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.
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