Chemical Biology of Cell Division - Revision - 2

细胞分裂的化学生物学 - 修订版 - 2

• 批准号: 10578031
• 负责人: TARUN M. KAPOOR
• 金额: $ 10.34万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10578031
• 关键词: Address; Anaphase; Biochemical; Cell division; Cells; Cellular biology; Centrosome; Chemicals; Complex; Cytokinesis; Cytoskeleton; Defect; Development; Disease; Ensure; Goals; Hour; Human; Image Analysis; Kinesin; Knowledge; Light; Link; Malignant Neoplasms; Methods; Microscopy; Microtubules; Mitosis; Molecular; Motor; PRC1 Protein; Pathway interactions; Permeability; Plus End of the Microtubule; Proteins; Proteomics; Recombinant Proteins; Research; Role; Shapes; Structure; Therapeutic; Time; Tubulin; Work; base; crosslink; experience; inhibitor; interdisciplinary approach; novel therapeutic intervention; novel therapeutics; protein function; protein protein interaction; quantitative imaging; response; self assembly

Project Summary/Abstract: Our long-term goal is to decipher the molecular mechanisms that ensure the proper assembly and function of the microtubule-based structures needed for error-free cell division. Essentially all the proteins required for cell division in human cells have now been identified. However, uncovering mechanisms has remained challenging as cell division is rapid and can be completed in <1 hour in human cells, with key steps taking only minutes. In addition, the microtubule-based structures needed for cell division require constant energy input to maintain shape and size, cannot be readily isolated in native forms and the protein-protein interactions critical for function can be transient and mitosis-specific. Finally, these structures are micrometer-sized and can be ~1000-times larger than their protein components. We take interdisciplinary approaches that can address these challenges and help dissect the dynamic self-assembly of these essential structures. We have: (i) Discovered and characterized selective cell-permeable chemical inhibitors of key proteins. These inhibitors can be powerful probes to examine cell division dynamics, as proteins can be inhibited or activated (via relief from inhibition), within minutes in living cells. To track the cellular responses to these fast perturbations we use state-of-the-art microscopy (e.g. lattice light-sheet microscopy) and quantitative image analysis methods. (ii) Developed and applied iCLASPI, a chemical proteomics approach to covalently ‘capture’ and profile transient and context-dependent protein-protein interactions in living cells. (iii) Analyzed the self-assembly of basic structural and functional motifs (e.g. bipolar microtubule arrays) with purified proteins. For these biochemical studies, we have assembled a ‘toolbox’ of recombinant proteins including isotypically-pure human tubulin, the augmin complex and key microtubule- associated motor and non-motor proteins. The proposed research, which benefits from our experience and expertise, will focus on anaphase and cytokinesis. These final stages of cell division can be difficult to study using approaches that cannot profile transient protein-protein interactions or do not provide precise temporal control over protein function. We will take interdisciplinary approaches to address the following gaps in our knowledge: (1) What are the roles of microtubule-severing proteins during the final stages of cell division? (2) How do PRC1, a non-motor protein that selectively crosslinks antiparallel microtubules, and kinesin-4, a microtubule plus-end directed motor protein, contribute to the assembly of the spindle midzone during anaphase? (3) What are the minimum number of proteins needed for microtubule-dependent microtubule formation, a key centrosome-independent microtubule nucleation pathway needed for cell division? Errors in cell division have been linked to diseases and developmental defects. Improper cell division has also been exploited in therapeutic strategies commonly used to treat diseases, such as cancer. Our work will not only shed light on fundamental cellular mechanisms but will also catalyze the development of new therapeutics.

项目摘要/摘要：我们的长期目标是破译分子机制，以确保 无错误的单元格所需的基于微管的结构的适当组装和功能。 基本上，现在已经鉴定出了人类细胞中细胞分裂所需的所有蛋白质。然而， 随着细胞分裂的速度迅速，可以在<1小时内完成，发现机制一直是挑战 人类细胞，关键步骤仅需几分钟。另外，细胞所需的基于微管的结构 除法需要恒定的能量输入以保持形状和大小，不能以天然形式容易隔离 并且对功能至关重要的蛋白质蛋白质相互作用可能是瞬时和有丝分裂特异性的。最后，这些 结构是微米大小的，可能比其蛋白质成分大约1000倍。我们接受 可以解决这些挑战并有助于剖析动态自我组装的跨学科方法 这些基本结构。我们有：（i）发现并表征了选择性的可渗透性化学物质 关键蛋白的抑制剂。这些抑制剂可能是检查细胞分裂动态的有力问题，因为 在活细胞中，可以在数分钟内抑制或激活蛋白质（通过缓解抑制作用）。跟踪 细胞对这些快速扰动的响应我们使用最先进的显微镜（例如晶格灯页） 显微镜）和定量图像分析方法。 （ii）开发和应用iClaspi，一种化学物质 蛋白质组学方法是共价“捕获”和剖面瞬态和上下文依赖性蛋白质 - 蛋白质蛋白 活细胞中的相互作用。 （iii）分析了基本结构和功能基序的自组装（例如双极 带有纯化蛋白的微管阵列）。对于这些生化研究，我们组装了一个“工具箱” 重组蛋白包括同种型纯化的人软素，Augmin复合物和关键微管 - 相关电动机和非运动蛋白。拟议的研究，从我们的经验和 专业知识将重点放在后期和细胞因子上。细胞分裂的最终阶段可能很难研究 使用无法介绍瞬时蛋白质 - 蛋白质相互作用或不提供精确临时性的方法 控制蛋白质功能。我们将采用跨学科的方法来解决我们的以下差距 知识：（1）在细胞分裂的最后阶段，微管蛋白的作用是什么？ （2） PRC1是一种选择性交联的反平行微管和驱动蛋白-4的非运动蛋白，A 微管加端的定向运动蛋白，有助于纺锤体中区的组装 后期？ （3）微管依赖性微管所需的最小蛋白质数量是多少 形成，细胞分裂所需的关键中心体无关的微管成核途径？错误 细胞分裂与疾病和发育缺陷有关。细胞分裂也不当 通常用于治疗疾病（例如癌症）的治疗策略。我们的工作不仅会 阐明了基本的细胞机制，但也将催化新疗法的发展。