Kinetochore oscillations in mitotic metaphase

在有丝分裂过程中，姐妹染色单体通过着丝粒（染色体上的蛋白质复合物）连接到从纺锤体两极延伸出来的微管上。当一对姐妹动粒建立这种配置时，它们围绕纺锤体的赤道平面振荡，并且这些振荡的幅度取决于驱动蛋白-8马达。在振荡期间，由于张力，姐妹动粒被分离，这已经被提出来调节纺锤体组装检查点。本项目的目的是研究动粒振荡的机制。我们将使用活细胞成像的裂变酵母细胞表达着丝粒2标记的GFP，以量化一对姐妹动粒的运动。驱动蛋白-8马达在振荡中的作用将通过比较野生型细胞和缺乏驱动蛋白-8的细胞中的动粒运动，以及通过定量GFP标记的驱动蛋白-8在微管上的动力学来研究。为了研究作用在动粒上的力，我们将通过使用激光烧蚀来切割纺锤体。我们将介绍一个理论模型，其中动粒运动是由微管聚合和解聚力驱动的，而驱动蛋白-8调节从聚合到解聚的转换。我们的初步实验和理论结果表明，在野生型动粒被发现附近的主轴中心，而在驱动蛋白-8的情况下，他们经常暂停附近的主轴极点，支持模型的初步假设。此外，该模型将确定振荡所需的条件并给出预测，这些预测将通过实验进行测试。例如，该模型将预测动粒间距离的动态，这是张力的读数。总之，了解动粒振荡的机制将阐明细胞如何调节姐妹动粒之间的张力。

During mitosis, sister chromatids connect to microtubules extending from the opposite spindle poles via kinetochores, protein complexes on the chromosome. When a pair of sister kinetochores establishes this configuration, they oscillate around the equatorial plane of the spindle, and the amplitude of these oscillations depends on kinesin-8 motors. During the oscillations sister kinetochores are separated due to tension, which has been proposed to regulate the spindle assembly checkpoint. The aim of this project is to study the mechanism of kinetochore oscillations. We will use live-cell imaging of fission yeast cells expressing centromere 2 labeled with GFP in order to quantify the movements of one pair of sister kinetochores. The role of kinesin-8 motors in the oscillations will be studied by comparing the kinetochore movements in wild-type cells and in those lacking kinesin-8, as well as by quantification of the dynamics of GFP-tagged kinesin-8 on the microtubules. To study the forces acting on the kinetochores, we will cut the spindle by using laser ablation. We will introduce a theoretical model in which kinetochore movement is driven by microtubule polymerization and depolymerization forces, whereas kinesin-8 regulates the switch from polymerization to depolymerization. Our preliminary experimental and theoretical results show that in wild type kinetochores are found near the spindle center, whereas in the absence of kinesin-8 they frequently pause near the spindle poles, supporting the preliminary assumptions of the model. In addition, the model will identify the conditions required for the oscillations and give predictions, which will be tested experimentally. For example, the model will predict the dynamics of the inter-kinetochore distance, which is a read-out of tension. In conclusion, understanding the mechanism of kinetochore oscillations will elucidate how the cell regulates the tension between sister kinetochores.