Molecular Mechanisms of Kinetochore-Microtubule Attachment Via the Ndc80 Complex

The fundamental property of living systems is their ability to consume resources from their environment in order to reproduce, heritably passing on their genetic program to their offspring. At the level of an individual cell, the irreducible unit of self-replicating life, this propagation occurs through cell division. While we have a relatively good grasp of nature's information storage mechanism, the DNA-encoded genome, the means for its distribution via the mechanics of a living cell remains mysterious. Eukaryotic cells entrust the partitioning of the duplicated genome to the mitotic spindle, a self-organizing supra-molecular machine primarily composed of dynamic microtubule filaments. The problem of delivering exactly one copy of each chromosome to each daughter cell must be solved an enormous number of times over the course of a human lifetime: errors result in aneuploidy, which can lead to birth defects and enhance the progression of cancer./nThe kinetochore is a network of protein complexes which assembles on centromeric chromatin to act as the connection point between chromosomes and the microtubules that segregate them into daughter cells. The kinetochore ensures proper bi-orientation of all chromosomes (i.e. that each sister chromatid is attached to microtubules emanating from opposite spindle poles) through the activity of the spindle assembly checkpoint (SAC), and also couples chromosome movement to microtubule dynamics, most dramatically when microtubule depolymerization provides the driving force for pulling apart sister chromatids during anaphase. Thus, the kinetochore serves to generate and integrate both chemical and mechanical processes at the cellular scale; both functions are necessary for mitosis to succeed./nThe vertebrate kinetochore, the primary subject of this thesis, is composed of over one hundred different proteins, presenting a system of daunting complexity. Over the previous two decades genetic, biochemical, and cell biological analyses in multiple organisms have provided an almost complete parts list, demonstrating that most key kinetochore subunits are conserved in all eukaryotes and are also generally organized into the same sets of complexes, suggesting that the core functions and architecture of the kinetochore is conserved throughout the tree of life. Despite this triumph of biological analysis, physical, mechanistic explanations of how the kinetochore fulfills both its chemical and mechanical roles are absent, largely due to a lack of understanding of how kinetochore components are organized (and reorganized) in space and time as mitosis proceeds./nThis thesis addresses the core problem of how kinetochores engage dynamic microtubule filaments from a molecular mechanistic perspective. I focus my analysis on the Ndc80 complex, which has been identified as the primary conserved direct interface between chromosomes and the microtubule surface. Through structural and biochemical experiments I uncovered the mechanism by which the human complex engages the microtubule and how this interaction can be regulated, with implications for coupling chromosome motions to microtubule dynamics and signaling proper chromosome bi-orientation. I also investigated the structure of the Mis12 linker complex and its interaction with Ndc80, demonstrating a conserved architecture of this interface between yeast and humans despite low sequence similarity. Finally, I probed how the Ndc80 complex and the Dam1 complex could coordinate their microtubule-binding activities in budding yeast using bioinformatics. I synthesize these results into scale three-dimensional models of the kinetochore, giving rise to precise hypotheses about kinetochore organization and function that can be tested in vivo, providing a step forward on the path towards a complete biophysical model of mitosis.

Advisor: Nogales, Eva