Kinetochore Dynamic Structure at Super-Resolution Accuracy and Mechanical Stiffness Revealed in vitro by Combined Tirf and Optical Tweezers

Abstract

The kinetochore is a protein complex that interfaces spindle microtubules with chromosomal 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, and exert force to the centromere DNA. The significance of the tension in silencing the spindle assembly checkpoint signal remains a central unsolved question in mitosis. Recent advances in isolating kinetochore particles from budding yeast enables the application of direct and controlled mechanical perturbation in vitro. Meanwhile, by fluorescently labeling kinetochores particles, the subunits can be accurately localized. We used a combined instrument of optical tweezers, total internal reflection fluorescence microscopy, and differential interference contract microscopy to measure the super-resolution structural change induced by an external tension exerted by attached microtubule. In addition to confirming the linear structural organization, the stiffness of kinetochore subunits is also directly quantified in this work. This information is useful to reveal the role of intra-kinetochore stretch in the mechanism of generating spindle assembly checkpoint signaling.