A unified model for the dynamics of ATP-independent ultrafast contraction.

In nature, several ciliated protists possess the remarkable ability to execute ultrafast motions using protein assemblies called myonemes, which contract in response to Ca2+ ions. Existing theories, such as actomyosin contractility and macroscopic biomechanical latches, do not adequately describe these systems, necessitating development of models to understand their mechanisms. In this study, we image and quantitatively analyze the contractile kinematics observed in two ciliated protists (Vorticella sp. and Spirostomum sp.), and, based on the mechanochemistry of these organisms, we propose a minimal mathematical model that reproduces our observations as well as those published previously. Analyzing the model reveals three distinct dynamic regimes, differentiated by the rate of chemical driving and the importance of inertia. We characterize their unique scaling behaviors and kinematic signatures. Besides providing insights into Ca2+-powered myoneme contraction in protists, our work may also inform the rational design of ultrafast bioengineered systems such as active synthetic cells.

Force by minus-end motors Dhc1 and Klp2 collapses the S. pombe spindle after laser ablation.

A microtubule-based machine called the mitotic spindle segregates chromosomes when eukaryotic cells divide. In the fission yeast Schizosaccharomyces pombe, which undergoes closed mitosis, the spindle forms a single bundle of microtubules inside the nucleus. During elongation, the spindle extends via antiparallel microtubule sliding by molecular motors. These extensile forces from the spindle are thought to resist compressive forces from the nucleus. We probe the mechanism and maintenance of this force balance via laser ablation of spindles at various stages of mitosis. We find that spindle pole bodies collapse toward each other after ablation, but spindle geometry is often rescued, allowing spindles to resume elongation. Although this basic behavior has been previously observed, many questions remain about the phenomenon's dynamics, mechanics, and molecular requirements. In this work, we find that previously hypothesized viscoelastic relaxation of the nucleus cannot explain spindle shortening in response to laser ablation. Instead, spindle collapse requires microtubule dynamics and is powered by the minus-end-directed motor proteins dynein Dhc1 and kinesin-14 Klp2, but it does not require the minus-end-directed kinesin Pkl1.

Automated tracking of S. pombe spindle elongation dynamics.

The mitotic spindle is a microtubule-based machine that pulls the two identical sets of chromosomes to opposite ends of the cell during cell division. The fission yeast Schizosaccharomyces pombe is an important model organism for studying mitosis due to its simple, stereotyped spindle structure and well-established genetic toolset. S. pombe spindle length is a useful metric for mitotic progression, but manually tracking spindle ends in each frame to measure spindle length over time is laborious and can limit experimental throughput. We have developed an ImageJ plugin that can automatically track S. pombe spindle length over time and replace manual or semi-automated tracking of spindle elongation dynamics. Using an algorithm that detects the principal axis of the spindle and then finds its ends, we reliably track the length of the spindle as the cell divides. The plugin integrates with existing ImageJ features, exports its data for further analysis outside of ImageJ and does not require any programming by the user. Thus, the plugin provides an accessible tool for quantification of S. pombe spindle length that will allow automatic analysis of large microscopy data sets and facilitate screening for effects of cell biological perturbations on mitotic progression.

The mitotic spindle is a biological machine that pulls the two identical sets of DNA to opposite ends of the cell during cell division. Incorrect cell division can result in serious issues like cancer and miscarriages. Schizosaccharomyces pombe (S. pombe), a kind of yeast, is commonly used to study cell division because its mitotic spindle is essentially linear in shape and its DNA sequence is well known, allowing for more complex experiments. To measure how well a cell divides, we measure the length of the spindle over time, but this can be tedious to do by hand for many cell images. We have developed software that interfaces with ImageJ (a common image analysis tool) that automatically tracks the length of S. pombe spindles over time and can replace manual tracking. Our software calculates the spindle's lines of symmetry, while allows us to accurately measure the length and track the ends over time. It integrates with existing ImageJ features, exports its data for further analysis outside of ImageJ, and does not require any programming by the user. Thus, the plugin provides an accessible tool for measuring S. pombe spindle length that will allow automatic analysis of large microscopy data sets and facilitate screening for effects of defects in cell division. This will facilitate the study of the basic fundamental process of how cells divide, and could have significant long term medical impacts.

Single-molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC).

Resolving single fluorescent molecules in the presence of high fluorophore concentrations remains a challenge in single-molecule biophysics that limits our understanding of weak molecular interactions. Total internal reflection fluorescence (TIRF) imaging, the workhorse of single-molecule fluorescence microscopy, enables experiments at concentrations up to about 100 nM, but many biological interactions have considerably weaker affinities, and thus require at least one species to be at micromolar or higher concentration. Current alternatives to TIRF often require three-dimensional confinement, and thus can be problematic for extended substrates, such as cytoskeletal filaments. To address this challenge, we have demonstrated and applied two new single-molecule fluorescence microscopy techniques, linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC), for imaging the processive motion of molecular motors myosin V and VI along actin filaments. Both technologies will allow imaging in the presence of higher fluorophore concentrations than TIRF microscopy. They will enable new biophysical measurements of a wide range of processive molecular motors that move along filamentous tracks, such as other myosins, dynein, and kinesin. A particularly salient application of these technologies will be to examine chemomechanical coupling by directly imaging fluorescent nucleotide molecules interacting with processive motors as they traverse their actin or microtubule tracks.

Mitosis: Augmin-based bridges keep kinetochores in line.

A recent study highlights the indispensability of the augmin complex for the construction of mitotic spindle bridging fibers, which in turn support accurate chromosome attachment and segregation.

Laser ablation reveals the impact of Cdc15p on the stiffness of the contractile ring.

The mechanics that govern the constriction of the contractile ring remain poorly understood yet are critical to understanding the forces that drive cytokinesis. We used laser ablation in fission yeast cells to unravel these mechanics focusing on the role of Cdc15p as a putative anchoring protein. Our work shows that the severed constricting contractile ring recoils to a finite point leaving a gap that can heal if less than ∼1 µm. Severed contractile rings in Cdc15p-depleted cells exhibit an exaggerated recoil, which suggests that the recoil is limited by the anchoring of the ring to the plasma membrane. Based on a physical model of the severed contractile ring, we propose that Cdc15p impacts the stiffness of the contractile ring more than the viscous drag.

Detailed tuning of structure and intramolecular communication are dispensable for processive motion of myosin VI.

Dimeric myosin VI moves processively hand-over-hand along actin filaments. We have characterized the mechanism of this processive motion by measuring the impact of structural and chemical perturbations on single-molecule processivity. Processivity is maintained despite major alterations in lever arm structure, including replacement of light chain binding regions and elimination of the medial tail. We present kinetic models that can explain the ATP concentration-dependent processivities of myosin VI constructs containing either native or artificial lever arms. We conclude that detailed tuning of structure and intramolecular communication are dispensable for processive motion, and further show theoretically that one proposed type of nucleotide gating can be detrimental rather than beneficial for myosin processivity.

Cytoskeletal biophysics: Passive crosslinker adapts to keep microtubule bundles on track.

Assembly of the mitotic spindle requires dynamic adaptation and coordination among an array of motors and crosslinkers. A new study demonstrates in vitro how the mitotic crosslinker PRC1 can tune its behavior to regulate the speed of microtubule sliding.

Viscoelastic Relaxation of the Nuclear Envelope Does Not Cause the Collapse of the Spindle After Ablation in S. pombe.

A large molecular machine called the mitotic spindle is responsible for accurate chromosome segregation in eukaryotic cells. The spindle consists of protein filaments known as microtubules and microtubule-associated proteins such as motors and crosslinkers, which help impart its organization. In the case of the fission yeast S. pombe, these form a single bundle inside the nucleus. During spindle elongation, sliding by motor proteins provides an internal source of extensile forces, which are resisted by the compressive forces of the nuclear envelope. To probe the sources of this force balance, we cut the spindle using focused laser light at various stages of spindle elongation. We find that the spindle pole bodies collapse toward each other post-ablation. While this basic behavior has been previously observed, many questions remain about the timing, mechanics, and molecular requirements of this phenomenon. Here, we quantify the time scale of the relaxation and probe its underlying mechanism. We demonstrate that viscoelastic relaxation of the nuclear envelope cannot explain this phenomenon and provide evidence of active forces as the underlying mechanism.

K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15.

The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes during cell division. In mammalian cells, microtubule bundles called kinetochore fibers (k-fibers) connect chromosomes to the spindle poles. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here we investigate the physical and molecular basis of k-fiber bundle cohesion. We detach k-fibers from poles by laser ablation-based cutting, thus revealing the contribution of pole-localized forces to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced by ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15-microtubule binding reduces the mechanical integrity of k-fibers. In contrast, inhibition of its motor activity but not its microtubule binding ability, i.e., locking Kif15 into a rigor state, does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule cross-linker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors.

Knitting Ripples.

Ripples are common in both biological systems and human clothes. Knitters have long exploited the ability of fabric to curl out of plane, by either removing or adding stitches to the material as it is created. Here, we present two knitting patterns for scarves to illustrate how ripples can arise through such additive processes.

The Spindle: Integrating Architecture and Mechanics across Scales.

The spindle segregates chromosomes at cell division, and its task is a mechanical one. While we have a nearly complete list of spindle components, how their molecular-scale mechanics give rise to cellular-scale spindle architecture, mechanics, and function is not yet clear. Recent in vitro and in vivo measurements bring new levels of molecular and physical control and shed light on this question. Highlighting recent findings and open questions, we introduce the molecular force generators of the spindle, and discuss how they organize microtubules into diverse architectural modules and give rise to the emergent mechanics of the mammalian spindle. Throughout, we emphasize the breadth of space and time scales at play, and the feedback between spindle architecture, dynamics, and mechanics that drives robust function.

The chromokinesin Klp3a and microtubules facilitate acentric chromosome segregation.

Although poleward segregation of acentric chromosomes is well documented, the underlying mechanisms remain poorly understood. Here, we demonstrate that microtubules play a key role in poleward movement of acentric chromosome fragments generated in Drosophila melanogaster neuroblasts. Acentrics segregate with either telomeres leading or lagging in equal frequency and are preferentially associated with peripheral bundled microtubules. In addition, laser ablation studies demonstrate that segregating acentrics are mechanically associated with microtubules. Finally, we show that successful acentric segregation requires the chromokinesin Klp3a. Reduced Klp3a function results in disorganized interpolar microtubules and shortened spindles. Normally, acentric poleward segregation occurs at the periphery of the spindle in association with interpolar microtubules. In klp3a mutants, acentrics fail to localize and segregate along the peripheral interpolar microtubules and are abnormally positioned in the spindle interior. These studies demonstrate an unsuspected role for interpolar microtubules in driving acentric segregation.

Mapping Load-Bearing in the Mammalian Spindle Reveals Local Kinetochore Fiber Anchorage that Provides Mechanical Isolation and Redundancy.

Active forces generated at kinetochores move chromosomes, and the dynamic spindle must robustly anchor kinetochore fibers (k-fibers) to bear this load. The mammalian spindle bears the load of chromosome movement far from poles, but we do not know where and how-physically and molecularly-this load distributes across the spindle. In part, this is because probing spindle mechanics in live cells is difficult. Yet answering this question is key to understanding how the spindle generates and responds to force and performs its diverse mechanical functions. Here, we map load-bearing across the mammalian spindle in space-time and dissect local anchorage mechanics and mechanism. To do so, we laser-ablate single k-fibers at different spindle locations and in different molecular backgrounds and quantify the immediate relaxation of chromosomes, k-fibers, and microtubule speckles. We find that load redistribution is locally confined in all directions: along the first 3-4 µm from kinetochores, scaling with k-fiber length, and laterally within ∼2 µm of k-fiber sides, without detectable load sharing between neighboring k-fibers. A phenomenological model suggests that dense, transient crosslinks to the spindle along k-fibers bear the load of chromosome movement but that these connections do not limit the timescale of spindle reorganization. The microtubule crosslinker NuMA is needed for the local load-bearing observed, whereas Eg5 and PRC1 are not detectably required, suggesting specialization in mechanical function. Together, the data and model suggest that NuMA-mediated crosslinks locally bear load, providing mechanical isolation and redundancy while allowing spindle fluidity. These features are well suited to support robust chromosome segregation.

Force on spindle microtubule minus ends moves chromosomes.

The spindle is a dynamic self-assembling machine that coordinates mitosis. The spindle's function depends on its ability to organize microtubules into poles and maintain pole structure despite mechanical challenges and component turnover. Although we know that dynein and NuMA mediate pole formation, our understanding of the forces dynamically maintaining poles is limited: we do not know where and how quickly they act or their strength and structural impact. Using laser ablation to cut spindle microtubules, we identify a force that rapidly and robustly pulls severed microtubules and chromosomes poleward, overpowering opposing forces and repairing spindle architecture. Molecular imaging and biophysical analysis suggest that transport is powered by dynein pulling on minus ends of severed microtubules. NuMA and dynein/dynactin are specifically enriched at new minus ends within seconds, reanchoring minus ends to the spindle and delivering them to poles. This force on minus ends represents a newly uncovered chromosome transport mechanism that is independent of plus end forces at kinetochores and is well suited to robustly maintain spindle mechanical integrity.

Future challenges in single-molecule fluorescence and laser trap approaches to studies of molecular motors.

Single-molecule analysis is a powerful modern form of biochemistry, in which individual kinetic steps of a catalytic cycle of an enzyme can be explored in exquisite detail. Both single-molecule fluorescence and single-molecule force techniques have been widely used to characterize a number of protein systems. We focus here on molecular motors as a paradigm. We describe two areas where we expect to see exciting developments in the near future: first, characterizing the coupling of force production to chemical and mechanical changes in motors, and second, understanding how multiple motors work together in the environment of the cell.

Engineered myosin VI motors reveal minimal structural determinants of directionality and processivity.

Myosins have diverse mechanical properties reflecting a range of cellular roles. A major challenge is to understand the structural basis for generating novel functions from a common motor core. Myosin VI (M6) is specialized for processive motion toward the (-) end of actin filaments. We have used engineered M6 motors to test and refine the "redirected power stroke" model for (-) end directionality and to explore poorly understood structural requirements for processive stepping. Guided by crystal structures and molecular modeling, we fused artificial lever arms to the catalytic head of M6 at several positions, retaining varying amounts of native structure. We found that an 18-residue alpha-helical insert is sufficient to reverse the directionality of the motor, with no requirement for any calmodulin light chains. Further, we observed robust processive stepping of motors with artificial lever arms, demonstrating that processivity can arise without optimizing lever arm composition or mechanics.

Rapid membrane fusion of individual virus particles with supported lipid bilayers.

Many enveloped viruses employ low-pH-triggered membrane fusion during cell penetration. Solution-based in vitro assays in which viruses fuse with liposomes have provided much of our current biochemical understanding of low-pH-triggered viral membrane fusion. Here, we extend this in vitro approach by introducing a fluorescence assay using single particle tracking to observe lipid mixing between individual virus particles (influenza or Sindbis) and supported lipid bilayers. Our single-particle experiments reproduce many of the observations of the solution assays. The single-particle approach naturally separates the processes of membrane binding and membrane fusion and therefore allows measurement of details that are not available in the bulk assays. We find that the dynamics of lipid mixing during individual Sindbis fusion events is faster than 30 ms. Although neither virus binds membranes at neutral pH, under acidic conditions, the delay between membrane binding and lipid mixing is less than half a second for nearly all virus-membrane combinations. The delay between binding and lipid mixing lengthened only for Sindbis virus at the lowest pH in a cholesterol-dependent manner, highlighting the complex interaction between lipids, virus proteins, and buffer conditions in membrane fusion.