Matthew R. Stachowiak

Mechanism of Cytokinetic Contractile Ring Constriction in Fission Yeast

Cytokinesis involves constriction of a contractile actomyosin ring. The mechanisms generating ring tension and setting the constriction rate remain unknown because the organization of the ring is poorly characterized, its tension was rarely measured, and constriction is coupled to other processes. To isolate ring mechanisms, we studied fission yeast protoplasts, in which constriction occurs without the cell wall. Exploiting the absence of cell wall and actin cortex, we measured ring tension and imaged ring organization, which was dynamic and disordered. Computer simulations based on the amounts and biochemical properties of the key proteins showed that they spontaneously self-organize into a tension-generating bundle. Together with rapid component turnover, the self-organization mechanism continuously reassembles and remodels the constricting ring. Ring constriction depended on cell shape, revealing that the ring operates close to conditions of isometric tension. Thus, the fission yeast ring sets its own tension, but other processes set the constriction rate.

Kinetics of stress fibers

Stress fibers are contractile cytoskeletal structures, tensile actomyosin bundles which allow sensing and production of force, provide cells with adjustable rigidity and participate in various processes such as wound healing. The stress fiber is possibly the best characterized and most accessible multiprotein cellular contractile machine. Here we develop a quantitative model of the structure and relaxation kinetics of stress fibers. The principal experimentally known features are incorporated. The fiber has a periodic sarcomeric structure similar to muscle fibers with myosin motor proteins exerting contractile force by pulling on actin filaments. In addition the fiber contains the giant spring-like protein titin. Actin is continuously renewed by exchange with the cytosol leading to a turnover time of several minutes. In order that steady state be possible, turnover must be regulated. Our model invokes simple turnover and regulation mechanisms: actin association and dissociation occur at filament ends, while actin filament overlap above a certain threshold in the myosin-containing regions augments depolymerization rates. We use the model to study stress fiber relaxation kinetics after stimulation, as observed in a recent experimental study where some fiber regions were contractile and others expansive. We find that two distinct episodes ensue after stimulation: the turnover–overlap system relaxes rapidly in seconds, followed by the slow relaxation of sarcomere lengths in minutes. For parameter values as they have been characterized experimentally, we find the long time relaxation of sarcomere length is set by the rate at which actin filaments can grow or shrink in response to the forces exerted by the elastic and contractile elements. Consequently, the stress fiber relaxation time scales inversely with both titin spring constant and the intrinsic actin turnover rate. The models predicted sarcomere velocities and contraction–expansion kinetics are in good quantitative agreement with experiment.

Recoil after severing reveals stress fiber contraction mechanisms.

Stress fibers are cellular contractile actomyosin machines central to wound healing, shear stress response, and other processes. Contraction mechanisms have been difficult to establish because stress fibers in cultured cells typically exert isometric tension and present little kinetic activity. In a recent study, living cell stress fibers were severed with laser nanoscissors and recoiled several mum over approximately 5 s. We developed a quantitative model of stress fibers based on known components and available structural information suggesting periodic sarcomeric organization similar to striated muscle. The model was applied to the severing assay and compared to the observed recoil. We conclude that the sarcomere force-length relation is similar to that of muscle with two distinct regions on the ascending limb and that substantial external drag forces act on the recoiling fiber corresponding to effective cytosolic viscosity approximately 10(4) times that of water. This may originate from both nonspecific and specific interactions. The model predicts highly nonuniform contraction with caps of collapsed sarcomeres growing at the severed ends. A directly measurable signature of external drag is that cap length and recoil distance increase at intermediate times as t(1/2). The severing data is consistent with this prediction.

α-Actinin links extracellular matrix rigidity-sensing contractile units with periodic cell-edge retractions

During cell migration, the cell edge undergoes periodic protrusion–retraction cycles. Quantitative analyses of the forces at the cell edge that drive these cycles are provided. We show that α-actinin links local contractile units and the global actin flow forces at the cell edge and present a novel model based on these results.

Self-Organization of Myosin II in Reconstituted Actomyosin Bundles

Cells assemble a variety of bundled actomyosin structures in the cytoskeleton for activities such as cell-shape regulation, force production, and cytokinesis. Although these linear structures exhibit varied architecture, two common organizational themes are a punctate distribution of myosin II and distinct patterns of actin polarity. The mechanisms that cells use to assemble and maintain these organizational features are poorly understood. To study these, we reconstituted actomyosin bundles in vitro that contained only actin filaments and myosin II. Upon addition of ATP, the bundles contracted and the uniformly distributed myosin spontaneously reorganized into discrete clusters. We developed a mathematical model in which the motion of myosin II filaments is governed by the polarities of the actin filaments with which they interact. The model showed that the assembly of myosins into clusters is driven by their tendency to migrate to locations with zero net actin filament polarity. With no fitting parameters, the predicted distribution of myosin cluster separations was in close agreement with our experiments, including a -3/2 power law decay for intermediate length scales. Thus, without an organizing template or accessory proteins, a minimal bundle of actin and myosin has the inherent capacity to self-organize into a heterogeneous banded structure.

A mechanical-biochemical feedback loop regulates remodeling in the actin cytoskeleton

Significance Cellular activities are regulated by signaling pathways in which information is transduced biochemically. Increasingly it is appreciated that regulation also involves mechanical signaling, where mechanical information is mechanotransduced into biochemical information. However, little is understood about the cooperation of mechanical and biochemical signaling in mixed pathways. We identified a pathway where the two types of signaling work in harmony to remodel actomyosin stress fibers in the cell’s cytoskeleton. We present evidence that expansion or contraction of fibers alters the actin filament overlap, a mechanical signal that is mechanotransduced into actin assembly or disassembly, which in turn alters the overlap. A mathematical model accurately describes our measurements and shows that this mechanical-biochemical feedback loop synchronizes actin remodeling with fiber length changes. Cytoskeletal actin assemblies transmit mechanical stresses that molecular sensors transduce into biochemical signals to trigger cytoskeletal remodeling and other downstream events. How mechanical and biochemical signaling cooperate to orchestrate complex remodeling tasks has not been elucidated. Here, we studied remodeling of contractile actomyosin stress fibers. When fibers spontaneously fractured, they recoiled and disassembled actin synchronously. The disassembly rate was accelerated more than twofold above the resting value, but only when contraction increased the actin density to a threshold value following a time delay. A mathematical model explained this as originating in the increased overlap of actin filaments produced by myosin II-driven contraction. Above a threshold overlap, this mechanical signal is transduced into accelerated disassembly by a mechanism that may sense overlap directly or through associated elastic stresses. This biochemical response lowers the actin density, overlap, and stresses. The model showed that this feedback mechanism, together with rapid stress transmission along the actin bundle, spatiotemporally synchronizes actin disassembly and fiber contraction. Similar actin remodeling kinetics occurred in expanding or contracting intact stress fibers but over much longer timescales. The model accurately described these kinetics, with an almost identical value of the threshold overlap that accelerates disassembly. Finally, we measured resting stress fibers, for which the model predicts constant actin overlap that balances disassembly and assembly. The overlap was indeed regulated, with a value close to that predicted. Our results suggest that coordinated mechanical and biochemical signaling enables extended actomyosin assemblies to adapt dynamically to the mechanical stresses they convey and direct their own remodeling.

Self-Organization in Reconstituted Actomyosin Bundles

Actomyosin bundles such as muscle myofibrils, stress fibers and cytokinetic rings are used by cells to exert force, contribute to the cells structural integrity and accomplish morphological change. In muscle and some types of stress fibers actin, myosin and other components are organized into sarcomeric repeat units but in many other cases the structure is far more disordered. The mechanisms whereby such disordered architectures produce tension are not established. Here we used mathematical modeling to quantitatively understand the behavior of reconstituted in vitro actomyosin bundles consisting solely of F-actin and myosin thick filaments. In the presence of ADP, actin and myosin formed stable (>1 hour), non-tensile bundles ∼5-50 microns long anchored between polystyrene beads. The myosin is initially uniformly distributed along the bundles. Upon addition of ATP, the bundles contracted and became taut while the myosin reorganized into discrete clusters. We developed a mathematical model to account for this behavior. The random actin filament locations and polarities leads to a random net actin polarity at different locations along the bundle. We found the self-organization of myosins into clusters is driven by the tendency of myosin to migrate to zeros of the polarity profile. In agreement with experiment myosin clusters develop over ∼10 s by myosin translation. We calculate the distribution of myosin cluster separations and predict the mean cluster separation increases with actin filament length. Thus, a minimal bundle of actin and myosin alone has the inherent capacity to self-organize into a heterogeneous structure exhibiting morphological similarity to tension-producing cellular actomyosin structures such as stress fibers.