A Nanoscale Blueprint of the Human Kinetochore and the Functional Limits of Its Design

Cellular duplication is the cornerstone of all life, allowing organisms to replicate and develop through the creation of genetically identical cells. Proper duplication requires cells to accurately segregate copies of their DNA so that the resulting sister cells each receive a full complement of the genome. In eukaryotes, the segregation process is accomplished by the kinetochore, a large multi-protein structure built upon the centromeric DNA of chromosomes. To segregate chromosomes with high fidelity, the kinetochore executes three principal functions: (1) spindle assembly checkpoint (SAC) signaling, where unattached kinetochores pause the cell cycle until they attach to the spindle; (2) error correction, where kinetochores destabilize incorrect attachments that may otherwise lead to missegregation; and (3) force generation, where kinetochores physically move chromosomes by coupling to the energy of microtubule depolymerization. To implement these functions, the kinetochore incorporates a large, interconnected network of multicopy proteins. While the biochemical and structural properties of many of these proteins are known, a mechanistic understanding of how these proteins integrate to give rise to the emergent functions of chromosome segregation is lacking. To achieve such insights, the number and spatial arrangement, or ‘architecture’, of kinetochore proteins must be solved. In this thesis, I develop a Förster Resonance Energy Transfer-based microscopy method to map the nanoscale organization of human kinetochore proteins relative to its ~20 microtubule attachments. I focus my studies on the Ndc80 complex (Ndc80C), the central microtubule-binding molecule of the kinetochore with important roles in all its principal functions. I find that Ndc80C molecules cluster during microtubule attachment despite being recruited by centromeric proteins which are separated by ≥10 nm. Furthermore, Ndc80C clustering depends on microtubule attachment and reduces significantly in unattached kinetochores. I also discover that Ndc80C clustering increases with centromeric tension and provide evidence that this correlation relies on the ability of centromeric tension to promote Ndc80C binding. Additionally, I show that Ndc80C clustering persists when its centromeric receptors are significantly diminished, suggesting a ‘lawn-like’ model of human kinetochore function. I synthesize these data into an architectural framework of human kinetochore-microtubule attachments, highlighting the potential functional implications of this architecture. In a second project, I investigate the architecture/function relationship of the human kinetochore by asking: what is the minimum number of the human kinetochore’s ~250 Ndc80C molecules needed for proper chromosome segregation? To answer this question, I perform a series of ‘stress tests’ to determine the failure limits of kinetochore function by titrating human kinetochores with microtubule-binding mutants of Ndc80C. These tests reveal that the SAC is activated in a sigmoidal, switch-like manner when the number of functional Ndc80C molecules falls below 60%. This all-or-nothing response suggests that the human kinetochore rapidly extinguishes SAC signaling beyond a threshold number of microtubule-bound Ndc80C molecules. Additionally, severe chromosome alignment and segregation errors occur when kinetochores contain as little as 20% of a non-regulatable, strong-binding Ndc80C mutant. Thus, the kinetochore achieves proper chromosome segregation through a remarkably acute ability to fine-tune the attachment strength of Ndc80C. Overall, these stress tests demonstrate that the human kinetochore incorporates an excess of Ndc80C, allowing it to operate with definable margins of error. As a final summary, my work highlights the reciprocal relationship between kinetochore architecture and function, each influencing the other to ultimately converge on the principles of accurate chromosome segregation.PhDBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/167966/1/akukreja_1.pd