Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes1,2. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules3. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B4,5. 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 microtubules6. Moreover, tension increases the lifetimes of the reconstituted attachments directly, via a catch bond-like mechanism that does not require Aurora B7-10. 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.
Selectin-ligand interactions mediate the tethering and rolling of circulating leukocytes on vascular surfaces during inflammation and immune surveillance. To support rolling, these interactions are thought to have rapid off-rates that increase slowly as wall shear stress increases. However, the increase of off-rate with force, an intuitive characteristic named slip bonds, is at odds with a shear threshold requirement for selectin-mediated cell rolling. As shear drops below the threshold, fewer cells roll and those that do roll less stably and with higher velocity. We recently demonstrated a low force regime where the off-rate of P-selectin interacting with P-selectin glycoprotein ligand-1 (PSGL-1) decreased with increasing force. This counter-intuitive characteristic, named catch bonds, might partially explain the shear threshold phenomenon. Because L-selectin-mediated cell rolling exhibits a much more pronounced shear threshold, we used atomic force microscopy and flow chamber experiments to determine off-rates of L-selectin interacting with their physiological ligands and with an antibody. Catch bonds were observed at low forces for L-selectin-PSGL-1 interactions coinciding with the shear threshold range, whereas slip bonds were observed at higher forces. These catch-slip transitional bonds were also observed for L-selectin interacting with endoglycan, a newly identified PSGL-1-like ligand. By contrast, only slip bonds were observed for L-selectin-antibody interactions. These findings suggest that catch bonds contribute to the shear threshold for rolling and are a common characteristic of selectin-ligand interactions.Selectins are a family of adhesion molecules that contribute to leukocyte trafficking during inflammation and tissue injury and to lymphocyte homing (1-3). E-selectin is expressed on activated endothelial cells; L-selectin is expressed on leukocytes, and P-selectin is expressed on activated endothelial cells and platelets. L-and P-selectins interact in a related manner with P-selectin glycoprotein ligand-1 (PSGL-1), 1 a mucin expressed on leukocytes. These interactions mediate tethering and rolling of circulating leukocytes on endothelial cells, platelets, and adherent leukocytes under flow. Selectin-ligand interactions are rapid and transient. In addition, the mechanically stressful environment in the circulation imposes forces on selectin-ligand bonds, which affect their dissociation rates.Bell suggested that applied force could accelerate bond dissociation, because work done by the force could lower the energy barrier between the bound and free states (4). Dembo et al. (5,6) conversely envisioned that force could also decelerate bond dissociation by deforming the molecules such that they locked more tightly. These two types of behavior are named slip and catch bonds, respectively. Bonds whose off-rates are independent of force are called ideal bonds (5, 6). Since the first experimental determination of the relationship between force and off-rate (7), many studies found slip bond behavior (7-18...
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 lasertrapping 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 kinetochoremicrotubule interface and also by destabilizing the kinetochoreattached microtubule tip.
Receptor-ligand bonds that mediate cell adhesion are often subjected to forces that regulate their dissociation via modulating off-rates. Off-rates control how long receptor-ligand bonds last and how much force they withstand. One should therefore be able to determine off-rates from either bond lifetime or unbinding force measurements. However, substantial discrepancies exist between the force dependence of off-rates derived from the two types of measurements even for the same interactions, e.g., selectins dissociating from their ligands, which mediate the tethering and rolling of leukocytes on vascular surfaces during inflammation and immune surveillance. We used atomic force microscopy to measure survival times of P-selectin dissociating from P-selectin glycoprotein ligand 1 or from an antibody in both bond lifetime and unbinding force experiments. By a new method of data analysis, we showed that the discrepancies resulted from the assumption that off-rates were functions of force only. The off-rates derived from forced dissociation data depended not only on force but also on the history of force application. This finding provides a new paradigm for understanding how force regulates receptor-ligand interactions.
Interactions between surface-anchored receptors and ligands mediate cell-cell and cell-environment communications in many biological processes. Molecular interactions across two apposing cell membrane are governed by two-dimensional (2D) kinetics, which are physically distinct from and biologically more relevant than three-dimensional (3D) kinetics with at least one interacting molecular species in the fluid phase. Here we review two assays for measuring 2D binding kinetics: the adhesion frequency assay and the thermal fluctuation assay. The former measures the binding frequency as a function of contact duration and extracts the force-free 2D kinetics parameters by nonlinearly fitting the data with a probabilistic model. The latter detects bond formation/dissociation by monitoring the reduction/resumption of thermal fluctuations of a force sensor. Both assays are mechanically based and operate at the level of mostly single molecular interaction, which requires ultrasensitive force techniques. Characterization of one such technique, the biomembrane force probe, is presented.
Production of healthy gametes requires a reductional meiosis I division in which replicated sister chromatids co-migrate, rather than separating as in mitosis or meiosis II. Fusion of sister kinetochores during meiosis I may underlie sister chromatid co-migration in diverse organisms, but direct evidence for such fusion has been lacking. Here we studied native kinetochore particles isolated from yeast using laser trapping and quantitative fluorescence microscopy. 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 co-migration, a conserved feature of meiosis that is fundamental to Mendelian inheritance.
Sport fishermen keep tension on their lines to prevent hooked fish from releasing. A molecular version of this angler’s 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. While 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 apply to kinetochores as well.
In single-molecule mechanics experiments the molecular elasticity is usually measured from the deformation in response to a controlled applied force, e.g., via an atomic force microscope cantilever. We have tested the validity of an alternative method based on a recently developed theory. The concept is to measure the change in thermal fluctuations of the cantilever tip with and without its coupling to a rigid surface via the molecule. The new method was demonstrated by its application to the elasticity measurements of L- and P-selectin complexed with P-selectin glycoprotein ligand-1 or their respective antibodies, which showed values comparable to those measured from the slope of the force-extension curve. L- and P-selectin were found to behave as nearly linear springs capable of sustaining large forces and strains without sudden unfolding. The measured spring constants of approximately 4 and approximately 1 pN/nm for L- and P-selectin, respectively, suggest that a physiological force of approximately 100 pN would result in an approximately 200% strain for the respective selectins.
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