We work to determine the fundamental principles underlying the operation of molecular machines that give cells the remarkable ability to segregate their chromosomes during cell division. Various force-sensitive interactions are essential for mitotic fidelity, and are therefore critical to our understanding of aneuploidy and genomic instability. Over the last 5 years, we have developed molecular tools, equipment, and expertise to quantitatively and rigorously address central questions about the role of force in chromosome segregation: (1) How do the macromolecular complexes that constitute human kinetochores travel with dynamic microtubule ends under load? (2) How do individual microtubule-associated proteins with no motor activity glide along microtubules under dragging force? (3) How does tension applied to the centromeric chromatin meshwork shape the spatial phosphorylation gradients that orchestrate assembly of the kinetochores and their binding to microtubules? We approach these problems using reductionist approaches and innovative in vitro assays that reconstruct these interactions at multiple scales, and analyze our findings with advanced theoretical modeling. (1) To recreate force-sensitive interactions between microtubules and human kinetochores, we developed a novel approach for generating macromolecular kinetochore subcomplexes using inducible protein-fusion scaffolds. When isolated from mitotic HeLa cells, these particles exhibit key physiological properties of native kinetochores, including their persistent association with dynamic microtubule ends. This breakthrough will enable us for the first time to study the motility of native human kinetochore complexes, driving forward our biophysical analysis of kinetochore load-bearing. (2) We will investigate the force sensitivity of individual microtubule-binding proteins at the single-molecule and ensemble levels using an advanced force spectroscopy approach. We have implemented a highly sensitive dual-trap, three-bead assay employing an ultrafast force-clamp that allows us to pull on a single non-motor molecule diffusing on the microtubule wall, imitating the forces these kinetochore-bound molecules experience during chromosome motions. This approach will provide unique molecular-mechanical insights into the friction-generating interface that allows the kinetochore to glide along microtubule, while preventing it from slipping from microtubule ends. (3) We will seek to understand how mechanical deformations shape chemical gradients formed within the chemo- mechanical meshworks, such as of the centromeric chromatin. Previously, we reconstructed a non-linear Aurora B kinase/phosphatase bi-stable switch using soluble components. In a proof-of-principle study, we will embed these enzymatic components into a flexible meshwork to test whether its deformations can control formation of distinct phosphorylation patterns. Spatio-temporal regulation of the phosphorylation status of kinetochore protei...