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

描述（由申请人提供）：我之前的训练是物理学，我在加州大学伯克利分校卡洛斯·布斯塔曼特的指导下获得了生物物理学博士学位。在那里，我进行了单分子生物物理学的研究，使用光学镊子机械地展开生物分子。在我的博士后学习期间，我正在过渡到细胞生物学领域，在哈佛医学院的Timothy Mitchison的指导下工作。最终，我想在学术环境下领导一个跨学科的研究小组：我的目标是将新的技能和知识（生物学）与旧的技能和知识（物理学）结合起来，研究主轴中机械力的产生和检测。在技术层面上，NIH独立之路奖将为我提供必要的支持，使我能够完全独立于现代基因工程和生物传感器的开发，并帮助我进一步开发分子试剂和生物物理技术，对我们对细胞分裂的机制理解产生影响。尽管参与细胞分裂的分子越来越多，但人们对支配纺锤体功能的潜在机械原理知之甚少。在某种程度上，这是由于难以将机械力应用于分子易于控制的哺乳动物系统。该研究的目的是了解机械力是如何在哺乳动物着丝点上产生的，以及它如何影响着丝点的运动和检查点化学。我开发了一种新颖而简单的方法，将外部可控的机械力应用于哺乳动物细胞的纺锤体和着丝点。我首先用这种方法研究了机械力如何调节主轴尺寸和主轴杆的力学化学；出乎意料的是，这项研究表明，只有在着丝点附近产生的力才会影响着丝点的行为。在这里，我的具体目标是#1)确定纺锤体力如何转化为施加在着丝点上的力，并测试是否，以及如何，#2)着丝点运动和#3)着丝点相关检查点动力学对机械力作出反应。在指导（K99）阶段，我将通过激光切割不同位置的着丝点纤维来完成目标#1，以绘制它们在纺锤体中的固定位置；这将产生纺锤体的受力图，并提供一个机械框架来测试机械力是否以及如何调节着丝点的运动和化学。针对目标#2和#3，我将生成报告细胞系，并测量着丝点运动和检查点动力学如何响应自然力波动（染色体振荡）和药理学扰动。在独立阶段（R00），我将测量着丝点运动（Aim #2）和检查点动力学（Aim #3）如何响应不同的外力扰动，并将这些响应与着丝点内部变形相关联，以探索张力响应的分子机制。作为共同导师，爱德华·萨蒙将提供着丝点、检查点和成像方面的专业知识。作为贡献者，Alexey Khodjakhov和Rudolf Oldenbourg将分别提供激光切割主轴的设置和专业知识，结合旋转盘共聚焦荧光成像和PolScope成像。该研究有望为着丝点机械化学及其在染色体运动和分离中的作用提供重要的新见解。

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.