Chemical Biology of Cell Division

项目概要/摘要：我们的长期目标是破译确保基因组安全的分子机制。 正确的组装和功能的微管为基础的结构所需的无错误的细胞分裂。 基本上，人类细胞中细胞分裂所需的所有蛋白质现在都已被鉴定出来。然而，在这方面， 揭示机制仍然具有挑战性，因为细胞分裂是快速的，并且可以在<1小时内完成。 人类细胞，关键步骤只需几分钟。此外，细胞所需的基于微管的结构 分裂需要恒定的能量输入来维持形状和大小，不能以天然形式容易地分离 并且对于功能至关重要的蛋白质-蛋白质相互作用可以是瞬时的和有丝分裂特异性的。最后这些 结构是微米大小的，可以比它们的蛋白质成分大1000倍。我们采取 跨学科的方法，可以解决这些挑战，并帮助剖析动态自组装的 这些重要的结构。我们有：（一）发现和特点的选择性细胞渗透化学 关键蛋白质的抑制剂。这些抑制剂可以是检查细胞分裂动力学的有力探针， 在活细胞中，蛋白质可以在几分钟内被抑制或激活（通过解除抑制）。跟踪 细胞对这些快速扰动的反应，我们使用最先进的显微镜（例如晶格光片 显微术）和定量图像分析方法。(ii)开发并应用化学品iCLASPI 蛋白质组学方法来共价“捕获”和分析瞬时和上下文依赖的蛋白质-蛋白质 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 微管阵列）。对于这些生物化学研究，我们已经组装了一个“工具箱”， 重组蛋白，包括同种型纯的人微管蛋白、augmin复合物和关键微管蛋白， 相关的马达和非马达蛋白。拟议的研究，这得益于我们的经验和 专业知识，将集中在后期和胞质分裂。这些细胞分裂的最后阶段可能很难研究 使用不能描绘瞬时蛋白质-蛋白质相互作用或不能提供精确的时间信息的方法， 控制蛋白质功能。我们将采取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，微管切割蛋白的作用是什么？（二） PRC 1是一种选择性交联反平行微管的非运动蛋白，而驱动蛋白4是一种 微管加末端定向马达蛋白，有助于纺锤体中间区的组装， 后期？(3)微管依赖性微管所需的最少蛋白质数量是多少 形成，一个关键的中心体独立的微管成核途径所需的细胞分裂？中的错误 细胞分裂与疾病和发育缺陷有关。不适当的细胞分裂 在通常用于治疗疾病如癌症的治疗策略中利用。我们的工作不仅 揭示了基本的细胞机制，但也将促进新疗法的发展。

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.