Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B

The Dam1 complex, regulated by aurora B phosphorylation, confers a more stable microtubule association for the Ndc80 complex at kinetochores (see also related paper by Lampert et al. in this issue)

VTT-006, an anti-mitotic compound, binds to the Ndc80 complex and suppresses cancer cell growth <i>in vitro</i>.

Hec1 (Highly expressed in cancer 1) resides in the outer kinetochore where it works to facilitate proper kinetochore-microtubule interactions during mitosis. Hec1 is overexpressed in various cancers and its expression shows correlation with high tumour grade and poor patient prognosis. Chemical perturbation of Hec1 is anticipated to impair kinetochore-microtubule binding, activate the spindle assembly checkpoint (spindle checkpoint) and thereby suppress cell proliferation. In this study, we performed high-throughput screen to identify novel small molecules that target the Hec1 calponin homology domain (CHD), which is needed for normal microtubule attachments. 4 million compounds were first virtually fitted against the CHD, and the best hit molecules were evaluated in vitro. These approaches led to the identification of VTT-006, a 1,2-disubstituted-tetrahydro-beta-carboline derivative, which showed binding to recombinant Ndc80 complex and modulated Hec1 association with microtubules in vitro. VTT-006 treatment resulted in chromosome congression defects, reduced chromosome oscillations and induced loss of inter-kinetochore tension. Cells remained arrested in mitosis with an active spindle checkpoint for several hours before undergoing cell death. VTT-006 suppressed the growth of several cancer cell lines and enhanced the sensitivity of HeLa cells to Taxol. Our findings propose that VTT-006 is a potential anti-mitotic compound that disrupts M phase, impairs kinetochore-microtubule interactions, and activates the spindle checkpoint

Anaphase A: Disassembling Microtubules Move Chromosomes toward Spindle Poles

The separation of sister chromatids during anaphase is the culmination of mitosis and one of the most strikingly beautiful examples of cellular movement. It consists of two distinct processes: Anaphase A, the movement of chromosomes toward spindle poles via shortening of the connecting fibers, and anaphase B, separation of the two poles from one another via spindle elongation. I focus here on anaphase A chromosome-to-pole movement. The chapter begins by summarizing classical observations of chromosome movements, which support the current understanding of anaphase mechanisms. Live cell fluorescence microscopy studies showed that poleward chromosome movement is associated with disassembly of the kinetochore-attached microtubule fibers that link chromosomes to poles. Microtubule-marking techniques established that kinetochore-fiber disassembly often occurs through loss of tubulin subunits from the kinetochore-attached plus ends. In addition, kinetochore-fiber disassembly in many cells occurs partly through ‘flux’, where the microtubules flow continuously toward the poles and tubulin subunits are lost from minus ends. Molecular mechanistic models for how load-bearing attachments are maintained to disassembling microtubule ends, and how the forces are generated to drive these disassembly-coupled movements, are discussed

Working strokes produced by curling protofilaments at disassembling microtubule tips can be biochemically tuned and vary with species

The disassembly of microtubules can generate force and drive intracellular motility. During mitosis, for example, chromosomes remain persistently attached via kinetochores to the tips of disassembling microtubules, which pull the sister chromatids apart. According to the conformational wave hypothesis, such force generation requires that protofilaments curl outward from the disassembling tips to exert pulling force directly on kinetochores. Rigorously testing this idea will require modifying the mechanical and energetic properties of curling protofilaments, but no way to do so has yet been described. Here, by direct measurement of working strokes generated in vitro by curling protofilaments, we show that their mechanical energy output can be increased by adding magnesium, and that yeast microtubules generate larger and more energetic working strokes than bovine microtubules. Both the magnesium and species-dependent increases in work output can be explained by lengthening the protofilament curls, without any change in their bending stiffness or intrinsic curvature. These observations demonstrate how work output from curling protofilaments can be tuned and suggest evolutionary conservation of the amount of curvature strain energy stored in the microtubule lattice

An automated two-dimensional optical force clamp for single molecule studies.

We constructed a next-generation optical trapping instrument to study the motility of single motor proteins, such as kinesin moving along a microtubule. The instrument can be operated as a two-dimensional force clamp, applying loads of fixed magnitude and direction to motor-coated microscopic beads moving in vitro. Flexibility and automation in experimental design are achieved by computer control of both the trap position, via acousto-optic deflectors, and the sample position, using a three-dimensional piezo stage. Each measurement is preceded by an initialization sequence, which includes adjustment of bead height relative to the coverslip using a variant of optical force microscopy (to +/-4 nm), a two-dimensional raster scan to calibrate position detector response, and adjustment of bead lateral position relative to the microtubule substrate (to +/-3 nm). During motor-driven movement, both the trap and stage are moved dynamically to apply constant force while keeping the trapped bead within the calibrated range of the detector. We present details of force clamp operation and preliminary data showing kinesin motor movement subject to diagonal and forward loads

Paired microtubules growing with a shared load

&lt;p&gt;During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing &lt;em&gt;in vitro&lt;/em&gt; are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubule pairs without any free fit parameters. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling k-fiber bundles with three or more microtubules, as found in many eukaryotes.&lt;/p&gt;&lt;p&gt;Funding provided by: National Institutes of Health&lt;br&gt;Crossref Funder Registry ID: https://ror.org/01cwqze88&lt;br&gt;Award Number: &lt;/p&gt;&lt;p&gt;Funding provided by: Howard Hughes Medical Institute&lt;br&gt;Crossref Funder Registry ID: https://ror.org/006w34k90&lt;br&gt;Award Number: &lt;/p&gt;&lt;p&gt;The data here was collected using the 'dual-trap assay,' which is based on our previously developed single-trap (force-clamp) assay (&lt;a href="https://elifesciences.org/reviewed-preprints/89467#c1"&gt;Akiyoshi et al., 2010&lt;/a&gt;; &lt;a href="https://elifesciences.org/reviewed-preprints/89467#c52"&gt;Miller et al., 2016&lt;/a&gt;; &lt;a href="https://elifesciences.org/reviewed-preprints/89467#c68"&gt;Sarangapani et al., 2013&lt;/a&gt;), in which dynamic microtubules are grown from stabilized seeds bound to a biotinylated coverslip. Using the single laser trap, we attach an individual bead decorated with isolated yeast kinetochores to the growing plus-end of a single microtubule. A computer then continuously measures the bead position and adjusts the trap to exert a precise, constant level of tension on the microtubule via the kinetochore-decorated bead. Under this persistent feedback-controlled tension, kinetochore-beads typically track with the microtubule tips even as the tips stochastically grow and shorten.&lt;/p&gt; &lt;p&gt;Our new dual-trap assay uses two separate laser trapping microscopes, located adjacent to one another in the same room and connected to a single computer. On each of the two instruments, we attach a kinetochore-decorated bead to a dynamic microtubule plus-end. The computer then simultaneously monitors and controls the forces on both microtubules. Rather than keeping the force constant on each microtubule, the computer adjusts the forces dynamically to simulate an elastic coupling of both plus-ends to a single shared load. Thus far, we have simulated only purely elastic couplers, where both coupling springs are linear (Hookean) with stiffness, &lt;em&gt;κ&lt;/em&gt;.&lt;/p&gt; &lt;p&gt;To begin a dual-trap experiment, we first choose the spring stiffness, &lt;em&gt;κ&lt;/em&gt;, and the total shared load, &lt;em&gt;F&lt;sub&gt;TOT&lt;/sub&gt;&lt;/em&gt;, which are kept constant. After a kinetochore-decorated bead is attached to a growing plus-end on each of the two instruments, feedback-control is initiated and the two plus-ends are arbitrarily considered to be parallel, with tips side-by-side (i.e., both at &lt;em&gt;x&lt;sub&gt;1&lt;/sub&gt;&lt;/em&gt; = &lt;em&gt;x&lt;sub&gt;2&lt;/sub&gt;&lt;/em&gt; = 0) and sharing the load equally (&lt;em&gt;F&lt;sub&gt;1&lt;/sub&gt;&lt;/em&gt; = &lt;em&gt;F&lt;sub&gt;2&lt;/sub&gt;&lt;/em&gt; = ½·&lt;em&gt;F&lt;sub&gt;TOT&lt;/sub&gt;&lt;/em&gt;). Because microtubule growth is intrinsically variable, the two microtubules subsequently grow at different speeds. The computer then dynamically monitors the bead positions and adjusts the forces, &lt;em&gt;F&lt;sub&gt;1&lt;/sub&gt;&lt;/em&gt; and &lt;em&gt;F&lt;sub&gt;2&lt;/sub&gt;&lt;/em&gt;, according to the elastic coupling model. For purely elastic couplers, the force difference across the two microtubules equals the tip separation, (&lt;em&gt;x&lt;sub&gt;2&lt;/sub&gt;&lt;/em&gt; – &lt;em&gt;x&lt;sub&gt;1&lt;/sub&gt;&lt;/em&gt;), multiplied by the coupling stiffness (&lt;em&gt;κ&lt;/em&gt;). When one microtubule grows more quickly than the other, tension on the leading (faster-growing) microtubule decreases, and tension on the lagging (slower-growing) microtubule increases to maintain a constant total force on the pair, &lt;em&gt;F&lt;sub&gt;TOT&lt;/sub&gt;&lt;/em&gt;. For all experiments, &lt;em&gt;F&lt;sub&gt;TOT&lt;/sub&gt;&lt;/em&gt; = 8 pN.&lt;/p&gt

Paired microtubules growing with a shared load

&lt;p&gt;During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing &lt;em&gt;in vitro&lt;/em&gt; are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubule pairs without any free fit parameters. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling k-fiber bundles with three or more microtubules, as found in many eukaryotes.&lt;/p&gt;&lt;p&gt;Funding provided by: National Institutes of Health&lt;br&gt;Crossref Funder Registry ID: https://ror.org/01cwqze88&lt;br&gt;Award Number: &lt;/p&gt;&lt;p&gt;Funding provided by: Howard Hughes Medical Institute&lt;br&gt;Crossref Funder Registry ID: https://ror.org/006w34k90&lt;br&gt;Award Number: &lt;/p&gt;&lt;p&gt;The data here was collected using the 'dual-trap assay,' which is based on our previously developed single-trap (force-clamp) assay (&lt;a href="https://elifesciences.org/reviewed-preprints/89467#c1"&gt;Akiyoshi et al., 2010&lt;/a&gt;; &lt;a href="https://elifesciences.org/reviewed-preprints/89467#c52"&gt;Miller et al., 2016&lt;/a&gt;; &lt;a href="https://elifesciences.org/reviewed-preprints/89467#c68"&gt;Sarangapani et al., 2013&lt;/a&gt;), in which dynamic microtubules are grown from stabilized seeds bound to a biotinylated coverslip. Using the single laser trap, we attach an individual bead decorated with isolated yeast kinetochores to the growing plus-end of a single microtubule. A computer then continuously measures the bead position and adjusts the trap to exert a precise, constant level of tension on the microtubule via the kinetochore-decorated bead. Under this persistent feedback-controlled tension, kinetochore-beads typically track with the microtubule tips even as the tips stochastically grow and shorten.&lt;/p&gt; &lt;p&gt;Our new dual-trap assay uses two separate laser trapping microscopes, located adjacent to one another in the same room and connected to a single computer. On each of the two instruments, we attach a kinetochore-decorated bead to a dynamic microtubule plus-end. The computer then simultaneously monitors and controls the forces on both microtubules. Rather than keeping the force constant on each microtubule, the computer adjusts the forces dynamically to simulate an elastic coupling of both plus-ends to a single shared load. Thus far, we have simulated only purely elastic couplers, where both coupling springs are linear (Hookean) with stiffness, &lt;em&gt;κ&lt;/em&gt;.&lt;/p&gt; &lt;p&gt;To begin a dual-trap experiment, we first choose the spring stiffness, &lt;em&gt;κ&lt;/em&gt;, and the total shared load, &lt;em&gt;F&lt;sub&gt;TOT&lt;/sub&gt;&lt;/em&gt;, which are kept constant. After a kinetochore-decorated bead is attached to a growing plus-end on each of the two instruments, feedback-control is initiated and the two plus-ends are arbitrarily considered to be parallel, with tips side-by-side (i.e., both at &lt;em&gt;x&lt;sub&gt;1&lt;/sub&gt;&lt;/em&gt; = &lt;em&gt;x&lt;sub&gt;2&lt;/sub&gt;&lt;/em&gt; = 0) and sharing the load equally (&lt;em&gt;F&lt;sub&gt;1&lt;/sub&gt;&lt;/em&gt; = &lt;em&gt;F&lt;sub&gt;2&lt;/sub&gt;&lt;/em&gt; = ½·&lt;em&gt;F&lt;sub&gt;TOT&lt;/sub&gt;&lt;/em&gt;). Because microtubule growth is intrinsically variable, the two microtubules subsequently grow at different speeds. The computer then dynamically monitors the bead positions and adjusts the forces, &lt;em&gt;F&lt;sub&gt;1&lt;/sub&gt;&lt;/em&gt; and &lt;em&gt;F&lt;sub&gt;2&lt;/sub&gt;&lt;/em&gt;, according to the elastic coupling model. For purely elastic couplers, the force difference across the two microtubules equals the tip separation, (&lt;em&gt;x&lt;sub&gt;2&lt;/sub&gt;&lt;/em&gt; – &lt;em&gt;x&lt;sub&gt;1&lt;/sub&gt;&lt;/em&gt;), multiplied by the coupling stiffness (&lt;em&gt;κ&lt;/em&gt;). When one microtubule grows more quickly than the other, tension on the leading (faster-growing) microtubule decreases, and tension on the lagging (slower-growing) microtubule increases to maintain a constant total force on the pair, &lt;em&gt;F&lt;sub&gt;TOT&lt;/sub&gt;&lt;/em&gt;. For all experiments, &lt;em&gt;F&lt;sub&gt;TOT&lt;/sub&gt;&lt;/em&gt; = 8 pN.&lt;/p&gt

Tension Directly Stabilizes Reconstituted Kinetochore-Microtubule Attachments

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for \u3e30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, via a catch bond-like mechanism that does not require Aurora B. Based on these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation