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: 10601048
• 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/10601048
• 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; Creativeness; Diffusion; 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; molecular assembly/self assembly; molecular mechanics; novel strategies; operation; particle; prevent; scaffold; segregation; 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，为细胞提供 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 对于有丝分裂保真度，因此对于我们理解非整倍性和基因组不稳定性至关重要。过去 5 多年来，我们开发了分子工具、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）构成染色体的大分子复合物是如何作用的？ 人类动粒在负载下随动态微管末端移动？ (2)个体微管如何关联 没有运动活性的蛋白质在拖曳力的作用下沿着微管滑动？ （3）如何施加张力 着丝粒染色质网络形成空间磷酸化梯度，协调组装 动粒及其与微管的结合？我们使用还原论方法来解决这些问题 创新的体外测定可以在多个尺度上重建这些相互作用，并利用先进的技术分析我们的发现 理论建模。 (1) 为了重建微管和人类动粒之间的力敏感相互作用，我们 开发了一种使用诱导蛋白融合生成大分子着丝粒亚复合物的新方法 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 首次研究人类天然着丝粒复合体的运动性，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在 使用先进的力谱方法进行单分子和整体水平。我们实施了高度 灵敏的双陷阱、三珠测定采用超快力夹，使我们能够拉动单个非电机 分子在微管壁上扩散，模仿这些动粒结合分子在微管壁上所经历的力 染色体运动。这种方法将为摩擦产生提供独特的分子力学见解 该界面允许着丝粒沿着微管滑动，同时防止其从微管末端滑落。 （3）我们将寻求了解机械变形如何塑造化学梯度内形成的化学梯度 机械网络，例如着丝粒染色质。之前，我们重建了一个非线性的Aurora B 使用可溶性成分的激酶/磷酸酶双稳态开关。在原理验证研究中，我们将嵌入这些 将酶组分放入柔性网络中，以测试其变形是否可以控制不同的形成 磷酸化模式。着丝粒蛋白磷酸化状态的时空调节对于 微管附着的纠错机制。因此，我们的研究结果将提供关于此的新知识 基本过程，并促进复杂化学机械系统的新发现。

项目成果

CLASP2 recognizes tubulins exposed at the microtubule plus-end in a nucleotide state-sensitive manner.

CLASP2 以核苷酸状态敏感的方式识别暴露在微管正端的微管蛋白。

• 发表时间: 2023-01-04
• 影响因子: 13.6
• 作者: Luo, Wangxi;Demidov, Vladimir;Shen, Qi;Girão, Hugo;Chakraborty, Manas;Maiorov, Aleksandr;Ataullakhanov, Fazly I;Lin, Chenxiang;Maiato, Helder;Grishchuk, Ekaterina L
• 通讯作者: Grishchuk, Ekaterina L

Higher-order protein assembly controls kinetochore formation.

高阶蛋白质组装控制着丝粒的形成。

• 发表时间: 2024-01
• 影响因子: 21.3
• 作者: Sissoko, Gunter B;Tarasovetc, Ekaterina V;Marescal, Océane;Grishchuk, Ekaterina L;Cheeseman, Iain M
• 通讯作者: Cheeseman, Iain M

Molecular density-accelerated binding-site maturation underlies CENP-T-dependent kinetochore assembly.

分子密度加速的结合位点成熟是 CENP-T 依赖性动粒组装的基础。

• 发表时间: 2024-02-26
• 作者: Tarasovetc, Ekaterina V;Sissoko, Gunter B;Mukhina, Anna S;Maiorov, Aleksandr;Ataullakhanov, Fazoil I;Cheeseman, Iain M;Grishchuk, Ekaterina L
• 通讯作者: Grishchuk, Ekaterina L

Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins.

微管结合蛋白的超快力钳光谱。

• 发表时间: 2022
• 作者: Tripathy, Suvranta K;Demidov, Vladimir M;Gonchar, Ivan V;Wu, Shaowen;Ataullakhanov, Fazly I;Grishchuk, Ekaterina L
• 通讯作者: Grishchuk, Ekaterina L