The primary goal of mitosis is to make two genetically identical copies of the dividing cell. To achieve this goal, the dividing cell must segregate exactly one copy of each chromosome into each daughter. Even a single error in chromosome segregation results in aneuploidy, which in turn leads to a plethora of defects, from cell death to tumorigenesis. Therefore, to accomplish accurate chromosome segregation, the eukaryotic cell uses two highly sophisticated systems: the kinetochore and the Spindle Assembly Checkpoint (SAC). The kinetochore is a multi- protein machine that moves and segregates each chromosome. If it is unable to do so, the kinetochore activates the SAC. The SAC is a signaling cascade that generates a diffusible checkpoint complex that arrests cell division. Extensive research has compiled a nearly complete list of proteins and activities necessary for the two systems. However, fundamental questions regarding each remain unanswered. How does the kinetochore seamlessly integrate the disparate molecular mechanisms that generate chromosome movement and activate the SAC? How does the cell calibrate SAC signaling output to maximize accurate chromosome segregation, but minimize the duration of mitosis? The most significant challenge in defining the molecular mechanisms of kinetochore function is its highly complex protein architecture. My lab reconstructed the nanoscale protein architecture of the kinetochore in budding yeast by developing an array of fluorescence microscopy techniques. We used this knowledge to undertake `architecture-function' analyses of the yeast kinetochore. Our work reveals how kinetochore architecture shapes functional mechanisms. Our next goal is to define how the architecture of the much more complex, human kinetochore shapes emergent mechanisms of force generation and SAC activation. The most significant challenge in studying the biochemical design of the SAC is our inability to measure the thermodynamic rate constants governing its signaling reactions. This is because these complex reactions are localized within the nanoscopic kinetochore. To circumvent this challenge, we designed the ?eSAC?: an ectopic, quantifiable, and controllable, SAC activator. Preliminary characterization of the biochemical design of the SAC provides an elegant model to explain how the human cell optimizes the SAC signaling cascade. We will use the eSAC to quantify biochemical steps in the SAC cascade, reconstitute key steps to study them at the thermodynamic and structural level, and then synthesize a detailed mathematical model to completely establish the mechanistic platform describing the SAC. Our integrative analyses of the two systems will thus elucidate their respective functional designs, and reveal how they cooperate to ensure accurate chromosome segregation.
Accurate inheritance of the genome by the two daughters of cell division is essential for their survival and proper function. The proposed research will study the molecular and biochemical bases of two highly complex systems that ensure accurate genome inheritance. The insights obtained from this research will elucidate why cancerous cells make frequent mistakes in the process genome inheritance.
