AN IN VITRO SINGLE MOLECULE FLUORESCENCE ASSAY TO STUDY THE SPINDLE ASSEMBLY CHECKPOINT

Abstract

Dividing cells rely on a surveillance mechanism at kinetochores called the spindle assembly checkpoint, which emits a diffusible ‘wait’ anaphase signal when kinetochores are unattached or improperly attached to microtubules. This results in cell arrest, delaying anaphase until stable bioriented kinetochore-microtubule attachments are formed. The evolutionarily conserved spindle assembly checkpoint kinase, Mps1, has been shown to phosphorylate the core kinetochore component, Spc105, leading to recruitment of the Bub1-Bub3 checkpoint complex. Further phosphorylation of Bub1 by Mps1 also results in the recruitment of the checkpoint protein Mad1 leading to generation of the ‘wait’ anaphase signal. Attachment of microtubules to kinetochores results in the silencing of the spindle assembly checkpoint signal. However, how attachment directly results in checkpoint silencing continues to be debated. Studying the checkpoint and how it is silenced has proven a challenge due to the difficulty of directly and unambiguously distinguishing the microtubule attachment status and levels of checkpoint proteins on individual kinetochores in vivo. In this dissertation, I developed a fluorescence-based approach to observe the association of checkpoint proteins with single kinetochore particles in vitro, where the kinetochore-microtubule attachment status can be controlled and its effect on checkpoint protein levels can be directly observed. Mps1 is most upstream of the checkpoint signaling cascade and is localized to the core Ndc80 component of the kinetochore. The Ndc80 sub-complex is a major microtubule-binding core component of the kinetochore. Recent studies on checkpoint silencing have focused on Mps1 and how it responds to kinetochore-microtubule attachment since it is most upstream of the signaling cascade and is implicated in the checkpoint. I isolated native budding yeast kinetochore particles carrying fluorescent tags on a core Mtw1 kinetochore component and Mps1. I then examined the kinetochores in the presence of microtubules using a custom-built multi-color Total Internal Reflection Fluorescence (TIRF) microscopy system and single particle assays. I show that the spindle assembly checkpoint can be studied in vitro, at the single particle level. The assays I develop also set the stage for checkpoint silencing to be understood.