Force generation and detection in the spindle: the case of the kinetochore

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DESCRIPTION (provided by applicant): My previous training is in physics and I obtained my Ph.D. in biophysics under the supervision of Carlos Bustamante at University of California, Berkeley. There, I conducted research in single-molecule biophysics, using optical tweezers to mechanically unfold biomolecules. During my postdoctoral fellowship, I am transitioning to the field of cell biology, working under the guidance of Timothy Mitchison at Harvard Medical School. Ultimately, I want to lead an interdisciplinary research group in an academic setting: I aim to combine new skills and knowledge (biology) with old ones (physics) to study mechanical force generation and detection in the spindle. On a technical level, the NIH Pathway to Independence Award would provide necessary support for me to become fully independent with modern genetic engineering and biosensor development, and help me further develop the molecular reagents and biophysical techniques needed to have an impact on our mechanistic understanding of cell division. Despite a growing list of molecules involved in cell division, little is known about the underlying mechanical principles that govern spindle function. In part, this is due to the difficulty of applying mechanical force to molecularly tractable mammalian systems. The goal of the proposed research is to understand how mechanical force is generated on mammalian kinetochores and how it affects kinetochore motility and checkpoint chemistry. I have developed a novel - and simple - method to apply externally controllable mechanical forces to the spindle and kinetochores in mammalian cells. I first used this method to study how mechanical force regulates spindle size and spindle pole mechanochemistry; unexpectedly, this study suggested that only forces generated relatively near the kinetochore affect its behavior. Herein, my specific aims are to #1) determine how spindle forces translate to forces applied on kinetochores, and test whether, and how, #2) kinetochore motility and #3) kinetochore associated checkpoint dynamics respond to mechanical force. During the mentored (K99) phase, I will accomplish Aim #1 by laser cutting kinetochore-fibers at different locations to map where they are anchored in the spindle; this will yield a force map of the spindle and provide a mechanical framework to test whether, and how, mechanical force regulates kinetochore motility and chemistry. Towards Aims #2 and #3, I will then generate reporter cell lines, and measure how kinetochore motility and checkpoint dynamics respond to natural force fluctuations (chromosome oscillations) and pharmacological perturbations. During the independent phase (R00), I will measure how kinetochore motility (Aim #2) and checkpoint dynamics (Aim #3) respond to different external force perturbations, and correlate these responses with intra-kinetochore deformations to probe the molecular mechanism of tension response. As a co-mentor, Edward Salmon will provide kinetochore, checkpoint, and imaging expertise. As contributors, Alexey Khodjakhov and Rudolf Oldenbourg will provide setups and expertise for laser cutting spindles, combined with spinning disk confocal fluorescence imaging and PolScope imaging, respectively. The proposed research promises to provide significant new insight into kinetochore mechanochemistry and its role in chromosome movement and segregation. PUBLIC HEALTH RELEVANCE: Cell division is responsible for reproduction, growth, development, and continuous organism renewal and repair. During cell division, chromosomes must be accurately segregated as errors can lead to cancer and birth defects. The results of this study will provide insight into how mechanical forces guide chromosome movement and segregation, and may as such provide new targets for cancer therapeutics.