Microtubule bundles in the mitotic spindle: probing how mechanical and functional robustness emerge from molecular architecture

项目摘要/摘要 有丝分裂纺锤体是一种基于微管的机器，它将染色体分离成两个新的 当细胞分裂时，子代细胞。准确的主轴功能至关重要：错误会导致额外或丢失 染色体，这与癌症、出生缺陷和流产有关。主轴功能需要 生物化学和力学的强大耦合。然而，理解这种自组织机器是如何 在正确的时间在正确的地点产生所需的部队仍然是一个挑战。我们的长期目标是 确定纺锤的微米级机械性能如何从分子尺度的生物化学中显现出来。 我们的重点是微管束，它在纺锤体和其他组织中提供组织和支撑刚性 基于微管的结构。我们不知道捆绑分子赋予了什么材料性质 纺锤体束，它们的分子特性如何允许它们这样做，或者这些新兴的机械 属性针对生物功能进行了调整。为了解决这些问题，我们将采取量化措施 读数束如何对改变力学的扰动作出反应。我们采取多系统的方法来 了解哺乳动物动粒纤维(k纤维)中附着和分离的束力学 染色体；在分裂酵母中，S.pombe纺锤体，其刻板的组织有助于探索 特定的交联剂性质会影响整个维管束；在体外，我们可以更精确地控制。 我们的方法被组织成两个计划：(1)探索分子和机械组织 纺锤体微管束，以及(2)控制纺锤体微管束改变功能 机械师。在计划1中，我们将确定k光纤组织如何平衡竞争对手的机械 灵活适应变化的稳健力传递对染色体分离的约束 纺锤体形态。我们还将开发新的工具来测量纺锤体内微管之间的力 确定这些束状物如何有效传递力以实现其机械功能。在……里面 程序2，我们将确定微管交联剂的几何和力学性质如何 适用于特定功能的捆绑包规模属性。我们将创造工程交联剂，其 我们将控制机械和几何特性，并用它们来构建重组的微管束 在体外，并在体内改变束的性质。通过测量这些束对分子尺度的响应 变化，我们将确定如何出现微米级的特性。 总之，这项拟议的工作将描绘出分子尺度的部件如何赋予纺锤体束以特性 这平衡了相互竞争的机械约束。从长远来看，这种方法可能会带来对 如何利用改变细胞的“建筑代码”来针对微管结构在 疾病，或建造新的建筑。这种方法可以扩展到理解 纺锤体外的微管束结构，如纤毛和轴突。

Project Summary/Abstract The mitotic spindle is a microtubule-based machine that segregates chromosomes into two new daughter cells when cells divide. Accurate spindle function is critical: mistakes lead to extra or missing chromosomes, which are associated with cancer, birth defects, and miscarriage. Spindle function requires robust coupling of biochemistry and mechanics. Yet, understanding how this self-organizing machine generates the required forces in the right place at the right time remains a challenge. Our long term goal is to determine how micron-scale mechanical properties of the spindle emerge from molecular-scale biochemistry. We focus on microtubule bundles, which provide organization and underpin rigidity in the spindle and in other microtubule-based structures. We do not understand what material properties bundling molecules impart to spindle bundles, how their molecular properties allow them to do so, or how these emergent mechanical properties are tuned for biological functions. To address these questions, we will measure quantitative readouts of how bundles respond to perturbations that alter mechanics. We take a multi-system approach to understanding bundle mechanics in mammalian kinetochore-fibers (k-fibers), which attach and segregate chromosomes; in fission yeast S. pombe spindles, whose stereotyped organization facilitates probing how specific crosslinker properties affect bundles overall; and in vitro, where we have more precise control. Our approach is organized into two programs: (1) probing the molecular and mechanical organization of spindle microtubule bundles, and (2) controlling spindle microtubule bundles to alter function through novel mechanics. In Program 1, we will determine how k-fiber organization balances competing mechanical constraints of robust force-transmission for chromosome segregation with flexibility to adapt to changing spindle morphology. We will also develop new tools to measure force between microtubules within spindle bundles, determining how these bundles effectively transmit force to achieve their mechanical functions. In Program 2, we will determine how the geometric and mechanical properties of microtubule crosslinkers impart bundle-scale properties that are adapted to particular functions. We will create engineered crosslinkers whose mechanical and geometric properties we will control, and use them to build reconstituted microtubule bundles in vitro, and to alter bundle properties in vivo. By measuring the response of these bundles to molecular-scale changes, we will determine how micron-scale properties emerge. In sum, the proposed work will map how molecular scale parts impart spindle bundles with properties that balance competing mechanical constraints. In the long term, this approach may lead to new insight into how altering the cell’s “building code” can be harnessed to target microtubule architectures with key roles in disease, or to build novel architectures. This approach can extend to understand the emergent mechanics of microtubule bundle architectures beyond the spindle, such as in cilia and axons.