In Vitro Reconstitution of Chromosome Separation Using Optical Tweezers

为了确保每个子细胞在有丝分裂过程中继承完整的基因组，DNA形成了一个高度浓缩的结构-中期染色体。虽然中期染色体对真核生物具有重要意义，但其结构和动力学尚不完全清楚。本项目的目标是利用光学镊子了解中期染色体的结构和机制，并最终在体外重建染色单体分离。这将使我们对中期染色体的结构有新的认识，使我们能够理解染色体机械特性的分子决定因素，并使我们第一次有机会用分子分辨率实时观察染色体分离。我们的策略是使用染色体的位点特异性生物素标记，以便通过强生物素-亲和素相互作用将聚合物连接物连接到分离的中期染色体的着丝点上。然后，这些连接体的正交功能化自由端可以连接到由两个光学陷阱捕获的微珠上。这将使我们能够以与有丝分裂纺锤体完全相同的几何形状来检查中期染色体的机制。在接下来的步骤中，我们将通过特异性去除结构蛋白来操纵它们的分子组成，特别是脚手架蛋白凝缩蛋白I、凝缩蛋白II和粘聚蛋白，以解开它们对染色体结构、动力学和机械反应的特定影响。最后，我们打算建立姐妹染色单体的体外分离。虽然已知拓扑异构酶II和分离酶在染色单体分离中起主要作用，但我们的目标是回答这个问题，这些蛋白质是否足以使姐妹染色单体完全分离。将光学陷阱与共聚焦显微镜相结合，将使我们能够使用单分子荧光显微镜实时跟踪分子分辨率的过程。该项目采用了一种全新的方法来理解中期染色体的结构和动力学，并有望取得突出的发现，这不仅将影响分子生物学，而且将为染色单体分离提供新的生物物理学视角。

To ensure that each daughter cell inherits a full genome during mitotic cell division, the DNA forms a highly condensed structure – the metaphase chromosome. Although the metaphase chromosome is of fundamental relevance for eukaryotic life, its structure and dynamics are not yet fully resolved.The goal of this project is to use optical tweezers to understand the structure and mechanics of metaphase chromosomes and finally recreate chromatid separation in vitro. This will allow new insights into the structure of metaphase chromosomes, enables us to understand the molecular determinants for the mechanical properties of chromosomes and gives us – for the first time – the opportunity to observe chromosome separation in real time with molecular resolution. Our strategy is to use site specific biotin labelling of chromosomes in order to attach polymer linkers to the kinetochores of isolated metaphase chromosomes via the strong biotin-avidin interaction. The orthogonally functionalized free end of these linkers can then be attached to microbeads which are caught by two optical traps. This will permit us to examine the mechanics of the metaphase chromosome in the exact same geometry as in the mitotic spindle. In a subsequent step we will manipulate their molecular composition by specifically removing structural proteins, in particular the scaffolding proteins condensin I, condensin II and cohesin, in order to disentangle their specific influence on the structure, the dynamics and the mechanical response of the chromosome. Finally we intend to establish the separation of sister chromatids in vitro. While it is known that the enzymes topoisomerase II and separase play a major role in chromatid separation, we aim to answer the question, whether these proteins alone are sufficient to allow full separation of the sister chromatids. Combining optical traps with confocal microscopy will allow us to follow the process in real time with molecular resolution using single molecule fluorescence microscopy.This project uses a completely new approach to the understanding of the structure and dynamics of metaphase chromosomes and promises outstanding findings, which will impact not only molecular biology, but also give a novel biophysical perspective on chromatid separation.