Defining the mechanisms underlying the mammalian kinetochore's structural integrity under force

Project Summary/Abstract At cell division, the cell must accurately segregate its chromosomes to its two daughter cells. The kinetochore connects spindle microtubules to chromosomes and plays both physical and biochemical roles: it must resist and transmit microtubule forces to move chromosomes, and process microtubule signals to control cell cycle progression. Errors in these processes can lead to disease and birth defects. The mammalian kinetochore consists of ~100 protein species, with innermost kinetochore proteins binding DNA and outermost ones binding microtubules ~100nm away. Parallel protein linkages connect DNA and microtubules. While much is known about the mammalian kinetochore’s architecture, molecular composition and biochemistry, much less is known about its mechanics. Yet, the kinetochore has a key mechanical function – segregating chromosomes – and spends much of its life under force. To what extent and how the mammalian kinetochore maintains its structural integrity under force remain unclear. A major challenge has been in measuring kinetochore structure in live cells, under different cellular forces, given the kinetochore’s small size and rapid movements. Our recent approach development measuring inner and outer kinetochore shape changes with high spatiotemporal resolution in vivo during normal mitosis brings this question within reach. Here, I propose to test physical and molecular models for how mammalian kinetochores maintain their structure under spindle forces at metaphase. I will do so using super resolution live imaging and computational shape analysis of inner (CENP-A) and outer (Hec1) kinetochore proteins in PtK2 cells. In Aim 1, I will test the hypothesis that centromere stability contributes to kinetochore structural maintenance. I will do so by decreasing centromere stiffness genetically using an inducible Condensin II knock out cell line and measuring the impact on resulting kinetochore deformations. In Aim 2, I will test the hypothesis that lateral reinforcement between parallel kinetochore protein linkages contributes to kinetochore structural integrity. I will do so by replacing an inner kinetochore protein with a mutant which disrupts crosslinking between parallel linkages, and also by inducing an acute, partial loss of this candidate protein. In Aim 3, I will test the hypothesis that incorrect, merotelic attachments give rise to the most dramatic kinetochore deformations at metaphase. I will do so by enriching for merotelic attachments and asking if more kinetochores then take on large deformations. Together, this work will help define the mechanisms underlying mammalian kinetochore structural maintenance (Aim 1 and Aim 2) and the impact of incorrect attachments on kinetochore structure (Aim 3). Looking forward, this will help us elucidate how the kinetochore’s functions emerge from its structure, and how its malfunctions can emerge from structural failures. Through this work, I will get training in cellular biophysics, computational image analysis, molecular approaches, and how to rigorously answer questions, mentor and communicate science. These will help me towards my goal of being a group leader in industry or academia.