Biomechanics of molecular machines and multiscale non-linear systems

分子机器和多尺度非线性系统的生物力学

• 批准号: 10397656
• 负责人: Ekaterina L Grishchuk
• 金额: $ 64.35万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10397656
• 关键词: Address; Aneuploidy; Automobile Driving; Binding; Binding Proteins; Biological Assay; Biomechanics; Cell division; Cells; Chemicals; Chimeric Proteins; Chromatin; Chromosome Segregation; Chromosomes; Closure by clamp; Complex; Diffuse; Equipment; Exhibits; Fostering; Friction; Genomic Instability; Health; Hela Cells; Human; Individual; Kinetochores; Knowledge; Macromolecular Complexes; Mechanics; Microtubule-Associated Proteins; Microtubules; Mission; Mitotic; Molecular; Molecular Machines; Motion; Motor Activity; Neuronal Plasticity; Pattern; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Process; Property; Proteins; Public Health; Regulation; Research; Role; Shapes; Spectrum Analysis; Subcellular structure; System; Testing; Theoretical model; Time; Travel; United States National Institutes of Health; Weight-Bearing state; Work; aurora B kinase; biophysical analysis; cell motility; experience; flexibility; improved; in vitro Assay; innovation; insight; novel strategies; operation; particle; prevent; scaffold; single molecule; spatiotemporal; tool

We work to determine the fundamental principles underlying the operation of molecular machines that give cells the remarkable ability to segregate their chromosomes during cell division. Various force-sensitive interactions are essential for mitotic fidelity, and are therefore critical to our understanding of aneuploidy and genomic instability. Over the last 5 years, we have developed molecular tools, equipment, and expertise to quantitatively and rigorously address central questions about the role of force in chromosome segregation: (1) How do the macromolecular complexes that constitute human kinetochores travel with dynamic microtubule ends under load? (2) How do individual microtubule-associated proteins with no motor activity glide along microtubules under dragging force? (3) How does tension applied to the centromeric chromatin meshwork shape the spatial phosphorylation gradients that orchestrate assembly of the kinetochores and their binding to microtubules? We approach these problems using reductionist approaches and innovative in vitro assays that reconstruct these interactions at multiple scales, and analyze our findings with advanced theoretical modeling. (1) To recreate force-sensitive interactions between microtubules and human kinetochores, we developed a novel approach for generating macromolecular kinetochore subcomplexes using inducible protein-fusion scaffolds. When isolated from mitotic HeLa cells, these particles exhibit key physiological properties of native kinetochores, including their persistent association with dynamic microtubule ends. This breakthrough will enable us for the first time to study the motility of native human kinetochore complexes, driving forward our biophysical analysis of kinetochore load-bearing. (2) We will investigate the force sensitivity of individual microtubule-binding proteins at the single-molecule and ensemble levels using an advanced force spectroscopy approach. We have implemented a highly sensitive dual-trap, three-bead assay employing an ultrafast force-clamp that allows us to pull on a single non-motor molecule diffusing on the microtubule wall, imitating the forces these kinetochore-bound molecules experience during chromosome motions. This approach will provide unique molecular-mechanical insights into the friction-generating interface that allows the kinetochore to glide along microtubule, while preventing it from slipping from microtubule ends. (3) We will seek to understand how mechanical deformations shape chemical gradients formed within the chemo- mechanical meshworks, such as of the centromeric chromatin. Previously, we reconstructed a non-linear Aurora B kinase/phosphatase bi-stable switch using soluble components. In a proof-of-principle study, we will embed these enzymatic components into a flexible meshwork to test whether its deformations can control formation of distinct phosphorylation patterns. Spatio-temporal regulation of the phosphorylation status of kinetochore proteins is central to the error correction mechanism of microtubule attachment. Hence, our findings will provide new knowledge about this fundamental process, and facilitate new discoveries about complex chemo-mechanical systems.

我们致力于确定分子机器运行的基本原理，分子机器赋予细胞 在细胞分裂过程中分离染色体的非凡能力。各种力敏感的相互作用是必不可少的 对于有丝分裂的保真度，因此对于我们理解非整倍体和基因组不稳定性是至关重要的。在过去5年中 多年来，我们已经开发了分子工具、设备和专业知识来定量和严格地处理中央 关于力在染色体分离中的作用的问题：(1)构成染色体的大分子复合体是如何 人类运动中枢在负荷下与动态微管末端一起运动？(2)单个微管如何与 没有运动活性的蛋白质在拖拉力的作用下沿着微管滑动？(3)张力如何作用于微管 着丝粒染色质网络形状空间磷酸化梯度，协调组装 动点及其与微管的结合？我们使用简化论的方法来处理这些问题 创新的体外试验在多个尺度上重建了这些相互作用，并分析了我们的发现 理论建模。(1)为了重现微管和人体运动中枢之间的力敏感相互作用，我们 开发了一种利用可诱导蛋白融合产生大分子动粒核亚复合体的新方法 脚手架。当从有丝分裂的HeLa细胞中分离出来时，这些颗粒表现出天然的关键生理特性 动点，包括它们与动态微管末端的持久联系。这一突破将使我们能够 首次研究天然人动粒复合体的运动性，推动了我们的生物物理分析 动粒承重。(2)我们将研究单个微管结合蛋白的力敏感性。 使用先进的力谱方法的单分子和系综能级。我们已经实施了高度的 灵敏的双捕捉器，三珠分析，使用超快的力钳，允许我们在单个非马达上拉动 分子在微管壁上扩散，模拟这些动粒结合的分子在 染色体运动。这种方法将为摩擦的产生提供独特的分子力学见解 允许动粒沿着微管滑动的界面，同时防止它从微管末端滑落。 (3)我们将试图了解机械变形如何影响化学-化学-化学过程中形成的化学梯度- 机械网络，如着丝粒染色质。之前，我们重建了一个非线性极光B 使用可溶性成分的激酶/磷酸酶双稳开关。在一项原则证明研究中，我们将嵌入这些 酶组分进入柔性网络，测试其变形是否能控制形成截然不同的 磷酸化模式。动粒蛋白磷酸化状态的时空调节是 微管附着的纠错机制。因此，我们的发现将提供有关这方面的新知识。 基本过程，并促进关于复杂化学机械系统的新发现。