THREE-DIMENSIONAL STRUCTURE AND FUNCTION OF THE MAMMALIAN KINETOCHORE

哺乳动物动粒的三维结构和功能

• 批准号: 7598341
• 负责人: BRUCE F MCEWEN
• 金额: $ 2.29万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598341
• 关键词: Anaphase; Biological Preservation; Cell Cycle Checkpoint; Cells; Cellular biology; Chromosome Segregation; Chromosomes; Classification; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer Vision Systems; Computers; Data; Data Set; Electrons; Freeze Substitution; Freezing; Funding; Grant; Hela Cells; Individual; Institution; Kinetochores; Knowledge; Laboratories; Methods; Microtubules; Mitosis; Mitotic; Mitotic spindle; Molecular; Molecular Conformation; Motivation; Noise; Numbers; Plus End of the Microtubule; Preparation; Procedures; Process; Protocols documentation; Range; Relative (related person); Reproducibility of Results; Research; Research Personnel; Resolution; Resources; Signal Transduction; Sorting - Cell Movement; Source; Specimen; Structural Models; Structure; Students; Ultramicrotomy; United States National Institutes of Health; Variant; Work; abstracting; base; density; electron tomography; interest; pressure; reconstruction; three dimensional structure; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT The mammalian kinetochore is a proteineous complexes that perform at least four functions that are vital for accurate chromosome segregation during mitosis: 1) attaching chromosomes to the mitotic spindle; 2) controlling the dynamics of kMTs; 3) generating force for chromosome alignment; and 4) generating a cell cycle checkpoint that delays anaphase onset until all chromosomes are attached to the mitotic spindle and aligned at the spindle equator. Intricate interactions between kinetochores and microtubules (MTs) are essential for all of these vital processes. Despite progress in identifying molecular components of the kinetochore, the underlying mechanisms of kinetochore function and its interactions with MTs remains largely obscure. This is in part due to the paucity of data concerning the 3D ultrastructure of the kinetochore. The only electron tomographic study was performed by the project PI over 10 years ago with chemically fixed specimens (McEwen et al, 1993). In 1998, McEwen and colleagues demonstrated a dramatic improvement in preservation of kinetochore ultrastructure using high-pressure freezing and freeze-substitution, but very little electron tomography was used in that study. Our long-term objective is to establish a high-quality, high-resolution structural model of the mammalian kinetochore in its unbound state and to determine structural changes that occur when key kinetochore components are removed. The next step is to refine our current structural model by reconstructing the kinetochore in the frozen-hydrated state. During the past year we have developed protocol that yield a high density of well-frozen mitotic cells and we are using high pressure-frozen specimens that have been freeze-substituted. This strategy allows a relatively quick and straightforward method of assessing specimen preparations and optimizing protocols before turning to the more technically demanding task of electron tomography of frozen-hydrated sections. The freeze-substituted material will also provide electron tomographic data sets with a higher signal-to-noise ratio and better quality high tilt data. We now have reproducible protocols for obtaining a high percentage of HeLa cells in mitosis using a procedure call mitotic shake off. Consistently good freezing was obtained using 15% BSA as a cryo protectant. We are currently confirming the reproducibility of the result and preparing cells for frozen-hydrated ultramicrotomy. In 2002 we began collaborating with the laboratory of Dr. Qiang Ji at RPI to develop automated tools for segmenting microtubules and their plus-ends from tomographic reconstructions of mammalian kinetochores. Motivation for this project came from our work to classify the plus ends of kinetochore microtubules according to their structural conformations (referred to above as the PtK project). These conformations are indicative of the dynamic state of the microtubules and knowledge of how the kinetochore controls microtubule dynamics is a critical issue in cell biology. Our general approach is to use the high throughput capabilities of modern electron tomography to collect a large enough date base of kinetochore microtubule plus ends to perform statistically meaningful analyses on how the conformations change with stage of mitosis, treatment with pharmacological agents, and knockdowns of key kinetochore molecular components. We are also interested in determining how well coordinated the plus-end conformations are on individual kinetochores. We would prefer to use computer classification methods to sort the conformations, but the reconstructions are too noisy and there is too much background structure to permit classification on kinetochore microtubules extracted directly from the raw tomographic reconstructions. There are also issues of variation due to orientation relative to the missing pyramid. To overcome these problems, we sought to develop an efficient automated segmentation method that would extract the microtubules, including their plus ends, from large numbers of tomographic reconstructions. Dr. Ji is an expert on computer vision. His graduate student, Ming Jing, has developed a multi-step segmentation method that uses the known cylindrical geometry of microtubules, and the range of feasible curvatures found at microtubule plus ends, as constraints on the segmentation process.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 摘要 哺乳动物动粒是一种蛋白质复合体，它至少具有四种功能，这些功能对于有丝分裂期间染色体的精确分离至关重要：1）将染色体附着到有丝分裂纺锤体上; 2）控制kMT的动力学; 3）产生染色体排列的力;和4）产生细胞周期检查点，其延迟后期开始，直到所有染色体都附着到有丝分裂纺锤体上并在纺锤体赤道处对齐。动粒和微管（MT）之间复杂的相互作用是所有这些重要过程所必需的。 尽管在确定动粒的分子组成方面取得了进展，但动粒功能及其与MT相互作用的潜在机制仍然很不清楚。 这部分是由于缺乏有关动粒的3D超微结构的数据。 唯一的电子断层扫描研究是由项目PI在10多年前使用化学固定样本进行的（麦克尤恩等人，1993年）。 在1998年，麦克尤恩和他的同事们证明了使用高压冷冻和冷冻替代在保存动粒超微结构方面的显着改善，但在该研究中很少使用电子断层扫描。 我们的长期目标是建立一个高质量，高分辨率的哺乳动物动粒在其未结合状态的结构模型，并确定当关键动粒组件被删除时发生的结构变化。 下一步是通过重建冷冻水合状态下的动粒来完善我们目前的结构模型。 在过去的一年里，我们开发了产生高密度冷冻良好的有丝分裂细胞的方案，并且我们正在使用已冷冻替代的高压冷冻标本。 这种策略允许一个相对快速和直接的方法来评估标本制备和优化协议，然后再转向技术要求更高的任务，冷冻水化切片的电子断层扫描。 冷冻取代的材料还将提供具有更高信噪比和更好质量的高倾斜数据的电子断层摄影数据集。我们现在有可重复的协议，获得高比例的HeLa细胞在有丝分裂使用程序调用 有丝分裂脱落. 使用15%BSA作为冷冻保护剂获得了一致良好的冷冻。 我们目前正在确认结果的重现性，并准备冷冻水化超微切片的细胞。 2002年，我们开始与RPI的Jiang Ji博士的实验室合作，开发自动化工具，用于从哺乳动物动粒的断层重建中分割微管及其加端。 这个项目的动机来自于我们的工作，根据它们的结构构象对动粒微管的正端进行分类（上面称为PtK项目）。 这些构象是微管的动态状态的指示，并且关于动粒如何控制微管动力学的知识是细胞生物学中的关键问题。 我们的一般方法是使用现代电子断层扫描的高通量能力，收集足够大的数据库的动粒微管加结束进行统计学上有意义的分析，如何构象变化与有丝分裂阶段，治疗药物，和击倒的关键动粒分子成分。 我们也有兴趣在确定如何协调的加端构象是对个人kinetochores。 我们更愿意使用计算机分类方法来排序的构象，但重建太嘈杂，有太多的背景结构，允许分类动粒微管直接从原始的断层重建提取。 还存在由于相对于缺失的金字塔的定向而引起的变化的问题。 为了克服这些问题，我们试图开发一种有效的自动分割方法，该方法将从大量的断层重建中提取微管，包括它们的正端。季博士是计算机视觉方面的专家。 他的研究生Ming Jing开发了一种多步分割方法，该方法使用微管的已知圆柱形几何形状，以及在微管加上末端发现的可行曲率范围，作为分割过程的约束条件。