Emergent mechanics of mammalian chromosome segregation

项目总结/摘要 两个大的大分子机器，纺锤体和动粒，协调染色体分离在细胞 师.它们功能的错误可能导致癌症和出生缺陷。虽然我们现在知道几乎所有的 哺乳动物纺锤体和动粒功能所需的分子，它们是如何共同产生这些 机器的自然物理特性仍然知之甚少。我们的长期目标是揭示 物理设计原理的鲁棒性和准确的哺乳动物染色体分离。成千上万的 纳米尺度的分子，推和拉，产生了纺锤体的纳米尺度的稳态结构， 机械师？数百个动粒分子如何协同工作来“计算”附着信号， 决策，并有力地抓住微管？这种知识差距之所以持续存在，是因为我们目前无法 在体外重建这些机器，而在体内缺乏工具。为了缩小这一差距，我们需要采取一些方法， 控制分子，并直接在哺乳动物细胞分裂中施加力量-这是我们最近开发的。 首先，我们的目标是定义主轴的涌现架构的机制和功能， 力学(i)根据我们的发现，我们将测试的想法，在主轴相反的应力是不是 需要给它一个稳定状态的结构，而是需要给它机械和功能 稳定此外，我们将定义在体内和体外如何主动和被动的力量有助于建立一个 稳态主轴(ii)为了揭示纺锤体特定稳态形状的功能，我们开发了一个 光遗传学方法来急性和局部控制纺锤体结构。我们将用它来测试给定的 通过有丝分裂在空间和时间上构建模块。(iii)为了确定稳定的潜在机制- 状态主轴的机械鲁棒性，我们将使用微针操纵主轴变形，我们 最近在哺乳动物细胞中进行了调整，并进行了建模。其次，我们的目标是定义动粒是如何 分子一起使它能够“计算”附着信息并牢固地抓住微管。(i)基于 我们最近的发现，动粒只需要几个结合的微管就可以允许后期，我们将 定量地重新连接动粒组成，以测试这种精致微管起源的模型 灵敏度(ii)利用我们开发的生物物理方法来去除并施加在体内动粒上的力， 我们将定义产生动粒的专门的、强有力的和动态的抓握的机制。 总之，这将提供一个框架，用于理解，定位和重新连接物理 染色体分离过程，用于基础和治疗目的。这一建议具有创新性， 它测试了关于分子和细胞尺度事件之间联系的新假设，并提供了 新的工具，重新布线分子规模的力量和组件，并直接探测力学内部划分 哺乳动物细胞更广泛地说，这项工作将作为理解紧急架构的平台， 各种高分子机器的力学和计算。

Project Summary/Abstract Two large macromolecular machines, the spindle and kinetochore, coordinate chromosome segregation at cell division. Errors in their function can lead to cancer and birth defects. While we now know nearly all the molecules required for mammalian spindle and kinetochore function, how they collectively give rise to these machines’ emergent physical properties remains poorly understood. Our long-term goal is to uncover the basic physical design principles of robust and accurate mammalian chromosome segregation. How do thousands of nm-scale molecules, pushing and pulling, give rise to the spindle’s m-scale steady-state architecture and mechanics? How do hundreds of kinetochore molecules work together to ‘compute’ attachment signals for decision-making, and to robustly grip microtubules? This knowledge gap persists because we cannot currently reconstitute these machines in vitro, and lack tools in vivo. To close this gap, we need approaches to finely control molecules and directly exert forces inside dividing mammalian cells – which we recently developed. First, we aim to define the mechanisms and functions of the spindle’s emergent architecture and mechanics. (i) Based on our findings, we will test the idea that opposing stresses in the spindle are not required to give it a steady-state structure, but are instead required to give it mechanical and functional stability. Further, we will define in vivo and in vitro how active and passive forces contribute to building a steady-state spindle. (ii) To uncover the function of the spindle’s specific steady-state shape, we developed an optogenetic approach to acutely and locally control spindle architecture. We will use it to test the role of given architectural modules in space and time through mitosis. (iii) To define the mechanisms underlying the steady- state spindle’s mechanical robustness, we will use microneedle manipulation to deform the spindle, which we recently adapted in mammalian cells, and modeling. Second, we aim to define how the kinetochore’s molecules together enable it to ‘compute' attachment information and robustly grip microtubules. (i) Based on our recent finding that only a few bound microtubules are needed for a kinetochore to allow anaphase, we will quantitatively rewire kinetochore composition to test models for the origin of this exquisite microtubule sensitivity. (ii) Using biophysical approaches we developed to remove and exert forces on kinetochores in vivo, we will define the mechanisms giving rise to the kinetochore’s specialized, strong and dynamic grip. Together, this will provide a framework for understanding, targeting, and rewiring the physical processes of chromosome segregation for both basic and therapeutic purposes. This proposal is innovative in that it tests new hypotheses about the connection between molecular and cellular-scale events, and provides new tools for rewiring molecular-scale forces and assemblies and directly probing mechanics inside dividing mammalian cells. More broadly, this work will serve as a platform for understanding the emergent architecture, mechanics and computation of diverse macromolecular machines.