Kinetochore oscillations in mitotic metaphase

在有丝分裂过程中，姐妹染色单体通过着丝点（染色体上的蛋白质复合体）连接到从相反纺锤极延伸出来的微管。当一对姐妹着丝点形成这种结构时，它们围绕纺锤的赤道面振荡，这些振荡的幅度取决于运动蛋白-8马达。在振荡过程中，姊妹着丝点由于张力而分离，这被认为是调节纺锤装配检查点的机制。本项目的目的是研究着丝点振荡的机制。我们将使用用GFP标记的表达着丝粒2的分裂酵母细胞的活细胞成像来量化一对姐妹着丝粒的运动。通过比较野生型细胞和缺乏kinesin-8的细胞中着丝点的运动，以及对微管上gfp标记的kinesin-8的动力学进行量化，将研究kinesin-8马达在振荡中的作用。为了研究作用在动片上的力，我们将用激光烧蚀法切割主轴。我们将介绍一个理论模型，其中着丝点的运动是由微管聚合和解聚力驱动的，而激酶-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，野生型着丝点在纺锤中心附近发现，而在没有激酶-8的情况下，它们经常在纺锤杆附近暂停，支持了模型的初步假设。此外，该模型将确定振荡所需的条件并给出预测，这些预测将进行实验测试。例如，该模型将预测着丝点间距离的动态，这是张力的读出。总之，了解着丝点振荡的机制将阐明细胞如何调节姐妹着丝点之间的张力。

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.