The kinesin-5 tail and bipolar minifilament domains are the origin of its microtubule crosslinking and sliding activity.

Kinesin-5 crosslinks and slides apart microtubules to assemble, elongate, and maintain the mitotic spindle. Kinesin-5 is a tetramer, where two N-terminal motor domains are positioned at each end of the motor, and the coiled-coil stalk domains are organized into a tetrameric bundle through the bipolar assembly (BASS) domain. To dissect the function of the individual structural elements of the motor, we constructed a minimal kinesin-5 tetramer (mini-tetramer). We determined the x-ray structure of the extended, 34-nm BASS domain. Guided by these structural studies, we generated active bipolar kinesin-5 mini-tetramer motors from Drosophila melanogastor and human orthologues which are half the length of native kinesin-5. We then used these kinesin-5 mini-tetramers to examine the role of two unique structural adaptations of kinesin-5: 1) the length and flexibility of the tetramer, and 2) the C-terminal tails which interact with the motor domains to coordinate their ATPase activity. The C-terminal domain causes frequent pausing and clustering of kinesin-5. By comparing microtubule crosslinking and sliding by mini-tetramer and full-length kinesin-5, we find that both the length and flexibility of kinesin-5 and the C-terminal tails govern its ability to crosslink microtubules. Once crosslinked, stiffer mini-tetramers slide antiparallel microtubules more efficiently than full-length motors.

Directly Measuring Forces within Reconstituted Active Microtubule Bundles.

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Two modes of PRC1-mediated mechanical resistance to kinesin-driven microtubule network disruption.

The proper organization of the microtubule-based spindle during cell division requires the collective activity of many different proteins. These include non-motor microtubule-associated proteins (MAPs), whose functions include crosslinking microtubules to regulate filament sliding rates and assemble microtubule arrays. One such protein is PRC1, an essential MAP that has been shown to preferentially crosslink overlapping antiparallel microtubules at the spindle midzone. PRC1 has been proposed to act as a molecular brake, but insight into the mechanism of how PRC1 molecules function cooperatively to resist motor-driven microtubule sliding and to allow for the formation of stable midzone overlaps remains unclear. Here, we employ a modified microtubule gliding assay to rupture PRC1-mediated microtubule pairs using surface-bound kinesins. We discovered that PRC1 crosslinks always reduce bundled filament sliding velocities relative to single-microtubule gliding rates and do so via two distinct emergent modes of mechanical resistance to motor-driven sliding. We term these behaviors braking and coasting, where braking events exhibit substantially slowed microtubule sliding compared to coasting events. Strikingly, braking behavior requires the formation of two distinct high-density clusters of PRC1 molecules near microtubule tips. Our results suggest a cooperative mechanism for PRC1 accumulation when under mechanical load that leads to a unique state of enhanced resistance to filament sliding and provides insight into collective protein ensemble behavior in regulating the mechanics of spindle assembly.

The Mitotic Crosslinking Protein PRC1 Acts Like a Mechanical Dashpot to Resist Microtubule Sliding.

Cell division in eukaryotes requires the regulated assembly of the spindle apparatus. The proper organization of microtubules within the spindle is driven by motor proteins that exert forces to slide filaments, whereas non-motor proteins crosslink filaments into higher-order motifs, such as overlapping bundles. It is not clear how active and passive forces are integrated to produce regulated mechanical outputs within spindles. Here, we employ simultaneous optical trapping and total internal reflection fluorescence (TIRF) microscopy to directly measure the frictional forces produced by the mitotic crosslinking protein PRC1 that resist microtubule sliding. These forces scale with microtubule sliding velocity and the number of PRC1 crosslinks but do not depend on overlap length or PRC1 density within overlaps. Our results suggest that PRC1 ensembles act similarly to a mechanical dashpot, producing significant resistance against fast motions but minimal resistance against slow motions, allowing for the integration of diverse motor activities into a single mechanical outcome.

Substrate-Enzyme Interactions in Intramembrane Proteolysis: γ-Secretase as the Prototype.

Intramembrane-cleaving proteases (I-CLiPs) catalyze the hydrolysis of peptide bonds within the transmembrane regions of membrane protein substrates, releasing bioactive fragments that play roles in many physiological and pathological processes. Based on their catalytic mechanism and nucleophile, I-CLiPs are classified into metallo, serine, aspartyl, and glutamyl proteases. Presenilin is the most prominent among I-CLiPs, as the catalytic subunit of γ-secretase (GS) complex responsible for cleaving the amyloid precursor protein (APP) and Notch, as well as many other membrane substrates. Recent cryo-electron microscopy (cryo-EM) structures of GS provide new details on how presenilin recognizes and cleaves APP and Notch. First, presenilin transmembrane helix (TM) 2 and 6 are dynamic. Second, upon binding to GS, the substrate TM helix is unwound from the C-terminus, resulting in an intermolecular ß-sheet between the substrate and presenilin. The transition of the substrate C-terminus from α-helix to ß-sheet is proposed to expose the scissile peptide bond in an extended conformation, leaving it susceptible to protease cleavage. Despite the astounding new insights in recent years, many crucial questions remain unanswered regarding the inner workings of γ-secretase, however. Key unanswered questions include how the enzyme recognizes and recruits substrates, how substrates are translocated from an initial docking site to the active site, how active site aspartates recruit and coordinate catalytic water, and the nature of the mechanisms of processive trimming of the substrate and product release. Answering these questions will have important implications for drug discovery aimed at selectively reducing the amyloid load in Alzheimer's disease (AD) with minimal side effects.

The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding.

Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.

High-resolution imaging reveals how the spindle midzone impacts chromosome movement.

In the spindle midzone, microtubules from opposite half-spindles form bundles between segregating chromosomes. Microtubule bundles can either push or restrict chromosome movement during anaphase in different cellular contexts, but how these activities are achieved remains poorly understood. Here, we use high-resolution live-cell imaging to analyze individual microtubule bundles, growing filaments, and chromosome movement in dividing human cells. Within bundles, filament overlap length marked by the cross-linking protein PRC1 decreases during anaphase as chromosome segregation slows. Filament ends within microtubule bundles appear capped despite dynamic PRC1 turnover and submicrometer proximity to growing microtubules. Chromosome segregation distance and rate are increased in two human cell lines when microtubule bundle assembly is prevented via PRC1 knockdown. Upon expressing a mutant PRC1 with reduced microtubule affinity, bundles assemble but chromosome hypersegregation is still observed. We propose that microtubule overlap length reduction, typically linked to pushing forces generated within filament bundles, is needed to properly restrict spindle elongation and position chromosomes within daughter cells.

The mechanics of microtubule networks in cell division.

The primary goal of a dividing somatic cell is to accurately and equally segregate its genome into two new daughter cells. In eukaryotes, this process is performed by a self-organized structure called the mitotic spindle. It has long been appreciated that mechanical forces must be applied to chromosomes. At the same time, the network of microtubules in the spindle must be able to apply and sustain large forces to maintain spindle integrity. Here we consider recent efforts to measure forces generated within microtubule networks by ensembles of key proteins. New findings, such as length-dependent force generation, protein clustering by asymmetric friction, and entropic expansion forces will help advance models of force generation needed for spindle function and maintaining integrity.

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends.

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human ß-tubulin isotype III (TUBB3), as well as heterodimers with one of two ß-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

Measuring Pushing and Braking Forces Generated by Ensembles of Kinesin-5 Crosslinking Two Microtubules.

The proper organization of the microtubule-based mitotic spindle is proposed to depend on nanometer-sized motor proteins generating forces that scale with a micron-sized geometric feature, such as microtubule overlap length. However, it is unclear whether such regulation can be achieved by any mitotic motor protein. Here, we employ an optical-trap- and total internal reflection fluorescence (TIRF)-based assay to show that ensembles of kinesin-5, a conserved mitotic motor protein, can push apart overlapping antiparallel microtubules to generate a force whose magnitude scales with filament overlap length. We also find that kinesin-5 can produce overlap-length-dependent "brake-like" resistance against relative microtubule sliding in both parallel and antiparallel geometries, an activity that has been suggested by cell biological studies but had not been directly measured. Together, these findings, along with numerical simulations, reveal how a motor protein can function as an analog converter, "reading" simple geometric and dynamic features in cytoskeletal networks to produce regulated force outputs.

Dynamic regulation of transcription factors by nucleosome remodeling.

The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes.

Reconstitution of the augmin complex provides insights into its architecture and function.

Proper microtubule nucleation during cell division requires augmin, a microtubule-associated hetero-octameric protein complex. In current models, augmin recruits γ-tubulin, through the carboxyl terminus of its hDgt6 subunit to nucleate microtubules within spindles. However, augmin's biochemical complexity has restricted analysis of its structural organization and function. Here, we reconstitute human augmin and show that it is a Y-shaped complex that can adopt multiple conformations. Further, we find that a dimeric sub-complex retains in vitro microtubule-binding properties of octameric complexes, but not proper metaphase spindle localization. Addition of octameric augmin complexes to Xenopus egg extracts promotes microtubule aster formation, an activity enhanced by Ran-GTP. This activity requires microtubule binding, but not the characterized hDgt6 γ-tubulin-recruitment domain. Tetrameric sub-complexes induce asters, but activity and microtubule bundling within asters are reduced compared with octameric complexes. Together, our findings shed light on augmin's structural organization and microtubule-binding properties, and define subunits required for its function in organizing microtubule-based structures.

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.

Torque measurement at the single-molecule level.

Methods for exerting and measuring forces on single molecules have revolutionized the study of the physics of biology. However, it is often the case that biological processes involve rotation or torque generation, and these parameters have been more difficult to access experimentally. Recent advances in the single-molecule field have led to the development of techniques that add the capability of torque measurement. By combining force, displacement, torque, and rotational data, a more comprehensive description of the mechanics of a biomolecule can be achieved. In this review, we highlight a number of biological processes for which torque plays a key mechanical role. We describe the various techniques that have been developed to directly probe the torque experienced by a single molecule, and detail a variety of measurements made to date using these new technologies. We conclude by discussing a number of open questions and propose systems of study that would be well suited for analysis with torsional measurement techniques.

Underwound DNA under tension: structure, elasticity, and sequence-dependent behaviors.

DNA melting under torsion plays an important role in a wide variety of cellular processes. In the present Letter, we have investigated DNA melting at the single-molecule level using an angular optical trap. By directly measuring force, extension, torque, and angle of DNA, we determined the structural and elastic parameters of torsionally melted DNA. Our data reveal that under moderate forces, the melted DNA assumes a left-handed structure as opposed to an open bubble conformation and is highly torsionally compliant. We have also discovered that at low forces melted DNA properties are highly dependent on DNA sequence. These results provide a more comprehensive picture of the global DNA force-torque phase diagram.

Direct measurements of torque during Holliday junction migration.

DNA experiences torsional stress resulting from the activities of motor enzymes and bound proteins. The mechanisms by which this torsional stress is dissipated to maintain DNA structural integrity are not fully known. Here, we show that a Holliday junction can limit torsion by coupling rotation to translocation and torque to force. The torque required to mechanically migrate through individual junctions was found to be an order of magnitude smaller than that required to melt DNA. We also directly show that substantially more torque was required to migrate through even a single-base sequence heterology, which has important implications for the activity of junction-migrating enzymes.

Passive torque wrench and angular position detection using a single-beam optical trap.

The recent advent of angular optical trapping techniques has allowed for rotational control and direct torque measurement on biological substrates. Here we present a method that increases the versatility and flexibility of these techniques. We demonstrate that a single beam with a rapidly rotating linear polarization can be utilized to apply a constant controllable torque to a trapped particle without active feedback, while simultaneously measuring the particle angular position. In addition, this device can rapidly switch between a torque wrench and an angular trap. These features should make possible torsional measurements across a wide range of biological systems.

Discontinuities at the DNA supercoiling transition.

While slowly turning the ends of a single molecule of DNA at constant applied force, a discontinuity was recently observed at the supercoiling transition when a small plectoneme is suddenly formed. This can be understood as an abrupt transition into a state in which stretched and plectonemic DNA coexist. We argue that there should be discontinuities in both the extension and the torque at the transition and provide experimental evidence for both. To predict the sizes of these discontinuities and how they change with the overall length of DNA, we organize a phenomenological theory for the coexisting plectonemic state in terms of four parameters. We also test supercoiling theories, including our own elastic rod simulation, finding discrepancies with experiment that can be understood in terms of the four coexisting state parameters.

Abrupt buckling transition observed during the plectoneme formation of individual DNA molecules.

The response of single DNA molecules to externally applied forces and torques was directly measured using an angular optical trap. Upon overwinding, DNA buckled abruptly as revealed by a sharp extension drop followed by a torque plateau. When the DNA was held at the buckling transition, its extension hopped rapidly between two distinct states. Furthermore, the initial plectonemic loop absorbed approximately twice as much extension as was absorbed into the plectoneme upon each additional turn. The observed extension change after buckling and the postbuckling torque support a recent DNA elasticity model.

Nanofabricated quartz cylinders for angular trapping: DNA supercoiling torque detection.

We designed and created nanofabricated quartz cylinders well suited for torque application and detection in an angular optical trap. We made the cylinder axis perpendicular to the extraordinary axis of the quartz crystal and chemically functionalized only one end of each cylinder for attachment to a DNA molecule. We directly measured the torque on a single DNA molecule as it underwent a phase transition from B-form to supercoiled P-form.