Emergent mechanics of mammalian chromosome segregation

项目摘要/摘要 两个大的大分子机器，纺锤体和动粒，协调细胞中的染色体分离。 组织。它们的功能错误可能会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，它们如何共同产生这些 人们对机器新出现的物理特性仍知之甚少。我们的长期目标是揭开 稳健而准确的哺乳动物染色体分离的物理设计原则。数以千计的 纳米尺度的分子，推动和拉动，产生了主轴的m尺度稳态结构和 机械师？成百上千个动粒分子是如何协同工作来计算连接信号的 决策，并牢牢抓住微管？这种知识鸿沟之所以持续存在，是因为我们目前无法 在体外重建这些机器，在体内缺乏工具。为了缩小这一差距，我们需要采取措施 控制分子并在哺乳动物细胞分裂中直接施加力--这是我们最近开发的。 首先，我们的目标是定义主轴的紧急体系结构的机制和功能 机械师。(I)基于我们的发现，我们将测试主轴中的相反应力不是 需要给它一个稳定的结构，但相反需要给它机械和功能 稳定性。此外，我们将在体内和体外定义主动和被动力量如何有助于建立 稳态主轴。(Ii)为了揭示主轴特定稳态形状的功能，我们开发了一种 光遗传学方法，以精确和局部控制主轴结构。我们将使用它来测试给定的角色 空间和时间中的建筑模块通过有丝分裂。(Iii)界定稳定以下各项的机制- 陈述主轴的机械稳定性，我们将使用微针操作来使主轴变形，这是我们 最近适应了哺乳动物细胞，并进行了建模。其次，我们的目标是定义动粒是如何 分子结合在一起，使其能够“计算”连接信息，并有力地抓住微管。(I)基于 我们最近的发现是，动粒只需要几个结合的微管就可以进入后期，我们将 定量重新连接动粒成分以测试这一精致微管的起源模型 敏感度。(Ii)使用我们开发的生物物理方法在体内移除和施加动心的力， 我们将定义动毛虫产生专门的、强大的和动态的抓地力的机制。 总而言之，这将为理解、确定目标和重新布线物理 用于基本和治疗目的的染色体分离过程。这项建议在以下方面具有创新性 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 用于重新布线分子尺度的力和组件并直接探测内部划分的力学的新工具 哺乳动物细胞。更广泛地说，这项工作将作为理解紧急体系结构的平台， 各种大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.