What Silences the Spindle Checkpoint? A Single Particle Study

Abstract

Accurate mitosis depends on a surveillance system called the spindle assembly checkpoint. The checkpoint acts at kinetochores, which attach chromosomes to the dynamic tips of spindle microtubules. When a kinetochore is unattached or improperly attached, the checkpoint kinase Mps1 phosphorylates core kinetochore components, catalyzing the generation of a diffusible ‘wait’ signal to block anaphase chromosome segregation. When a kinetochore becomes properly attached, this signal is somehow silenced to allow anaphase to proceed. Two classes of models are proposed to explain how Mps1 is selectively silenced at microtubule-attached kinetochores. In competitive binding models, Mps1 and microtubules compete for binding to the kinetochore, either directly or allosterically, such that Mps1 is released upon kinetochore-microtubule attachment. In switch models, kinetochore attachment to the end of a microtubule inhibits Mps1 kinase activity without causing its release. By direct observation of single fluorescent kinetochores in vitro, we find that Mps1 levels are not reduced when a kinetochore binds laterally to the side of a microtubule, nor when it tracks processively with a disassembling tip. Thus, association of Mps1 with a kinetochore is not strictly competitive with microtubule attachment. Incorporating a fluorescent kinase sensor should allow us to test the switch models.