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: 10204551
• 负责人: Ekaterina L Grishchuk
• 金额: $ 48.95万
• 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/10204551
• 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作的基本原理，这些分子机器赋予细胞 在细胞分裂过程中分离染色体的非凡能力。各种力敏相互作用至关重要 对于有丝分裂保真度，因此对于我们了解非整倍性和基因组不稳定性至关重要。按最近5 多年来，我们已经开发了分子工具，设备和专业知识，以定量和严格地解决中央 关于力在染色体分离中的作用的问题：（1）构成染色体分离的大分子复合物是如何形成的 人类动粒在负载下以动态微管末端行进？(2)单个微管相关的 没有运动活性的蛋白质在拖曳力的作用下沿着沿着滑动？(3)张力是如何作用于 着丝粒染色质网络塑造空间磷酸化梯度，协调组装的蛋白质。 动粒和它们与微管的结合我们使用简化主义的方法来处理这些问题， 创新的体外试验，在多个尺度上重建这些相互作用，并分析我们的研究结果与先进的 理论建模(1)为了重现微管和人类动粒之间的力敏相互作用，我们 开发了一种新的方法，利用诱导蛋白融合产生大分子动粒亚复合物 脚手架当从有丝分裂的HeLa细胞中分离时，这些颗粒表现出天然细胞的关键生理特性。 动粒，包括它们与动态微管末端的持续关联。这一突破将使我们能够 第一次研究了人类动粒复合体的运动性，推动了我们对 动粒承重。(2)我们将研究单个微管结合蛋白的力敏感性， 单分子和合奏水平使用先进的力谱方法。我们实施了一项高度 灵敏的双陷阱，三珠检测采用超快速力钳，使我们能够拉上一个单一的非电机 在微管壁上扩散的分子，模仿这些运动舞蹈结合的分子在运动过程中所经历的力。 染色体运动这种方法将提供独特的分子力学见解的摩擦产生 界面，其允许动粒沿着微管滑动，同时防止其从微管末端滑动。 (3)我们将试图了解机械变形如何塑造化学梯度内形成的化学- 机械的网状结构，如着丝粒染色质的网状结构。以前，我们重建了一个非线性极光B 激酶/磷酸酶双稳态开关。在一项原理验证研究中，我们将嵌入这些 酶成分进入柔性网络，以测试其变形是否可以控制不同的细胞的形成。 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，我们的研究结果将提供有关这方面的新知识 基本过程，促进复杂化学机械系统的新发现。