis, adhesion and motility. The bundle emergence has been extensively related to specific actin regulators in vivo. Such dynamic modulation was also highlighted by biochemical reconstitution of the actin-network assembly, in bulk solution or with biomimetic devices. However, the question of how geometrical boundaries, such as those encountered in cells, affect the dynamic formation of highly ordered actin structures remains poorly studied. Here we demonstrate that the nucleation geometry in itself can be the principal determinant of actin-network architecture. We developed a micropatterning method that enables the spatial control of actin nucleation sites for in vitro assays. Shape, orientation and distance between nucleation regions control filament orientation and length, filament-filament interactions and filopodium-like bundle formation. Modelling of filament growth and interactions demonstrates that basic mechanical and probabilistic laws govern actin assembly in higher-order structures.

A "primer"-based mechanism underlies branched actin filament network formation and motility.

Cells use actin assembly to generate forces for membrane protrusions during movement [1] or, in the case of pathogens, to propel themselves in the host cells, in crude extracts [2], or in mixtures of actin and other purified proteins [3]. Significant progress has been made in understanding the mechanism of actin-based motility at a macroscopic level by using biomimetic systems in vitro [4-6]. Here, we combined such a system with evanescent wave microscopy to visualize Arp2/3-mediated actin network formation at single-actin-filament resolution. We found that actin filaments that we call "primers" determine the origin of the autocatalytic and propagative formation of the actin network. In the presence of capping protein, multiple "primers" generate independent networks that merge around the object to form an outer "shell" made of entangled and capped filaments. Simultaneously, newly created filaments on the surface of the particle initiate mechanical stress, which develops until symmetry breaking. Our results and extensive modeling support that the stress, which releases into propulsive forces [7], is controlled not by any specific orientation of actin filaments toward the nucleation sites but only by new monomers added near the load surface.

Increased cardiac index due to terbutaline treatment aggravates capillary-alveolar macromolecular leakage in oleic acid lung injury in dogs.

INTRODUCTION: We assessed the in vivo effects of terbutaline, a beta2-agonist assumed to reduce microvascular permeability in acute lung injury. METHODS: We used a recently developed broncho-alveolar lavage (BAL) technique to repeatedly measure (every 15 min. for 4 hours) the time-course of capillary-alveolar leakage of a macromolecule (fluorescein-labeled dextran) in 19 oleic acid (OA) lung injured dogs. BAL was performed in a closed lung sampling site, using a bronchoscope fitted with an inflatable cuff. Fluorescein-labeled Dextran (FITC-D70) was continuously infused and its concentration measured in plasma and BAL fluid. A two-compartment model (blood and alveoli) was used to calculate KAB, the transport rate coefficient of FITC-D70 from blood to alveoli. KAB was estimated every 15 minutes over 4 hours. Terbutaline intra-venous perfusion was started 90 min. after the onset of the injury and then continuously infused until the end of the experiment. RESULTS: In the non-treated injured group, the capillary-alveolar leakage of FITC-D70 reached a peak within 30 minutes after the OA injury. Thereafter the FITC-D70 leakage decreased gradually until the end of the experiment. Terbutaline infusion, started 90 min after injury, interrupted the recovery with an aggravation in FITC-D70 leakage. CONCLUSIONS: As cardiac index increased with terbutaline infusion, we speculate that terbutaline recruits leaky capillaries and increases FITC-D70 leakage after OA injury. These findings suggest that therapies inducing an increase in cardiac output and a decrease in pulmonary vascular resistances have the potential to heighten the early increase in protein transport from plasma to alveoli within the acutely injured lung.