Experimental and Computational Analysis of Merotelic Kinetochore Formation, Dynamics, and Correction

该奖项由2009年美国复苏和再投资法案（公法111-5）资助。动物细胞通过在每次有丝分裂时将染色体平均分配到两个子细胞中来保持正确的染色体数目。在有丝分裂进入后，每条染色体由两个姐妹染色单体组成，每个染色单体具有专门的蛋白质复合物，动粒（KT），其与有丝分裂纺锤体的微管（MT）相互作用。为了准确的染色体分离，两个姐妹KT必须与来自相反两极的MT相互作用。然而，可能发生错误的KT-MT相互作用。本项目的主要目的是研究部分KT取向，一种特定的KT错误附着，其中单个KT从两个纺锤体极而不是一个极结合MT。这种类型的错误连接代表了哺乳动物组织细胞中染色体错误分离的主要来源。然而，许多关于merotelic KT的形成，动力学和校正仍然是未知的。组织培养细胞将被用作模型系统，实验数据主要通过Cimini实验室的高分辨率显微镜和活细胞成像获得，将由Co-P.I. Gul Civelekoglu-Scholey博士和合作者Alex Mogilner博士，研究以下具体目标：1.有丝分裂纺锤体的几何形状对动粒错误附着的影响; 2.对减数分裂后期启动前的动粒行为和后期前的校正机制的分子和机制的理解; 3.染色体分离错误是由于染色体末端定向持续到后期。了解merotelic KT的形成，动力学和校正将阐明几个分子和机制方面的KT-MT附件，染色体动力学和分离。在这个项目中解决的具体生物学问题将通过整合实验方法与理论/计算建模方法进行研究。这种结合的方法提供了明显的优势：理论/计算建模允许定量测试的定性模型出现从实验数据和能力，使预测。然后，可以在进一步的实验中测试这些预测，以验证、改进或修改模型，然后重新开始这个过程。这创造了一个有效的反馈回路，其中实验结果为计算模型提供反馈，模型做出可以通过实验进行测试的预测，从而显着加速学习和发现过程。这里采用的实验-计算相结合的方法将产生各种有丝分裂过程的综合模型，并且对于更接近建立有丝分裂的系统级模型至关重要。拟议的项目将对教育和研究作出贡献。至少有三名研究生和几名本科生将参与这项研究项目。除了学习最先进的显微镜和细胞生物学方法，这些学生将有机会发展批判性，分析性，独立性和跨学科的思维能力。他们将学习如何从不同的角度分析生物学问题，并将学习如何整合不同研究领域的知识（即，实验和计算生物学）。强烈鼓励Cimini实验室的本科生积极参与正在进行的研究项目（一名当前学生是最近提交给高知名度期刊评审的两篇研究论文的合著者）。这种类型的培训可能会鼓励他们追求科学事业。这项工作的结果将定期在相关会议上发表，如美国细胞生物学学会年会、有丝分裂FASEB夏季研究会议、染色体动力学戈登会议以及染色体分离和非整倍性国际研讨会。此外，预计在该项目执行期间将在同行审查的期刊上发表若干出版物。最后，本研究期间获得的一些高分辨率图像和视频将提交给美国细胞生物学学会的图像和视频库。这个在线图像和视频库是一个开放的细胞图像和视频集合，它是教育工作者，研究人员和任何年龄和背景的学生的宝贵信息来源。

ABSTRACTThis award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Research. Animal cells maintain a correct chromosome number by equally partitioning their chromosomes into two daughter cells at each mitotic division. Upon mitotic entry, each chromosome is composed of two sister chromatids, each possessing a specialized protein complex, the kinetochore (KT), that interacts with microtubules (MTs) of the mitotic spindle. For accurate chromosome segregation, the two sister KTs must interact with MTs from opposite poles. However, erroneous KT-MT interactions can occur. The primary objective of this project is to study merotelic KT orientation, a specific KT mis-attachment in which a single KT binds MTs from both spindle poles rather than just one. This type of mis-attachment represents a major source of chromosome mis-segregation in mammalian tissue cells. However, much about merotelic KT formation, dynamics, and correction is still unknown. Tissue culture cells will be used as a model system and the experimental data, mostly obtained through high-resolution microscopy and live-cell imaging in the Cimini lab, will be complemented by quantitative and computational modeling, performed by the Co-P.I. Dr. Gul Civelekoglu-Scholey and collaborator Dr. Alex Mogilner, to investigate the following specific objectives: 1. Effect of mitotic spindle geometry on establishment of kinetochore mis-attachments; 2. Molecular and mechanistic understanding of merotelic kinetochore behavior before anaphase onset and the pre-anaphase correction mechanism; 3. Chromosome segregation errors due to the persistence of merotelic orientation into anaphase. Understanding merotelic KT formation, dynamics, and correction will elucidate several molecular and mechanistic aspects of KT-MT attachment, chromosome dynamics and segregation. The specific biological questions addressed in this project will be investigated by integrating the experimental approach with a theoretical / computational modeling approach. This combined approach offers obvious advantages: theoretical / computational modeling allows quantitative testing of qualitative models emerging from experimental data and the capacity to make predictions. Such predictions can then be tested in further experiments to validate, refine, or modify the model, and the process can start over. This creates an efficient feedback loop in which the experimental results feed the computational model, and the model makes predictions that can be tested experimentally, thus significantly accelerating the learning and discovery process. The combined experimental-computational approach undertaken here will generate comprehensive models of various mitotic processes and will be crucial to move one step closer to building a systems-level model of mitosis.Broader impacts. The proposed project will make contributions to both education and research. At least three graduate students and several undergraduate students will be involved in the research project. Besides learning state-of-the-art microscopy and cell biology methodologies, these students will have the opportunity to develop critical, analytical, independent, and interdisciplinary thinking skills. They will learn how to analyze biological problems from different perspectives and will learn how to integrate the knowledge arising from different fields of study (i.e., experimental and computational biology). Undergraduate studentsin the Cimini lab are strongly encouraged to be active participants in ongoing research projects (one current student is co-author on two research papers recently submitted for review in high profile journals). This type of training will likely encourage them to pursue a career in science. The findings of this work will be regularly presented at relevant meetings, such as the American Society for Cell Biology annual meeting, the Mitosis FASEB summer research meeting, the Chromosome Dynamics Gordon Conference, and the Chromosome Segregation and Aneuploidy International Workshop. In addition, several publications on peer-reviewed journals are expected to be produced during the course of this project. Finally, some of the high resolution images and videos obtained during this study will be submitted to the Image and Video Library of the American Society for Cell Biology. This on-line image and video library is an open access collection of images and videos of cells, and it is a valuable source of information for educators, researchers, and students of any age and background.