Asymmetric friction of nonmotor MAPs can lead to their directional motion in active microtubule networks.

Diverse cellular processes require microtubules to be organized into distinct structures, such as asters or bundles. Within these dynamic motifs, microtubule-associated proteins (MAPs) are frequently under load, but how force modulates these proteins' function is poorly understood. Here, we combine optical trapping with TIRF-based microscopy to measure the force dependence of microtubule interaction for three nonmotor MAPs (NuMA, PRC1, and EB1) required for cell division. We find that frictional forces increase nonlinearly with MAP velocity across microtubules and depend on filament polarity, with NuMA's friction being lower when moving toward minus ends, EB1's lower toward plus ends, and PRC1's exhibiting no directional preference. Mathematical models predict, and experiments confirm, that MAPs with asymmetric friction can move directionally within actively moving microtubule pairs they crosslink. Our findings reveal how nonmotor MAPs can generate frictional resistance in dynamic cytoskeletal networks via micromechanical adaptations whose anisotropy may be optimized for MAP localization and function within cellular structures.

Insights into the micromechanical properties of the metaphase spindle.

The microtubule-based metaphase spindle is subjected to forces that act in diverse orientations and over a wide range of timescales. Currently, we cannot explain how this dynamic structure generates and responds to forces while maintaining overall stability, as we have a poor understanding of its micromechanical properties. Here, we combine the use of force-calibrated needles, high-resolution microscopy, and biochemical perturbations to analyze the vertebrate metaphase spindle's timescale- and orientation-dependent viscoelastic properties. We find that spindle viscosity depends on microtubule crosslinking and density. Spindle elasticity can be linked to kinetochore and nonkinetochore microtubule rigidity, and also to spindle pole organization by kinesin-5 and dynein. These data suggest a quantitative model for the micromechanics of this cytoskeletal architecture and provide insight into how structural and functional stability is maintained in the face of forces, such as those that control spindle size and position, and can result from deformations associated with chromosome movement.

Geometric trade-off between contractile force and viscous drag determines the actomyosin-based motility of a cell-sized droplet.

Cell migration in confined environments is fundamental for diverse biological processes from cancer invasion to leukocyte trafficking. The cell body is propelled by the contractile force of actomyosin networks transmitted from the cell membrane to the external substrates. However, physical determinants of actomyosin-based migration capacity in confined environments are not fully understood. Here, we develop an in vitro migratory cell model, where cytoplasmic actomyosin networks are encapsulated into droplets surrounded by a lipid monolayer membrane. We find that the droplet can move when the actomyosin networks are bound to the membrane, in which the physical interaction between the contracting actomyosin networks and the membrane generates a propulsive force. The droplet moves faster when it has a larger contact area with the substrates, while narrower confinement reduces the migration speed. By combining experimental observations and active gel theory, we propose a mechanism where the balance between sliding friction force, which is a reaction force of the contractile force, and viscous drag determines the migration speed, providing a physical basis of actomyosin-based motility in confined environments.

Morphological growth dynamics, mechanical stability, and active microtubule mechanics underlying spindle self-organization.

The spindle is a dynamic intracellular structure self-organized from microtubules and microtubule-associated proteins. The spindle's bipolar morphology is essential for the faithful segregation of chromosomes during cell division, and it is robustly maintained by multifaceted mechanisms. However, abnormally shaped spindles, such as multipolar spindles, can stochastically arise in a cell population and cause chromosome segregation errors. The physical basis of how microtubules fail in bipolarization and occasionally favor nonbipolar assembly is poorly understood. Here, using live fluorescence imaging and quantitative shape analysis in Xenopus egg extracts, we find that spindles of varied shape morphologies emerge through nonrandom, bistable self-organization paths, one leading to a bipolar and the other leading to a multipolar phenotype. The bistability defines the spindle's unique morphological growth dynamics linked to each shape phenotype and can be promoted by a locally distorted microtubule flow that arises within premature structures. We also find that bipolar and multipolar spindles are stable at the steady-state in bulk but can infrequently switch between the two phenotypes. Our microneedle-based physical manipulation further demonstrates that a transient force perturbation applied near the assembled pole can trigger the phenotypic switching, revealing the mechanical plasticity of the spindle. Together with molecular perturbation of kinesin-5 and augmin, our data propose the physical and molecular bases underlying the emergence of spindle-shape variation, which influences chromosome segregation fidelity during cell division.

Micromechanics of the vertebrate meiotic spindle examined by stretching along the pole-to-pole axis.

The meiotic spindle is a bipolar molecular machine that is designed to segregate duplicated chromosomes toward the opposite poles of the cell. The size and shape of the spindle are considered to be maintained by a balance of forces produced by molecular motors and microtubule assembly dynamics. Several studies have probed how mechanical perturbations of the force balance affect the spindle structure. However, the spindle's response to a stretching force acting at the spindle pole and along its long axis, i.e., the direction in which chromosomes are segregated, has not been examined. Here, we describe a method to apply a stretching force to the metaphase spindle assembled in Xenopus egg extracts and measure the relationship between the force and the three-dimensional deformation of the spindle. We found that the spindle behaves as a Zener-type viscoelastic body when forces are applied at the spindle pole, generating a restoring force for several minutes. In addition, both the volume of the spindle and the tubulin density are conserved under the stretching force. These results provide insight into how the spindle size is maintained at metaphase.

PIGB maintains nuclear lamina organization in skeletal muscle of Drosophila.

The nuclear lamina (NL) plays various roles and participates in nuclear integrity, chromatin organization, and transcriptional regulation. Lamin proteins, the main components of the NL, form a homogeneous meshwork structure under the nuclear envelope. Lamins are essential, but it is unknown whether their homogeneous distribution is important for nuclear function. Here, we found that PIGB, an enzyme involved in glycosylphosphatidylinositol (GPI) synthesis, is responsible for the homogeneous lamin meshwork in Drosophila. Loss of PIGB resulted in heterogeneous distributions of B-type lamin and lamin-binding proteins in larval muscles. These phenotypes were rescued by expression of PIGB lacking GPI synthesis activity. The PIGB mutant exhibited changes in lamina-associated domains that are large heterochromatic genomic regions in the NL, reduction of nuclear stiffness, and deformation of muscle fibers. These results suggest that PIGB maintains the homogeneous meshwork of the NL, which may be essential for chromatin distribution and nuclear mechanical properties.

Locally and globally coupled oscillators in muscle.

At an intermediate activation level, striated muscle exhibits autonomous oscillations called SPOC, in which the basic contractile units, sarcomeres, oscillate in length, and various oscillatory patterns such as traveling waves and their disrupted forms appear in a myofibril. Here we show that these patterns are reproduced by mechanically connecting in series the unit model that explains characteristics of SPOC at the single-sarcomere level. We further reduce the connected model to phase equations, revealing that the combination of local and global couplings is crucial to the emergence of these patterns.

Confined-microtubule assembly shapes three-dimensional cell wall structures in xylem vessels.

Properly patterned deposition of cell wall polymers is prerequisite for the morphogenesis of plant cells. A cortical microtubule array guides the two-dimensional pattern of cell wall deposition. Yet, the mechanism underlying the three-dimensional patterning of cell wall deposition is poorly understood. In metaxylem vessels, cell wall arches are formed over numerous pit membranes, forming highly organized three-dimensional cell wall structures. Here, we show that the microtubule-associated proteins, MAP70-5 and MAP70-1, regulate arch development. The map70-1 map70-5 plants formed oblique arches in an abnormal orientation in pits. Microtubules fit the aperture of developing arches in wild-type cells, whereas microtubules in map70-1 map70-5 cells extended over the boundaries of pit arches. MAP70 caused the bending and bundling of microtubules. These results suggest that MAP70 confines microtubules within the pit apertures by altering the physical properties of microtubules, thereby directing the growth of pit arches in the proper orientation. This study provides clues to understanding how plants develop three-dimensional structure of cell walls.

Probing the mechanical architecture of the vertebrate meiotic spindle.

Accurate chromosome segregation during meiosis depends on the assembly of a microtubule-based spindle of proper shape and size. Current models for spindle-size control focus on reaction diffusion-based chemical regulation and balance in activities of motor proteins. Although several molecular perturbations have been used to test these models, controlled mechanical perturbations have not been possible. Here we report a piezoresistive dual cantilever-based system to test models for spindle-size control and examine the mechanical features, such as deformability and stiffness, of the vertebrate meiotic spindle. We found that meiotic spindles prepared in Xenopus laevis egg extracts were viscoelastic and recovered their original shape in response to small compression. Larger compression resulted in plastic deformation, but the spindle adapted to this change, establishing a stable mechanical architecture at different sizes. The technique we describe here may also be useful for examining the micromechanics of other cellular organelles.

Inter-sarcomere coordination in muscle revealed through individual sarcomere response to quick stretch.

The force generation and motion of muscle are produced by the collective work of thousands of sarcomeres, the basic structural units of striated muscle. Based on their series connection to form a myofibril, it is expected that sarcomeres are mechanically and/or structurally coupled to each other. However, the behavior of individual sarcomeres and the coupling dynamics between sarcomeres remain elusive, because muscle mechanics has so far been investigated mainly by analyzing the averaged behavior of thousands of sarcomeres in muscle fibers. In this study, we directly measured the length-responses of individual sarcomeres to quick stretch at partial activation, using micromanipulation of skeletal myofibrils under a phase-contrast microscope. The experiments were performed at ADP-activation (1 mM MgATP and 2 mM MgADP in the absence of Ca(2+)) and also at Ca(2+)-activation (1 mM MgATP at pCa 6.3) conditions. We show that under these activation conditions, sarcomeres exhibit 2 distinct types of responses, either "resisting" or "yielding," which are clearly distinguished by the lengthening distance of single sarcomeres in response to stretch. These 2 types of sarcomeres tended to coexist within the myofibril, and the sarcomere "yielding" occurred in clusters composed of several adjacent sarcomeres. The labeling of Z-line with anti-alpha-actinin antibody significantly suppressed the clustered sarcomere "yielding." These results strongly suggest that the contractile system of muscle possesses the mechanism of structure-based inter-sarcomere coordination.

Local body weight measurement of the spindle.

The spindle is a micron-sized chromosome segregation machine built from microtubules and many other proteins. In this issue of Developmental Cell, Biswas et al. (2021) use sophisticated imaging and Xenopus egg extracts to show that the spindle's mass density is only as much as the surrounding cytoplasm, contrary to popular belief.

RanGTP and the actin cytoskeleton keep paternal and maternal chromosomes apart during fertilization.

Zygotes require two accurate sets of parental chromosomes, one each from the mother and the father, to undergo normal embryogenesis. However, upon egg-sperm fusion in vertebrates, the zygote has three sets of chromosomes, one from the sperm and two from the egg. The zygote therefore eliminates one set of maternal chromosomes (but not the paternal chromosomes) into the polar body through meiosis, but how the paternal chromosomes are protected from maternal meiosis has been unclear. Here we report that RanGTP and F-actin dynamics prevent egg-sperm fusion in proximity to maternal chromosomes. RanGTP prevents the localization of Juno and CD9, egg membrane proteins that mediate sperm fusion, at the cell surface in proximity to maternal chromosomes. Following egg-sperm fusion, F-actin keeps paternal chromosomes away from maternal chromosomes. Disruption of these mechanisms causes the elimination of paternal chromosomes during maternal meiosis. This study reveals a novel critical mechanism that prevents aneuploidy in zygotes.

Nanoscopic changes in the lattice structure of striated muscle sarcomeres involved in the mechanism of spontaneous oscillatory contraction (SPOC).

Muscles perform a wide range of motile functions in animals. Among various types are skeletal and cardiac muscles, which exhibit a steady auto-oscillation of force and length when they are activated at an intermediate level of contraction. This phenomenon, termed spontaneous oscillatory contraction or SPOC, occurs devoid of cell membranes and at fixed concentrations of chemical substances, and is thus the property of the contractile system per se. We have previously developed a theoretical model of SPOC and proposed that the oscillation emerges from a dynamic force balance along both the longitudinal and lateral axes of sarcomeres, the contractile units of the striated muscle. Here, we experimentally tested this hypothesis by developing an imaging-based analysis that facilitates detection of the structural changes of single sarcomeres at unprecedented spatial resolution. We found that the sarcomere width oscillates anti-phase with the sarcomere length in SPOC. We also found that the oscillatory dynamics can be altered by osmotic compression of the myofilament lattice structure of sarcomeres, but they are unchanged by a proteolytic digestion of titin/connectin-the spring-like protein that provides passive elasticity to sarcomeres. Our data thus reveal the three-dimensional mechanical dynamics of oscillating sarcomeres and suggest a structural requirement of steady auto-oscillation.

Mechanically Distinct Microtubule Arrays Determine the Length and Force Response of the Meiotic Spindle.

The microtubule-based spindle is subjected to various mechanical forces during cell division. How the structure generates and responds to forces while maintaining overall integrity is unknown because we have a poor understanding of the relationship between filament architecture and mechanics. Here, to fill this gap, we combine microneedle-based quantitative micromanipulation with high-resolution imaging, simultaneously analyzing forces and local filament motility in the Xenopus meiotic spindle. We find that microtubules exhibit a compliant, fluid-like mechanical response at the middle of the spindle half, being distinct from those near the pole and the equator. A force altering spindle length induces filament sliding at this compliant array, where parallel microtubules predominate, without influencing equatorial antiparallel filament dynamics. Molecular perturbations suggest that kinesin-5 and dynein contribute to the spindle's local mechanical difference. Together, our data establish a link between spindle architecture and mechanics and uncover the mechanical design of this essential cytoskeletal assembly.

Length-dependent activation and auto-oscillation in skeletal myofibrils at partial activation by Ca2+.

The length-dependent activation of skeletal myofibrils was examined at the single-sarcomere level with phase-contrast microscopy at sarcomere length (SL) >2.2 microm. At the maximal activation by Ca(2+) (pCa 4.5) the active force linearly decreased with increasing SL, while at partial activation by Ca(2+) (pCa 6.1-6.5) the larger active force was generated at longer SL. Throughout these experiments, the distribution of SL was kept homogeneous upon activation. In addition, we found that the spontaneous oscillation of force and SL frequently occurs in the SL range 2.2-2.6 microm at pCa 6.1-6.2. Either changes in [Ca(2+)] or osmotic compression of the myofilament lattice induced by the addition of dextran T-500, affected both the length dependence of activation and the occurrence of auto-oscillation. These results suggest that the force-generating properties of sarcomeres in striated muscle are determined not only by [Ca(2+)], but also by the lattice spacing as a function of SL.

Chromatin as a nuclear spring.

The nucleus in eukaryotic cells is the site for genomic functions such as RNA transcription, DNA replication, and DNA repair/recombination. However, the nucleus is subjected to various mechanical forces associated with diverse cellular activities, including contraction, migration, and adhesion. Although it has long been assumed that the lamina structure, underlying filamentous mesh-work of the nuclear envelope, plays an important role in resisting mechanical forces, the involvement of compact chromatin in mechanical resistance has also recently been suggested. However, it is still unclear how chromatin functions to cope with the stresses. To address this issue, we studied the mechanical responses of human cell nuclei by combining a force measurement microscopy setup with controlled biochemical manipulation of chromatin. We found that nuclei with condensed chromatin possess significant elastic rigidity, whereas the nuclei with a decondensed chromatin are considerably soft. Further analyses revealed that the linker DNA and nucleosome-nucleosome interactions via histone tails in the chromatin act together to generate a spring-like restoring force that resists nuclear deformation. The elastic restoring force is likely to be generated by condensed chromatin domains, consisting of interdigitated or "melted" 10-nm nucleosome fibers. Together with other recent studies, it is suggested that chromatin functions not only as a "memory device" to store, replicate, and express the genetic information for various cellular functions but also as a "nuclear spring" to resist and respond to mechanical forces.

Analyzing the micromechanics of the cell division apparatus.

Cell division involves mechanical processes, such as chromosome transport and centrosome separation. Quantitative micromanipulation-based approaches have been central to dissecting the forces driving these processes. We highlight two biophysical assays that can be employed for such analyses. First, an in vitro "mini-spindle" assay is described that can be used to examine the collective mechanics of mitotic motor proteins cross-linking two microtubules. In the spindle, motor proteins (e.g., kinesin-5, kinesin-14, and dynein) can localize to overlapping microtubules that slide relative to each other, work as an ensemble, and equilibrate between cytoplasm and the microtubules. The "mini-spindle" assay can recapitulate these features and allows measurements of forces generated between adjacent microtubules and their dependence on filament orientation, sliding speed, overlap length, and motor protein density. Second, we describe a force-calibrated microneedle-based "whole-spindle" micromechanics assay. Microneedle-based micromanipulation can be a useful technique to examine cellular scale mechanics, but its use has been restricted by the difficulty in getting probes to penetrate the plasma membrane without disrupting cell physiology. As detailed here, the use of cell-free extracts prepared from metaphase-arrested Xenopus eggs can address this limitation. These micromanipulation studies also benefit from the use of frozen stocks of Xenopus egg extract. Together, these approaches can be used to decipher how micromechanics and biochemical activities ensure successful cell division.

Nonlinear force-length relationship in the ADP-induced contraction of skeletal myofibrils.

The regulatory mechanism of sarcomeric activity has not been fully clarified yet because of its complex and cooperative nature, which involves both Ca(2+) and cross-bridge binding to the thin filament. To reveal the mechanism of regulation mediated by the cross-bridges, separately from the effect of Ca(2+), we investigated the force-sarcomere length (SL) relationship in rabbit skeletal myofibrils (a single myofibril or a thin bundle) at SL > 2.2 microm in the absence of Ca(2+) at various levels of activation by exogenous MgADP (4-20 mM) in the presence of 1 mM MgATP. The individual SLs were measured by phase-contrast microscopy to confirm the homogeneity of the striation pattern of sarcomeres during activation. We found that at partial activation with 4-8 mM MgADP, the developed force nonlinearly depended on the length of overlap between the thick and the thin filaments; that is, contrary to the maximal activation, the maximal active force was generated at shorter overlap. Besides, the active force became larger, whereas this nonlinearity tended to weaken, with either an increase in [MgADP] or the lateral osmotic compression of the myofilament lattice induced by the addition of a macromolecular compound, dextran T-500. The model analysis, which takes into account the [MgADP]- and the lattice-spacing-dependent probability of cross-bridge formation, was successfully applied to account for the force-SL relationship observed at partial activation. These results strongly suggest that the cross-bridge works as a cooperative activator, the function of which is highly sensitive to as little as

Regulation of muscle contraction by Ca2+ and ADP: focusing on the auto-oscillation (SPOC).

A molecular motor in striated muscle, myosin II, is a non-processive motor that is unable to perform physiological functions as a single molecule and acts as an assembly of molecules. It is widely accepted that a myosin II motor is an independent force generator; the force generated at a steady state is usually considered to be a simple sum of those generated by each motor. This is the case at full activation (pCa < 5 in the presence of MgATP); however, we found that the myosin II motors show cooperative functions, i.e., non-linear auto-oscillation, named SPOC (SPontaneous Oscillatory Contraction), when the activation level is intermediate between those of contraction and relaxation (that is, at the intermediate level of pCa, 5-6, for cardiac muscle, or at the coexistence of MgATP, MgADP and inorganic phosphate (Pi) at higher pCa (> 7) for both skeletal and cardiac muscles). Here, we summarize the characteristics of SPOC phenomena, especially focusing on the physiological significance of SPOC in cardiac muscle. We propose a new concept that the auto-oscillatory property, which is inherent to the contractile system of cardiac muscle, underlies the molecular mechanism of heartbeat. Additionally, we briefly describe the dynamic properties of the thin filaments, i.e., the Ca(2+)-dependent flexibility change of the thin filaments, which may be the basis for the SPOC phenomena. We also describe a newly developed experimental system named "bio-nanomuscle," in which tension is asserted on a single reconstituted thin filament by interacting with crossbridges in the A-band composed of the thick filament lattice. This newly devised hybrid system is expected to fill the gap between the single-molecule level and the muscle system.