Insights into the role of phosphorylation on microtubule cross-linking by PRC1.

The mitotic spindle is composed of distinct networks of microtubules, including interpolar bundles that can bridge sister kinetochore fibers and bundles that organize the spindle midzone in anaphase. The cross-linking protein PRC1 can mediate such bundling interactions between antiparallel microtubules. PRC1 is a substrate of mitotic kinases including CDK/cyclin-B, suggesting that it can be phosphorylated in metaphase and dephosphorylated in anaphase.

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).

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.

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.

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.

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.

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.

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.

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.

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 β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Adsorption of CF4 on the internal and external surfaces of opened single-walled carbon nanotubes: a vibrational spectroscopy study.

Infrared spectroscopy has been used to make the first experimental discrimination between molecules bound by physisorption on the exterior surface of carbon single-walled nanotubes (SWNTs) and molecules bound in the interior. In addition, the selective displacement of the internally bound molecules has been observed as a second adsorbate is added. SWNTs were opened by oxidative treatment with O(3) at room temperature, followed by heating in a vacuum to 873 K.