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: 7357269
• 负责人: BRUCE F MCEWEN
• 金额: $ 2.81万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7357269
• 关键词: THREE; DIMENSIONAL; STRUCTURE; FUNCTION; MAMMALIAN

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. NIH R01 GM66270 ?Mammalian Kinetochore Control of Microtubule Dynamics?, 08/01/02 ? 07/31/06 Bruce F. McEwen, Wadsworth Center NSF MCB 0110821 ?3D Structure and Function of the Mammalian Kinetochore?, 09/01/01 ? 08/31/05. Bruce F. McEwen, Wadsworth Center ABSTRACT The mammalian kinetochore is a distinctive mat-like structure that forms at the primary constriction of mitotic and meiotic chromosomes Rieder and Salmon, 1998, Manly et al, 1999). It functions to attach chromosomes to spindle microtubules. In addition, the kinetochore has a major role in metaphase alignment of chromosomes at the spindle equator, the cell cycle checkpoint for entry into anaphase, and the anaphase segregation of replicate chromatids into nascent daughter cells. Despite progress in identifying molecular components of the kinetochore, the mechanism of kinetochore function 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 at that study. With improvements in instrumentation and technique, now is an opportune time to initiate a comprehensive study of kinetochore 3D ultrastructure. Initially we will perform this study with high-pressure frozen, freeze-substituted cells in order to optimize freezing conditions and obtain a relatively high-throughput for statistical analysis. However, it is imperative to also determine the structure of frozen-hydrated kinetochores because our earlier study demonstrated the sensitivity of kinetochore ultrastructure to specimen preparation conditions. The objective of this study 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. Present work is primarily with PTK cells, but as far as possible the model will be constructed based on tomographic reconstructions of frozen-hydrated mitotic HeLa cells. However, in order to develop specimen preparation protocols that yield a high density of well-frozen mitotic cells, we have been working initially work with 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. The PTK project was completed, and specimen preparation trials with HeLa cells are beginning. A short methods study using PHEM versus PBS buffer during conventional fixation is close to completion. It was found that PTK cells fixed in PHEM buffer yielded similar microtubule plus-end conformations as high pressure frozen PTK control cells. This was not the case with cells conventionally fixed in PBS buffer. Additional PTK cells treated with nocodazol and taxol were fixed in PHEM buffer and these cells are in the process of being compared to HPF nocodazol and taxol cells. Anaphase cells were also tested. All tomographic data has been collected and reconstructed. If results from these experiments confirm that PHEM produces results similar to HPF, then only PHEM prepared material will be included in the PTK study. This data is also being written up into a short methods paper, and it will be important for protocols used by collaborators using conventional fixation. Two presentations were made at the Microscopy and Microanalysis 2005 Meeting, Honolulu, HI, July 29 ? August 4, 2005: ? McEwen, B.F., Jiang, M., Zhang, W., Vandenbeldt, K., and Ji, Q. (2005) Model-based approach to automated segmentation of electron tomographic reconstructions. Microsc. Microanal. 11 (Suppl 2): 328CD. (Platform) ? Dong, Y., Meng, X., Vandenbeldt, K., Hergert, P. and McEwen, B.F. (2005) Ultrastructure of nocodazole-treated PtK1 kinetochore after high-pressure freezing and freeze-substitution. Microsc. Microanal. 11 (Suppl 2): 338CD. (Poster) A collaborative project with Dr. Katsumi Kitagawa from St. Jude Children?s Research Hospital was completed. Dr. Kitagawa was interested in determining whether the 17-AAG treatment affects the kinetochore or kinetochore binding. Both 17-AAG treated and control Hela cells were prepared using conventional EM techniques. Initial results indicate that there is little if any effect. A manuscript is in preparation. A collaboration with Dr. Bill Earnshaw?s lab (University of Edinburgh, Scotland) to examine the kinetochore of DT40 cells began in October of this year with the two week visit by Dr. Earnshaw?s technician Paola Vagnarelli. The advantage of the DT40 cell system is that creation of conditional mutations by killing the endogenous protein and placing a copy under a promoter on the tet-off system. This project is still in the initial stages but we were able to grow cells and examine them by conventional plastic embedded electron microscopy. We also have some high pressure frozen, freeze-substituted specimens. 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. During the past year we have debugged and refined the method. This culminated in writing user-friendly GUI that enables users to run the whole suite program modules as a unit or to run individual modules in any desired combination. For example, the user could chose to segment microtubules in a volume with concern about their plus ends. Although the tools were developed for our specific purposes, we feel that the methods are general and will benefit any one wishing to segment cytoskeletal fibers such as microtubules, actin and even intermediate filaments, from tomographic reconstruction. The following paper was written: ? Jiang, M., Ji, Q., and McEwen, B. (2005) Automated extraction of fine-features of kinetochore microtubules and plus ends from electron tomography. IEEE Computer Graphics and Applications (in press). Dr. McEwen gave an invited platform talk entitled ?Using electron tomography to investigate filamentous structures in prokaryotic and eukaryotic cells? at the Annual Meeting of the American Society for Microbiology in Atlanta, GA, June 6, 2005. Two presentations were made at the Microscopy and Microanalysis 2005 Meeting, Honolulu, HI, July 29 ? August 4, 2005: Platform: ? McEwen, B.F., Jiang, M., Zhang, W., Vandenbeldt, K., and Ji, Q. (2005) Model-based approach to automated segmentation of electron tomographic reconstructions. Microsc. Microanal. 11 (Suppl 2): 328CD. Poster: ? Dong, Y., Meng, X., Vandenbeldt, K., Hergert, P. and McEwen, B.F. (2005) Ultrastructure of nocodazole-treated PtK1 kinetochore after high-pressure freezing and freeze-substitution. Microsc. Microanal. 11 (Suppl 2): 338CD.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。 NIH R01 GM66270“哺乳动物动粒控制微管动力学”，2002 年 8 月 1 日？ 07/31/06 Bruce F. McEwen，沃兹沃斯中心 NSF MCB 0110821 “哺乳动物动粒的 3D 结构和功能”，2001 年 9 月 1 日？ 2005 年 8 月 31 日。 Bruce F. McEwen，沃兹沃斯中心 摘要 哺乳动物动粒是一种独特的垫状结构，形成于有丝分裂和减数分裂染色体的初级缢缩处（Rieder 和 Salmon，1998；Manly 等，1999）。其功能是将染色体附着到纺锤体微管上。此外，着丝粒在纺锤体赤道处染色体的中期排列、进入后期的细胞周期检查点以及复制染色单体进入新生子细胞的后期分离中发挥着重要作用。尽管在识别动粒分子成分方面取得了进展，但动粒功能的机制在很大程度上仍然不清楚。这部分是由于有关着丝粒 3D 超微结构的数据缺乏。唯一的电子断层扫描研究是 PI 项目在 10 多年前使用化学固定样本进行的（McEwen 等，1993）。 1998 年，McEwen 及其同事展示了使用高压冷冻和冷冻替代对动粒超微结构保存的显着改善，但该研究中很少使用电子断层扫描。随着仪器和技术的改进，现在是启动动粒 3D 超微结构全面研究的最佳时机。最初，我们将使用高压冷冻、冷冻替代细胞进行这项研究，以优化冷冻条件并获得相对高的统计分析通量。然而，还必须确定冷冻水合动粒的结构，因为我们早期的研究证明了动粒超微结构对样本制备条件的敏感性。本研究的目的是建立哺乳动物动粒在未结合状态下的高质量、高分辨率结构模型，并确定当关键动粒成分被去除时发生的结构变化。 目前的工作主要是针对 PTK 细胞，但模型将尽可能基于冷冻水合有丝分裂 HeLa 细胞的断层扫描重建来构建。然而，为了开发产生高密度冷冻良好有丝分裂细胞的样本制备方案，我们最初一直致力于使用已冷冻替代的高压冷冻样本。该策略允许在转向技术要求更高的冷冻水合切片电子断层扫描任务之前，采用相对快速和直接的方法来评估标本制备和优化方案。冷冻替代材料还将提供具有更高信噪比和质量更好的高倾斜数据的电子断层扫描数据集。我们现在拥有可重复的方案，可以使用“有丝分裂抖动”程序获得高比例的有丝分裂 HeLa 细胞。使用 15% BSA 作为冷冻保护剂可以获得一致的良好冷冻效果。我们目前正在确认结果的可重复性，并为冷冻水合超薄切片术准备细胞。 PTK项目已经完成，HeLa细胞的标本制备试验正在开始。在传统固定过程中使用 PHEM 与 PBS 缓冲液的简短方法研究已接近完成。结果发现，固定在 PHEM 缓冲液中的 PTK 细胞产生与高压冷冻 PTK 对照细胞相似的微管正端构象。对于传统固定在 PBS 缓冲液中的细胞来说，情况并非如此。将用诺考达唑和紫杉醇处理的其他 PTK 细胞固定在 PHEM 缓冲液中，并将这些细胞与 HPF 诺考达唑和紫杉醇细胞进行比较。还测试了后期细胞。 所有断层扫描数据均已收集并重建。如果这些实验的结果证实 PHEM 产生与 HPF 相似的结果，则只有 PHEM 制备的材料才会包含在 PTK 研究中。这些数据也被写入简短的方法论文中，这对于使用传统固定的合作者使用的协议非常重要。 7 月 29 日在夏威夷州檀香山举行的 2005 年显微镜和微量分析会议上做了两场演讲？ 2005 年 8 月 4 日：？ McEwen, B.F.、Jiang, M.、Zhang, W.、Vandenbeldt, K. 和 Ji, Q. (2005) 基于模型的电子断层扫描重建自动分割方法。显微镜。微肛门。 11（补充 2）：328CD。 （平台） ？ Dong, Y.、Meng, X.、Vandenbeldt, K.、Hergert, P. 和 McEwen, B.F. (2005) 高压冷冻和冷冻替代后诺考达唑处理的 PtK1 着丝粒的超微结构。显微镜。微肛门。 11（增补 2）：338CD。 （海报） 与圣裘德儿童研究医院 Katsumi Kitakawa 博士的合作项目已完成。 Kitakawa 博士有兴趣确定 17-AAG 治疗是否影响着丝粒或着丝粒结合。 17-AAG 处理和对照 Hela 细胞均使用常规 EM 技术制备。初步结果表明，即使有影响，影响也很小。手稿正在准备中。今年 10 月，Earnshaw 博士的技术员 Paola Vagnarelli 进行了为期两周的访问，开始与 Bill Earnshaw 博士的实验室（苏格兰爱丁堡大学）合作检查 DT40 细胞的动粒。 DT40细胞系统的优点是通过杀死内源蛋白并将拷贝置于tet-off系统的启动子下来创建条件突变。该项目仍处于初始阶段，但我们能够培养细胞并通过传统的塑料嵌入式电子显微镜检查它们。我们还有一些高压冷冻、冷冻替代标本。 2002年，我们开始与RPI的Qiang Ji博士实验室合作开发自动化工具，用于从哺乳动物动粒的断层扫描重建中分割微管及其末端。这个项目的动机来自于我们根据动粒微管的结构构象对正端进行分类的工作（上面称为 PtK 项目）。这些构象表明微管的动态状态，了解动粒如何控制微管动态是细胞生物学中的一个关键问题。我们的一般方法是利用现代电子断层扫描的高通量能力来收集足够大的着丝粒微管加末端的数据基础，以对构象如何随有丝分裂阶段、药物治疗和关键着丝粒分子成分的敲低而变化进行统计上有意义的分析。我们还对确定个体动粒上正端构象的协调程度感兴趣。我们更愿意使用计算机分类方法对构象进行排序，但重建噪声太大，并且背景结构太多，无法对直接从原始断层扫描重建中提取的动粒微管进行分类。由于相对于缺失金字塔的方向，还存在变化问题。为了克服这些问题，我们寻求开发一种有效的自动分割方法，该方法可以从大量断层扫描重建中提取微管，包括其正端。季博士是计算机视觉领域的专家。他的研究生 Ming Jing 开发了一种多步骤分割方法，该方法使用已知的微管圆柱几何形状以及在微管正端发现的可行曲率范围作为分割过程的约束。在过去的一年里，我们对该方法进行了调试和完善。最终编写了用户友好的 GUI，使用户能够将整个套件程序模块作为一个单元运行，或者以任何所需的组合运行单个模块。例如，用户可以选择对体积中的微管进行分段，并考虑其正端。尽管这些工具是为了我们的特定目的而开发的，但我们认为这些方法是通用的，并且对于任何希望通过断层扫描重建分割细胞骨架纤维（例如微管、肌动蛋白甚至中间丝）的人都将受益。 写了以下论文： ？ Jiang, M.、Ji, Q. 和 McEwen, B. (2005) 从电子断层扫描中自动提取动粒微管和正端的精细特征。 IEEE 计算机图形和应用（正在印刷中）。 McEwen博士做了题为“利用电子断层扫描研究原核和真核细胞中的丝状结构？”的邀请平台演讲。于 2005 年 6 月 6 日在佐治亚州亚特兰大举行的美国微生物学会年会上。在 7 月 29 日于夏威夷州檀香山举行的 2005 年显微镜和微量分析会议上做了两个演讲？ 2005 年 8 月 4 日：平台：？ McEwen, B.F.、Jiang, M.、Zhang, W.、Vandenbeldt, K. 和 Ji, Q. (2005) 基于模型的电子断层扫描重建自动分割方法。显微镜。微肛门。 11（补充 2）：328CD。 海报： ？ Dong, Y.、Meng, X.、Vandenbeldt, K.、Hergert, P. 和 McEwen, B.F. (2005) 高压冷冻和冷冻替代后诺考达唑处理的 PtK1 着丝粒的超微结构。显微镜。微肛门。 11（增补 2）：338CD。