THREE-DIMENSIONAL STRUCTURE AND FUNCTION OF THE MAMMALIAN KINETOCHORE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。Nih r01 gm66270 ？哺乳动物着丝点微管动力学控制？， 08/01/02 ？07/31/06 Bruce F. McEwen, Wadsworth Center NSF MCB 0110821 ？哺乳动物着丝点的三维结构和功能？9月1日？08/31/05。哺乳动物着丝点是一种独特的席状结构，形成于有丝分裂和减数分裂染色体的初级收缩处（Rieder和Salmon， 1998， Manly等，1999）。它的功能是将染色体连接到纺锤体微管上。此外，着丝点在纺锤体赤道染色体的中期排列、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起着重要作用。尽管在鉴定着丝点的分子成分方面取得了进展，但着丝点的功能机制仍然很不清楚。这部分是由于缺乏有关着丝点三维超微结构的数据。唯一的电子层析研究是由PI项目在10多年前用化学固定标本进行的（McEwen et al, 1993）。1998年，McEwen和他的同事们通过高压冷冻和冷冻取代证明了在保存着丝点超微结构方面的显著改进，但在该研究中很少使用电子断层扫描。随着仪器和技术的进步，现在是开始对着丝点三维超微结构进行全面研究的好时机。首先，我们将使用高压冷冻、冷冻替代的细胞进行这项研究，以优化冷冻条件，并获得相对高的统计分析通量。然而，确定冷冻水合着丝点的结构是必要的，因为我们早期的研究表明了着丝点超微结构对样品制备条件的敏感性。本研究的目的是建立一个高质量、高分辨率的哺乳动物着丝点非结合状态的结构模型，并确定当关键的着丝点成分被去除时发生的结构变化。目前的工作主要是PTK细胞，但该模型将尽可能基于冷冻水合有丝分裂HeLa细胞的层析重建。然而，为了开发能够产生高密度冷冻的有丝分裂细胞的标本制备方案，我们最初一直在使用高压冷冻的标本进行冷冻替代。该策略允许相对快速和直接的方法来评估样品制备和优化方案，然后再转向更技术要求高的冷冻水合切片电子断层扫描任务。冷冻替代材料还将提供具有更高信噪比和更高质量高倾角数据的电子层析数据集。我们现在有了可重复的方案，可以在有丝分裂中获得高比例的HeLa细胞，使用的程序称为？有丝分裂脱落？使用15%牛血清白蛋白作为冷冻保护剂，获得了持续良好的冷冻效果。我们目前正在确认结果的可重复性，并准备用于冷冻水合超显微切片的细胞。PTK项目已经完成，海拉细胞的标本制备试验正在开始。一项使用phm和PBS缓冲液进行常规固定的短方法研究即将完成。发现固定在PHEM缓冲液中的PTK细胞与高压冷冻PTK对照细胞产生相似的微管+端构象。这与常规固定在PBS缓冲液中的细胞不同。另外用诺可达唑和紫杉醇处理的PTK细胞固定在PHEM缓冲液中，这些细胞正在与HPF诺可达唑和紫杉醇细胞进行比较。后期细胞也进行了检测。所有层析数据均已收集并重建。如果这些实验的结果证实PHEM产生的结果与HPF相似，那么只有PHEM制备的材料将被纳入PTK研究。这些数据也被写进了一篇简短的方法论文中，这对于使用传统固定的合作者使用的协议将是重要的。在显微镜和微量分析2005年会议上做了两个报告，檀香山，HI， 7月29日？2005年8月4日：？McEwen， B.F, Jiang， M, Zhang， W., Vandenbeldt， K., and Ji， Q.（2005）基于模型的电子层析重建自动分割方法。Microsc。微肛门。11（增刊2）：328CD。(平台)?董耀文，张晓峰，张晓峰，张晓峰，张晓峰（2005）高压冷冻和冷冻取代处理后PtK1着丝点的超微结构。Microsc。微肛门。11（增刊2）：338CD。（海报）与圣犹达儿童基金会的Katsumi Kitagawa博士的合作项目？s研究医院建成。Kitagawa博士感兴趣的是确定17-AAG治疗是否影响着丝点或着丝点结合。17-AAG处理和对照的Hela细胞均采用常规电镜技术制备。初步结果表明，即使有效果，效果也微乎其微。手稿正在准备中。和比尔·恩肖博士合作？今年10月，随着恩肖？该公司的技术人员保拉·瓦格纳雷利说。DT40细胞系统的优势在于，通过杀死内源性蛋白并将一个副本放置在触发系统的启动子下，可以产生条件突变。这个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电子显微镜检查它们。我们也有一些高压冷冻，冷冻替代的标本。2002年，我们开始与RPI的Qiang Ji博士的实验室合作，开发用于从哺乳动物动点的断层扫描重建中分割微管及其正端的自动化工具。这个项目的动机来自于我们根据着丝点微管的结构构象对正端进行分类的工作（上述称为PtK项目）。这些构象表明了微管的动态状态，并且了解着丝点如何控制微管动力学是细胞生物学中的一个关键问题。我们的一般方法是使用现代电子断层扫描的高通量能力来收集足够大的着丝点微管+端数据库，对有丝分裂阶段、药物治疗和关键着丝点分子成分的敲低如何改变构象进行统计上有意义的分析。我们还对确定单个着丝点上的正端构象的协调性有多好感兴趣。我们更倾向于使用计算机分类方法对构象进行分类，但重建的构象噪声太大，并且有太多的背景结构，无法对直接从原始层析重建中提取的着丝点微管进行分类。也有变化的问题，由于方向相对于丢失的金字塔。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，从大量的层析重建中提取微管，包括它们的正端。纪博士是计算机视觉方面的专家。他的研究生Ming Jing开发了一种多步骤分割方法，该方法使用已知的微管圆柱形几何形状，以及在微管+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，我们对这个方法进行了调试和改进。这最终导致编写用户友好的GUI，使用户能够将整个程序模块套件作为一个单元运行，或者以任何期望的组合运行单个模块。例如，用户可以选择在一个体积中分割微管，并关注它们的正端。虽然这些工具是为我们的特定目的而开发的，但我们认为这些方法是通用的，并且将有利于任何希望从层析重建中分割细胞骨架纤维（如微管，肌动蛋白甚至中间细丝）的人。文章如下：？蒋，M, Ji， Q，和McEwen， B.（2005）从电子断层扫描中自动提取着丝点微管和正端精细特征。IEEE计算机图形学与应用（即将出版）。麦克尤恩博士做了一个名为？利用电子断层扫描研究原核和真核细胞中的丝状结构？2005年6月6日，在亚特兰大举行的美国微生物学会年会上。在显微镜和微量分析2005年会议上做了两个报告，檀香山，HI， 7月29日？2005年8月4日：平台：？McEwen， B.F, Jiang， M, Zhang， W., Vandenbeldt， K., and Ji， Q.（2005）基于模型的电子层析重建自动分割方法。Microsc。微肛门。11（增刊2）：328CD。海报:?董耀文，张晓峰，张晓峰，张晓峰，张晓峰（2005）高压冷冻和冷冻取代处理后PtK1着丝点的超微结构。Microsc。微肛门。11（增刊2）：338CD。

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.