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

简介(申请人提供)：我之前接受的是物理学方面的培训，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。在我的博士后研究期间，我正在过渡到细胞生物学领域，在哈佛医学院蒂莫西·米奇森的指导下工作。最终，我想在学术环境中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)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独立之路奖将为我完全独立于现代基因工程和生物传感器开发提供必要的支持，并帮助我进一步开发分子试剂和生物物理技术，以影响我们对细胞分裂的机械理解。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。在一定程度上，这是因为很难将机械力应用于分子上易于处理的哺乳动物系统。这项拟议研究的目标是了解机械力是如何在哺乳动物动粒上产生的，以及它是如何影响动粒运动和检查点化学的。我开发了一种新颖而简单的方法，将外部可控的机械力施加到哺乳动物细胞的纺锤体和动粒上。我首先用这种方法研究了机械力如何调节心轴的大小和心轴极点的机械力化学；出乎意料的是，这项研究表明，只有在动粒附近产生的力才会影响它的行为。在这里，我的具体目标是#1)确定主轴力如何转化为施加在动粒上的力，并测试#2)动粒运动和#3)动粒相关的检查点动力学是否以及如何响应机械力。在指导(K99)阶段，我将通过激光切割不同位置的动粒-纤维来实现目标#1，以绘制它们在主轴中锚定的位置；这将产生主轴的力图，并提供一个机械框架来测试机械力是否以及如何调节动粒的运动和化学。为了达到目标2和3，我将产生报告细胞系，并测量动粒运动性和检查点动力学如何对自然力波动(染色体振荡)和药物扰动做出反应。在独立阶段(R00)，我将测量动粒运动(Aim#2)和检查点动力学(Aim#3)对不同外力扰动的反应，并将这些反应与动粒内变形联系起来，以探索张力反应的分子机制。作为联合导师，Edward Salmon将提供动点、检查点和成像方面的专业知识。作为贡献者，Alexey Khodjakhov和Rudolf Oldenburg将分别结合旋转圆盘共聚焦荧光成像和PolScope成像，提供激光切割主轴的设置和专业知识。这项拟议的研究有望为动粒机械化及其在染色体运动和分离中的作用提供重要的新见解。 与公共卫生相关：细胞分裂负责生殖、生长、发育以及持续的有机体更新和修复。在细胞分裂过程中，染色体必须准确分离，因为错误可能会导致癌症和出生缺陷。这项研究的结果将提供关于机械力如何引导染色体移动和分离的见解，并因此可能为癌症治疗提供新的靶点。

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.