Kinesin and Myosin Motors Compete to Drive Rich Multi-Phase Dynamics in Programmable Cytoskeletal Composites

The cytoskeleton of biological cells relies on a diverse population of motors, filaments, and binding proteins acting in concert to enable non-equilibrium processes ranging from mitosis to chemotaxis. The cytoskeleton’s versatile reconfigurability, programmed by interactions between its constituents, make it a foundational active matter platform. However, current active matter endeavors are limited largely to single force-generating components acting on a single substrate – far from the composite cytoskeleton in live cells. Here, we engineer actin-microtubule composites, driven by kinesin and myosin motors and tuned by crosslinkers, that restructure into diverse morphologies from interpenetrating filamentous networks to de-mixed amorphous clusters. Our Fourier analyses reveal that kinesin and myosin compete to delay kinesin-driven restructuring and suppress de-mixing and flow, while crosslinking accelerates reorganization and promotes actin-microtubule correlations. The phase space of non-equilibrium dynamics falls into three broad classes– slow reconfiguration, fast advective flow, and multi-mode ballistic dynamics – with structure-dynamics relations described by the relative contributions of elastic and dissipative responses to motor-generated forces.

Structural basis of the super- and hyper-relaxed states of myosin II

Super-relaxation is a state of muscle thick filaments in which ATP turnover by myosin is much slower than that of myosin II in solution. This inhibited state, in equilibrium with a faster (relaxed) state, is ubiquitous and thought to be fundamental to muscle function, acting as a mechanism for switching off energy-consuming myosin motors when they are not being used. The structural basis of super-relaxation is usually taken to be a motif formed by myosin in which the two heads interact with each other and with the proximal tail forming an interacting-heads motif, which switches the heads off. However, recent studies show that even isolated myosin heads can exhibit this slow rate. Here, we review the role of head interactions in creating the super-relaxed state and show how increased numbers of interactions in thick filaments underlie the high levels of super-relaxation found in intact muscle. We suggest how a third, even more inhibited, state of myosin (a hyper-relaxed state) seen in certain species results from additional interactions involving the heads. We speculate on the relationship between animal lifestyle and level of super-relaxation in different species and on the mechanism of formation of the super-relaxed state. We also review how super-relaxed thick filaments are activated and how the super-relaxed state is modulated in healthy and diseased muscles.

Extent of myosin penetration within the actin cortex regulates cell surface mechanics

In animal cells, shape is mostly determined by the actomyosin cortex, a thin cytoskeletal network underlying the plasma membrane. Myosin motors generate tension in the cortex, and tension gradients result in cellular deformations. As such, many cell morphogenesis studies have focused on the mechanisms controlling myosin activity and recruitment to the cortex. Here, we demonstrate using super-resolution microscopy that myosin does not always overlap with actin at the cortex, but remains restricted towards the cytoplasm in cells with low cortex tension. We propose that this restricted penetration results from steric hindrance, as myosin minifilaments are considerably larger than the cortical actin meshsize. We identify myosin activity and actin network architecture as key regulators of myosin penetration into the cortex, and show that increasing myosin penetration increases cortical tension. Our study reveals that the spatial coordination of myosin and actin at the cortex regulates cell surface mechanics, and unveils an important mechanism whereby myosin size controls its action by limiting minifilament penetration into the cortical actin network. More generally, our findings suggest that protein size could regulate function in dense cytoskeletal structures.

Myosin motors that cannot bind actin leave their folded OFF state on activation of skeletal muscle

The myosin motors in resting skeletal muscle are folded back against their tails in the thick filament in a conformation that makes them unavailable for binding to actin. When muscles are activated, calcium binding to troponin leads to a rapid change in the structure of the actin-containing thin filaments that uncovers the myosin binding sites on actin. Almost as quickly, myosin motors leave the folded state and move away from the surface of the thick filament. To test whether motor unfolding is triggered by the availability of nearby actin binding sites, we measured changes in the x-ray reflections that report motor conformation when muscles are activated at longer sarcomere length, so that part of the thick filaments no longer overlaps with thin filaments. We found that the intensity of the M3 reflection from the axial repeat of the motors along the thick filaments declines almost linearly with increasing sarcomere length up to 2.8 µm, as expected if motors in the nonoverlap zone had left the folded state and become relatively disordered. In a recent article in JGP, Squire and Knupp challenged this interpretation of the data. We show here that their analysis is based on an incorrect assumption about how the interference subpeaks of the M3 reflection were reported in our previous paper. We extend previous models of mass distribution along the filaments to show that the sarcomere length dependence of the M3 reflection is consistent with <10% of no-overlap motors remaining in the folded conformation during active contraction, confirming our previous conclusion that unfolding of myosin motors on muscle activation is not due to the availability of local actin binding sites.

F-Actin Bending Facilitates Net Actomyosin Contraction By Inhibiting Expansion With Plus-End-Located Myosin Motors

We investigate whether a microscopic system of two semi-flexible actin filaments with an attached myosin motor can facilitate contraction. Based on energy minimisation, we derive and analyse a partial differential equation model for a two-filament-motor structure embedded within a dense, two-dimensional network. Our method enables calculation of the plane stress tensor, providing a measure for contractility. After deriving the model, we use a combination of asymptotic analysis and numerical solutions to show how F-actin bending facilitates net contraction as a myosin motor traverses two symmetric filaments. Myosin motors close to the minus-ends facilitate contraction, whereas motors close to the plus-ends facilitate expansion. The leading-order solution for rigid filaments exhibits polarity-reversal symmetry, such that the contractile and expansive components balance to zero. Surprisingly, after introducing bending the first-order correction to stress indicates expansion. However, numerical solutions show that filament bending induces a geometric asymmetry that brings the filaments closer to parallel as a myosin motor approaches their plus-ends. This decreases the effective spring force opposing motion of the motor, enabling it to move faster close to filament plus-ends. This reduces the contribution of expansive stress, giving rise to net contraction. Further numerical solutions confirm that this applies beyond the small bending regime considered in the asymptotic analysis. Our findings confirm that filament bending gives rise to microscopic-scale actomyosin contraction, and provides a possible explanation for network-scale contraction.

The regulatory light chain mediates inactivation of myosin motors during active shortening of cardiac muscle

The normal function of heart muscle depends on its ability to contract more strongly at longer length. Increased venous filling stretches relaxed heart muscle cells, triggering a stronger contraction in the next beat- the Frank-Starling relation. Conversely, heart muscle cells are inactivated when they shorten during ejection, accelerating relaxation to facilitate refilling before the next beat. Although both effects are essential for the efficient function of the heart, the underlying mechanisms were unknown. Using bifunctional fluorescent probes on the regulatory light chain of the myosin motor we show that its N-terminal domain may be captured in the folded OFF state of the myosin dimer at the end of the working-stroke of the actin-attached motor, whilst its C-terminal domain joins the OFF state only after motor detachment from actin. We propose that sequential folding of myosin motors onto the filament backbone may be responsible for shortening-induced de-activation in the heart.

Titin switches from an extensible spring to a mechanical rectifier upon muscle activation

In contracting striated muscle titin acts as a spring in parallel with the array of myosin motors in each half-sarcomere and could prevent the intrinsic instability of thousands of serially linked half-sarcomeres, if its stiffness, at physiological sarcomere lengths (SL), were ten times larger than reported. Here we define titin mechanical properties during tetanic stimulation of single fibres of frog muscle by suppressing myosin motor responses with Para-Nitro-Blebbistatin, which is able to freeze thick filament in the resting state. We discover that thin filament activation switches I-band titin spring from the large SL-dependent extensibility of the OFF-state to an ON-state in which titin acts as a SL-independent mechanical rectifier, allowing free shortening while opposing stretch with an effective stiffness 4 pN nm-1 per half-thick filament. In this way during contraction titin limits weak half-sarcomere elongation to a few % and, also, provides an efficient link for mechanosensing-based thick filament activation.

A coarse-grained approach to model the dynamics of the actomyosin cortex

The dynamics of the actomyosin machinery is at the core of many important biological processes. Several relevant cellular responses such as the rhythmic compression of the cell cortex are governed, at a mesoscopic level, by the nonlinear interaction between actin monomers, actin crosslinkers and myosin motors. Coarse grained models are an optimal tool to study actomyosin systems, since they can include processes that occur at long time and space scales, while maintaining the most relevant features of the molecular interactions. Here, we present a coarse grained model of a two-dimensional actomyosin cortex, adjacent to a three-dimensional cytoplasm. Our simplified model incorporates only well characterized interactions between actin monomers, actin cross- linkers and myosin, and it is able to reproduce many of the most important aspects of actin filament and actomyosin network formation, such as dynamics of polymerization and depolymerization, treadmilling, network formation and the autonomous oscilla- tory dynamics of actomyosin. Furthermore, the model can be used to predict the in vivo response of actomyosin networks to changes in key parameters of the system, such as alterations in the anchor of actin filaments to the cell cortex.

Imaging methods in mechanosensing: a historical perspective and visions for the future

Recently established microscopic techniques are shedding new light on fundamental questions regarding mechanotransduction. Evidence suggests that myosin motors and the actin cytoskeleton are the single largest aggregate producers and transmitters of force within the cell. We review the roots of this hypothesis and future directions of the field.

Reconstitution of contractile actomyosin rings in vesicles

One of the grand challenges of bottom-up synthetic biology is the development of minimal machineries for cell division. The mechanical transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the molecular scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theoretical modeling. By changing few key parameters, actin polymerization can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theoretical considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.