Michael P. Murrell

F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex

Here we develop a minimal model of the cell actomyosin cortex by forming a quasi-2D cross-linked filamentous actin (F-actin) network adhered to a model cell membrane and contracted by myosin thick filaments. Myosin motors generate both compressive and tensile stresses on F-actin and consequently induce large bending fluctuations, which reduces their effective persistence length to <1 μm. Over a large range of conditions, we show the extent of network contraction corresponds exactly to the extent of individual F-actin shortening via buckling. This demonstrates an essential role of buckling in breaking the symmetry between tensile and compressive stresses to facilitate mesoscale network contraction of up to 80% strain. Portions of buckled F-actin with a radius of curvature ∼300 nm are prone to severing and thus compressive stresses mechanically coordinate contractility with F-actin severing, the initial step of F-actin turnover. Finally, the F-actin curvature acquired by myosin-induced stresses can be further constrained by adhesion of the network to a membrane, accelerating filament severing but inhibiting the long-range transmission of the stresses necessary for network contractility. Thus, the extent of membrane adhesion can regulate the coupling between network contraction and F-actin severing. These data demonstrate the essential role of the nonlinear response of F-actin to compressive stresses in potentiating both myosin-mediated contractility and filament severing. This may serve as a general mechanism to mechanically coordinate contractility and cortical dynamics across diverse actomyosin assemblies in smooth muscle and nonmuscle cells.

Forcing cells into shape: the mechanics of actomyosin contractility

Actomyosin-mediated contractility is a highly conserved mechanism for generating mechanical stress in animal cells and underlies muscle contraction, cell migration, cell division and tissue morphogenesis. Whereas actomyosin-mediated contractility in striated muscle is well understood, the regulation of such contractility in non-muscle and smooth muscle cells is less certain. Our increased understanding of the mechanics of actomyosin arrays that lack sarcomeric organization has revealed novel modes of regulation and force transmission. This work also provides an example of how diverse mechanical behaviours at cellular scales can arise from common molecular components, underscoring the need for experiments and theories to bridge the molecular to cellular length scales.

Distribution of directional change as a signature of complex dynamics

Significance Since Einstein’s seminal work in 1905, the main means of characterizing stochastic processes has been the mean square displacement (MSD). However, this order parameter fails to capture many features of dynamics at the forefront of science today, ranging from glassy relaxation to active transport in biological cells. Although there have been several studies seeking to go beyond the MSD, such studies have not made full use of the information available in individual trajectories in two (or more) dimensions, as are now commonly obtained in particle tracking experiments. Here, we introduce an approach that quantifies directional properties of complex motions and discover striking correlations in a number of condensed phase systems. Analyses of random walks traditionally use the mean square displacement (MSD) as an order parameter characterizing dynamics. We show that the distribution of relative angles of motion between successive time intervals of random walks in two or more dimensions provides information about stochastic processes beyond the MSD. We illustrate the behavior of this measure for common models and apply it to experimental particle tracking data. For a colloidal system, the distribution of relative angles reports sensitively on caging as the density varies. For transport mediated by molecular motors on filament networks in vitro and in vivo, we discover self-similar properties that cannot be described by existing models and discuss possible scenarios that can lead to the elucidated statistical features.

Spreading Dynamics of Biomimetic Actin Cortices

Reconstituted systems mimicking cells are interesting tools for understanding the details of cell behavior. Here, we use an experimental system that mimics cellular actin cortices, namely liposomes developing an actin shell close to their inner membrane, and we study their dynamics of spreading. We show that depending on the morphology of the actin shell inside the liposome, spreading dynamics is either reminiscent of a bare liposome (in the case of a sparse actin shell) or of a cell (in the case of a continuous actin shell). We use a mechanical model that qualitatively accounts for the shape of the experimental curves. From the data on spreading dynamics, we extract characteristic times that are consistent with mechanical estimates. The mechanical characterization of such stripped-down experimental systems paves the way for a more complex design closer to a cell. We report here the first step in building an artificial cell and studying its mechanics.

How cells flow in the spreading of cellular aggregates

Significance Cellular aggregates are in vitro model of tumors. Deposited on adhesive substrates, they spread like liquid droplets with a monolayer expanding from the aggregate. We model spreading dynamics by balancing driving forces at the film periphery and viscous forces associated to the penetration of the cells from the (3D) aggregate into the (2D) film. By confocal microscopy, we observe this mechanism named “permeation” by tracking single cells. Using particle image velocimetry, we characterize the flow field versus substrate rigidity. If cells spread like a viscous liquid on stiff substrate, the flows become irregular, with formation of holes as the rigidity decreases. This work will shed light on the dynamics of tissue spreading occurring during cancer progression and embryonic development. Like liquid droplets, cellular aggregates, also called “living droplets,” spread onto adhesive surfaces. When deposited onto fibronectin-coated glass or polyacrylamide gels, they adhere and spread by protruding a cellular monolayer (precursor film) that expands around the droplet. The dynamics of spreading results from a balance between the pulling forces exerted by the highly motile cells at the periphery of the film, and friction forces associated with two types of cellular flows: (i) permeation, corresponding to the entry of the cells from the aggregates into the film; and (ii) slippage as the film expands. We characterize these flow fields within a spreading aggregate by using fluorescent tracking of individual cells and particle imaging velocimetry of cell populations. We find that permeation is limited to a narrow ring of width ξ (approximately a few cells) at the edge of the aggregate and regulates the dynamics of spreading. Furthermore, we find that the subsequent spreading of the monolayer depends heavily on the substrate rigidity. On rigid substrates, the migration of the cells in the monolayer is similar to the flow of a viscous liquid. By contrast, as the substrate gets softer, the film under tension becomes unstable with nucleation and growth of holes, flows are irregular, and cohesion decreases. Our results demonstrate that the mechanical properties of the environment influence the balance of forces that modulate collective cell migration, and therefore have important implications for the spreading behavior of tissues in both early development and cancer.

High-content imaging with micropatterned multiwell plates reveals influence of cell geometry and cytoskeleton on chromatin dynamics

Understanding the mechanisms underpinning cellular responses to microenvironmental cues requires tight control not only of the complex milieu of soluble signaling factors, extracellular matrix (ECM) connections and cell-cell contacts within cell culture, but also of the biophysics of human cells. Advances in biomaterial fabrication technologies have recently facilitated detailed examination of cellular biophysics and revealed that constraints on cell geometry arising from the cellular microenvironment influence a wide variety of human cell behaviors. Here, we create an in vitro platform capable of precise and independent control of biochemical and biophysical microenvironmental cues by adapting microcontact printing technology into the format of standard six- to 96-well plates to create MicroContact Printed Well Plates (μCP Well Plates). Automated high-content imaging of human cells seeded on μCP Well Plates revealed tight, highly consistent control of single-cell geometry, cytoskeletal organization, and nuclear elongation. Detailed subcellular imaging of the actin cytoskeleton and chromatin within live human fibroblasts on μCP Well Plates was then used to describe a new relationship between cellular geometry and chromatin dynamics. In summary, the μCP Well Plate platform is an enabling high-content screening technology for human cell biology and cellular engineering efforts that seek to identify key biochemical and biophysical cues in the cellular microenvironment.

Reconstitution of contractile actomyosin arrays.

Networks and bundles comprised of F-actin and myosin II generate contractile forces used to drive morphogenic processes in both muscle and nonmuscle cells. To elucidate the minimal requirements for contractility and the mechanisms underlying their contractility, model systems reconstituted from a known set of purified proteins in vitro are needed. Here, we describe two experimental protocols our lab has developed to reconstitute 1D bundles and quasi-2D networks of actomyosin that are amenable to quantitative biophysical measurement. These assays have enabled our discovery of the mechanisms of contractility in disordered actomyosin assemblies and of a mechanical feedback between contraction and F-actin severing.

Cooperation of dual modes of cell motility promotes epithelial stress relaxation to accelerate wound healing

Collective cell migration in cohesive units is vital for tissue morphogenesis, wound repair, and immune response. While the fundamental driving forces for collective cell motion stem from contractile and protrusive activities of individual cells, it remains unknown how their balance is optimized to maintain tissue cohesiveness and the fluidity for motion. Here we present a cell-based computational model for collective cell migration during wound healing that incorporates mechanochemical coupling of cell motion and adhesion kinetics with stochastic transformation of active motility forces. We show that a balance of protrusive motility and actomyosin contractility is optimized for accelerating the rate of wound repair, which is robust to variations in cell and substrate mechanical properties. This balance underlies rapid collective cell motion during wound healing, resulting from a tradeoff between tension mediated collective cell guidance and active stress relaxation in the tissue.