Myosin Clusters Research Articles

Polarity-driven three-dimensional spontaneous rotation of a cell doublet

Mechanical interactions between cells play a fundamental role in the self-organization of organisms. How these interactions drive coordinated cell movement in three dimensions remains unclear. Here we report that cell doublets embedded in a three-dimensional extracellular matrix undergo spontaneous rotations. We investigate the rotation mechanism and find that it is driven by a polarized distribution of myosin within cell cortices. The mismatched orientation of this polarized distribution breaks the doublet mirror symmetry. In addition, cells adhere at their interface through adherens junctions and with the extracellular matrix through focal contacts near myosin clusters. We use a physical theory describing the doublet as two interacting active surfaces to show that rotation is driven by myosin-generated gradients of active tension whose profiles are dictated by interacting cell polarity axes. We also show that three-dimensional shape symmetries are related to broken symmetries of the myosin distribution in cortices. To test for the rotation mechanism, we suppress myosin clusters using laser ablation and generate new myosin clusters by optogenetics. Our work clarifies how polarity-oriented active mechanical forces drive collective cell motion in three dimensions.

Limiting pool and actin architecture controls myosin cluster sizes in adherent cells

The actomyosin cytoskeleton generates mechanical forces that power important cellular processes, such as cell migration, cell division, and mechanosensing. Actomyosin self-assembles into contractile networks and bundles that underlie force generation and transmission in cells. A central step is the assembly of the myosin II filament from myosin monomers, regulation of which has been extensively studied. However, myosin filaments are almost always found as clusters within the cell cortex. While recent studies characterized cluster nucleation dynamics at the cell periphery, how myosin clusters grow on stress fibers remains poorly characterized. Here, we utilize a U2OS osteosarcoma cell line with endogenously tagged myosin II to measure the myosin cluster size distribution in the lamella of adherent cells. We find that myosin clusters can grow with Rho-kinase (ROCK) activity alone in the absence of myosin motor activity. Time-lapse imaging reveals that myosin clusters grow via increased myosin association to existing clusters, which is potentiated by ROCK-dependent myosin filament assembly. Enabling myosin motor activity allows further myosin cluster growth through myosin association that is dependent on F-actin architecture. Using a toy model, we show that myosin self-affinity is sufficient to recapitulate the experimentally observed myosin cluster size distribution, and that myosin cluster sizes are determined by the pool of myosin available for cluster growth. Together, our findings provide new insights into the regulation of myosin cluster sizes within the lamellar actomyosin cytoskeleton.

A role for myosin II clusters and membrane energy in cortex rupture for Dictyostelium discoideum.

Blebs, pressure driven protrusions of the cell membrane, facilitate the movement of eukaryotic cells such as the soil amoeba Dictyostelium discoideum, white blood cells and cancer cells. Blebs initiate when the cell membrane separates from the underlying cortex. A local rupture of the cortex, has been suggested as a mechanism by which blebs are initiated. However, much clarity is still needed about how cells inherently regulate rupture of the cortex in locations where blebs are expected to form. In this work, we examine the role of membrane energy and the motor protein myosin II (myosin) in facilitating the cell driven rupture of the cortex. We perform under-agarose chemotaxis experiments, using Dictyostelium discoideum cells, to visualize the dynamics of myosin and calculate changes in membrane energy in the blebbing region. To facilitate a rapid detection of blebs and analysis of the energy and myosin distribution at the cell front, we introduce an autonomous bleb detection algorithm that takes in discrete cell boundaries and returns the coordinate location of blebs with its shape characteristics. We are able to identify by microscopy naturally occurring gaps in the cortex prior to membrane detachment at sites of bleb nucleation. These gaps form at positions calculated to have high membrane energy, and are associated with areas of myosin enrichment. Myosin is also shown to accumulate in the cortex prior to bleb initiation and just before the complete disassembly of the cortex. Together our findings provide direct spatial and temporal evidence to support cortex rupture as an intrinsic bleb initiation mechanism and suggests that myosin clusters are associated with regions of high membrane energy where its contractile activity leads to a rupture of the cortex at points of maximal energy.

Robustness of protein: Using pH shifting and low speed shearing to partially recover conformation and dispersibility of myosin from pale, soft, exudative (PSE)-like chicken breast

An unresolved problem in the field of food protein is the poor dispersibility of pale, soft, exudative (PSE)-like meat protein. This study aimed to verify the structural and functional robustness of myosin. Two different models, namely, PSE-induced whole meat model and pH shifting plus temperature holding of protein solution model, were developed. A novel combination of pH shifting (from 5.6 to 7.0) and low speed shearing (400 rpm) treatment decreased myosin turbidity by 9.6% and 36.5%, respectively. After readjusting the pH to 7.0, z-average size decreased from 289.0 nm to 1563.6 nm–274.0 nm and 333.3 nm, respectively. The experimental groups exhibited excellent solubility like the normal group. Scanning electron microscopy revealed that large myosin clusters and aggregates decreased. The improvement in PSE myosin stability increased the absolute ζ-potential value from −13.1 mV to −15.5 mV under treatment. Raman analysis revealed that the increase in protein function was not only because of surface charge states induced by pH shifting but also because of conformation recovery as evidenced by the increase in α-helix content. Results indicated that pH shifting and proper low shearing could fulfill the robustness of myosin function by recovering conformation and improving solubility.

Sliding Filament and Fixed Filament Mechanisms Contribute to Tension of the Fission Yeast Cytokinetic Ring

A fundamental challenge in cell biology is understanding how cells generate contractile force from actomyosin machineries. The contractile ring is a vital actomyosin machine that generates tension and constricts and divides the cell during cytokinesis. Following discovery of the contractile ring decades ago, a sliding filament mechanism for ring tension was proposed similar to that in muscle (Schroeder, 1972). However, contractile rings lack the highly ordered periodic sarcomeric architecture of muscle, and it remains unknown how tension emerges from the disordered ring organization. Here, we developed a highly coarse-grained continuum model of the fission yeast cytokinetic ring, severely constrained by experimentally measured component amounts. The model incorporates the two myosin-II isoforms, Myo2 in membrane-anchored protein complexes called nodes, and Myp2 clusters assumed unanchored. We found that the model does indeed predict a sliding filament mechanism, but somewhat different to that envisaged by the pioneering studies of Schroeder and others. The sliding filament mechanism is a spatially and temporally homogeneous version of that in muscle: pairs of oppositely oriented actin filaments are made tense as they are pulled toward one another by myosin-II clusters through which they slide. The mechanism relies on anchoring of actin filament barbed ends to the plasma membrane, which enables filaments to become tense when pulled. A second fixed filament component is generated by chains of like-oriented actin filaments encircling the ring, independent of lateral anchoring. Cellular actomyosin contractile machines are intrinsically vulnerable to contractile instabilities. We also studied how such instabilities are controlled. We find that tension loss due to runaway catastrophic aggregation of the contractile components Myo2 and Myp2 is prevented by fast turnover and sufficiently low myosin cluster mobilities, enforced by lateral anchoring and crowding.

Active bundles of polar and bipolar filaments

Bundles of actin filaments and molecular motors of the myosin family are a common subcellular organizational motif. Typically, such bundles are under contractile stress resulting from interactions between the filaments and the motors. This holds in particular for contractile rings that appear in the late stages of cell division in animal cells and that cleave the mother into two daughter cells. It was recently shown that myosin organizes into regularly spaced clusters along rings in mammalian cells, whereas myosin clusters in fission yeast travel along the perimeter of actomyosin rings [Wollrab et al., Nat. Commun. 7, 11860 (2016)2041-172310.1038/ncomms11860]. A mechanism based on the association of the structurally polar actin filaments into bipolar structures was shown to provide a common explanation for both observations. Here, we analyze the dynamics of this mechanism in detail. We find a rich phase diagram depending on the actomyosin interaction strength and the stability of the bipolar structures. The system can notably organize into traveling waves. Furthermore, we identify the nature of the bifurcations connecting the various patterns as parameters are changed. Finally, we report experimental patterns observed in cytokinetic rings in fission yeast and link them to solutions of our dynamic equations. Our analysis highlights the possible role played by local polarity sorting of actin filaments for the dynamics and functionality of actomyosin networks.

Myosin Clusters of Finite Size Develop Contractile Stress in 1D Random Actin Arrays

Myosin-powered force generation and contraction in nonmuscle cells underlies many cell biological processes and is based on contractility of random actin arrays. This contractility must rely on a microscopic asymmetry, the precise mechanism of which is not completely clear. A number of models of mechanical and structural asymmetries in actomyosin contraction have been posited. Here, we examine a contraction mechanism based on a finite size of myosin clusters and anisotropy of force generation by myosin heads at the ends of the myosin clusters. We use agent-based numerical simulations to demonstrate that if average lengths of actin filaments and myosin clusters are similar, then the proposed microscopic asymmetry leads to effective contraction of random 1D actomyosin arrays. We discuss the model’s implication for mechanics of contractile rings and stress fibers.

Effects of heat treatment at two temperatures on the myosin cluster of bighead carp for gel formation

ABSTRACTThe α-helix of myosin decreased and the diameter of the myosin clusters increased monotonically with increasing heating time at 50 and 90°C. The clusters that formed during heating at 90°C were much larger than those that formed at 50°C. With increasing heating time, the G′ of the myosin heated at 50°C increased, but it decreased in the myosin heated at 90°C. The rheology data suggested that myosin heated at 50°C exhibited better gel formation than myosin that was heated at 90°C. The myosin gel network formed at 50°C was more consistent and homogeneous, whereas the gel formed 90°C was irregular and sturdy. Myosin heated at 50°C tended to form small, open clusters via heads, and the tails were outstretched, whereas the clusters that formed via myosin heads and tails at 90°C were larger and closed.

Still and rotating myosin clusters determine cytokinetic ring constriction.

The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo.

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.

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 cell's 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.

Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation.

While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin-myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin-myosin network in the lamellipodial/cell body transition zone.