Unravelling the chromosome
Unravelling the chromosome
prof.dr.ir. G.J.L. Wuite, prof.dr.ir. E.J.G. Peterman
Faculty of Science
The genome of the human body is subdivided into 46 chromosomes, the smallest of which contains a total DNA length of 16 mm. During cell division, the chromosome adopts a compacted X-shaped structure with a length of only a few micrometers. As such, the DNA gets compacted around four orders of magnitude in length. How this extremely robust structure is achieved has been a topic of debate for a few decades. Several proteins have been shown to have key functions in the formation and maintenance of this structure (see Chapter 1). However, due to the relatively small and dense structure of the chromosome and the limited control over the conditions that experimentalists have when dealing with living cells, it is very challenging to study the chromosomal architecture. Therefore, we set out to develop a novel methodology with the aim to study the complex structure of mitotic chromosomes in an environment where we have full control over experimental conditions. To this end, we decided to use the technique of optical tweezers, where highly focused laser beams can be used to grab micron-sized objects and apply forces to them. In our lab, this technique is frequently employed to study DNA molecules, by tethering them in between two micron-sized spheres that can be trapped with the optical tweezers. In this thesis a method is presented that allows for the optical manipulation of mitotic chromosomes. We have developed human cell lines that incorporated a specific linker (biotin) at the end of the chromosome arms (telomeres). After isolating the chromosomes from mitotic cells, we could attach the telomeres via strong biotin-streptavidin interactions to our streptavidin-coated microspheres. This enabled us to perform experiments where we probed the mechanical response of the chromosome to applied extensions. From these experiments, we learned that the chromosome is relatively soft for small extensions, but shows a dramatic increase in the force at higher extensions. The observed behavior is not consistent with any classical model that describes polymer (network) mechanics. Therefore, we proposed a new model to describe the chromosome's mechanical behavior. This model describes the chromosome as consisting of many elements or modes that successively stiffen. Moreover, we were able to investigate the role of a specific protein, TopoisomeraseIIα (TOP2A), in the chromosome structure. We found that upon depletion of TOP2A the chromosome shows a softer stiffening behavior. Interestingly, when we perturbed the structure of the chromosome by swelling it with high salt concentrations and then let it come back to its original shape, the control chromosomes did not show significant changes in their stiffness, but the TOP2A degraded chromosomes did. This indicated that TOP2A plays a structural role in chromosome architecture. Together, these results highlighted the capability of our novel method to determine mechanical properties of chromosomes under highly controlled conditions.
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