Multivalent Binding and Diffusion of Isolated Kinetochore Particles on Microtubule Lattice in Vitro

The kinetochore is a protein complex that connects spindle microtubules and centromere DNA. During mitosis, kinetochores establish correct bi-oriented attachment of spindle on sister chromatids, and mediate the segregation of sister chromatids in anaphase. Throughout this process, kinetochores remain attached on the plus ends of the microtubules, which undergo rapid depolymerization to effectively pull chromosomes apart. Moreover, kinetochores are able to exert force to the centromere DNA to overcome the viscous drag on the chromosome. How kinetochores harness the energy released from the microtubule depolymerization to do work is under active debate.Recent advances in isolating kinetochore particles enable the possibility to test how kinetochores interact with microtubule in vitro. Using fluorescently labeled kinetochores purified from budding yeast, we are able to observe the dynamic binding, diffusion, pausing, and detachment events of single kinetochore particles on microtubule lattice. Consistent with the stoichiometry knowledge of kinetochore architecture that each kinetochore contains multiple microtubule binding units, the Ndc80 complex, we find that the resident time increases and the diffusion decreases as the number of binding unit increases. The kinetics observation can be explained with a biased-diffusion model, from which the binding and detachment rate, diffusion constant, and pausing rate are extracted. Our observation suggests that biased-diffusion is a plausible mechanism for kinetochore to interact with shrinking microtubule tip. We also show that the binding affinity of kinetochore with microtubule is regulated by the phosphorylation state and the presence of Dam1 complex, as means to establish correct microtubule-kinetochore attachments adopted in vivo.