In Vitro Reconstitution of Chromosome Separation Using Optical Tweezers

• Applicant: Dr. Hannes Witt
• Subject Area: Biophysics
• Term: from 2020 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 438883799

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.

• DFG Programme: Research Fellowships
• International Connection: Netherlands
• Host: Dr. Gijs Wuite