Mechanical activities ensuring accurate chromosome segregation

PROJECT SUMMARY / ABSTRACT: A central aspect of cell division is the faithful segregation of chromosomes. Errors in chromosome segregation are the leading cause of miscarriages, and are associated with the vast majority of tumor cells. Despite this, we know very little about why this process is so defective in these circumstances, in large part because the molecules and mechanisms involved are not fully defined. A highly conserved protein complex, the kinetochore, physically attaches chromosomes to the spindle microtubules that are central to pulling chromosomes apart every time a cell divides. Doing this accurately requires kinetochores to remain persistently attached to a constantly changing substrate, the dynamically growing and shrinking tips of the spindle microtubules. They must also sense when they’re improperly attached and self-correct these errors. How kinetochores carry out these dynamic functions remains largely mysterious. We propose to investigate two key aspects of the kinetochore- microtubule interface. First, we know that the ability to sense tension (mechano-sensing) is vital for monitoring erroneous attachments of chromosomes to the spindle. Yet, how mechanical forces are sensed by and transmitted through this megadalton protein assembly is poorly understood. Second, the kinetochore’s ability to specifically recognize and bind to various microtubule tip structures is also crucial. How the microtubule side of this interface impacts kinetochore attachment fidelity is unknown, and has remained largely unstudied. Understanding these critical questions has proven challenging because of the lack of ways to measure and manipulate the kinetochore’s highly dynamic activities. Prior studies utilized mainly genetic and cell biological approaches, relying on downstream functional readouts that do not directly monitor protein activity. We use an innovative and fundamentally different strategy, combining cutting-edge biochemical and biophysical tools to reconstitute the activities of these protein machines in vitro. The ability to reconstitute these activities allows us to examine both sides of this interface, making our approach uniquely capable of addressing these fundamental questions in innovative ways. Capitalizing on my groundwork in which I identified the first factor, Stu2chTOG, required for a novel mechano-sensing pathway, we have now determined its exact kinetochore binding site via structural analysis and developed genetic tools allowing us to perturb and inducibly re-localize it to kinetochores. We will use these tools to expand insight into the mechanisms of mechano-sensing and extend our knowledge of the factors involved. We will also examine which kinetochore activities are affected by alterations to its microtubule substrate. We have identified numerous tubulin mutations that specifically disrupt chromosome segregation, and our reconstitution-based assays now give us the unique ability to understand this mechanistically. This approach constitutes the first direct examination of the microtubule side of this interface. I anticipate this work will greatly increase our understanding of how kinetochores work to segregate chromosomes and how defects arise in human disease.