Krishna K. Sarangapani

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 as the result of 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 >30 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, through a catch bond-like mechanism that does not require Aurora B. On the basis of 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.

Catch and release: How do kinetochores hook the right microtubules during mitosis?

Sport fishermen keep tension on their lines to prevent hooked fish from releasing. A molecular version of this anglers trick, operating at kinetochores, ensures accuracy during mitosis: the mitotic spindle attaches randomly to chromosomes and then correctly bioriented attachments are stabilized due to the tension exerted on them by opposing microtubules. Incorrect attachments, which lack tension, are unstable and release quickly, allowing another chance for biorientation. Stabilization of molecular interactions by tension also occurs in other physiological contexts, such as cell adhesion, motility, hemostasis, and tissue morphogenesis. Here, we review models for the stabilization of kinetochore attachments with an eye toward emerging models for other force-activated systems. Although attention in the mitosis field has focused mainly on one kinase-based mechanism, multiple mechanisms may act together to stabilize properly bioriented kinetochores and some principles governing other tension-sensitive systems may also apply to kinetochores.

Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms

During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser-trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms: by weakening the kinetochore-microtubule interface and also by destabilizing the kinetochore-attached microtubule tip.

Sister kinetochores are mechanically fused during meiosis I in yeast

Production of healthy gametes requires a reductional meiosis I division in which replicated sister chromatids comigrate, rather than separate as in mitosis or meiosis II. Fusion of sister kinetochores during meiosis I may underlie sister chromatid comigration in diverse organisms, but direct evidence for such fusion has been lacking. We used laser trapping and quantitative fluorescence microscopy to study native kinetochore particles isolated from yeast. Meiosis I kinetochores formed stronger attachments and carried more microtubule-binding elements than kinetochores isolated from cells in mitosis or meiosis II. The meiosis I–specific monopolin complex was both necessary and sufficient to drive these modifications. Thus, kinetochore fusion directs sister chromatid comigration, a conserved feature of meiosis that is fundamental to Mendelian inheritance. The meiosis I–specific monopolin complex helps to keep sister chromatids together in the first meiotic division. Monopolin masterfully manages meiosis Biologists have wondered for decades how replicated sister chromatids, which normally separate during mitotic cell division, instead comigrate during the first meiotic division, meiosis I. This process segregates chromosomal homologs and is needed to produce haploid gametes after the second, more mitosis-like, meiotic division. One hypothesis for sister chromatid comigration suggests that meiosis I–specific factors directly cross-link the sister kinetochores that attach each sister chromatid to dynamic microtubule tips. Yeast possesses a putative kinetochore cross-linker, known as monopolin, but monopolins precise role during meiosis I is unknown. Sarangapani et al. isolated functional meiotic kinetochores from yeast cells. They reconstituted kinetochore activity in vitro and found that monopolin causes kinetochore fusion and underlies the sister chromatid comigration seen in meiosis I. Science, this issue p. 248

Direct measurement of the strength of microtubule attachment to yeast centrosomes

Laser trapping is used to manipulate single attached microtubules in vitro. Direct mechanical measurement shows that attachment of microtubule minus ends to yeast spindle pole bodies is extraordinarily strong.

Measuring kinetochore-microtubule interaction in vitro.

Many proteins and protein complexes perform sophisticated, regulated functions in vivo. Many of these functions can be recapitulated using in vitro reconstitution, which serves as a means to establish unambiguous cause-effect relationships, for example, between a protein and its phosphorylating kinase. Here, we describe a protocol to purify kinetochores, the protein complexes that attach chromosomes to microtubules during mitosis, and quantitatively assay their microtubule-binding characteristics. Our assays, based on DIC imaging and laser trapping microscopy, are used to measure the attachment of microtubules to kinetochores and the load-bearing capabilities of those attachments. These assays provide a platform for studying kinase disruption of kinetochore-microtubule attachments, which is believed to be critical for correcting erroneous kinetochore-spindle attachments and thereby avoiding chromosome missegregation. The principles of our approach should be extensible to studies of a wide range of force-bearing interactions in biology.