Micromechanical studies on chromosomes reveal interplay between chromatin, SMC complexes, protein networks and mitotic, meiotic, and centromeric stiffness

Biggs, Ronald Joseph

doi:https://doi.org/10.21985/n2-pe9x-ha21

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Micromechanical studies on chromosomes reveal interplay between chromatin, SMC complexes, protein networks and mitotic, meiotic, and centromeric stiffness

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The purpose of my thesis research has been to understand the formation of mitotic chromosome structure by using chromosome micromanipulation. Folding mitotic chromosomes from their loose interphase form into their individualized, compacted form is required for easy handling of the chromosomes for cell division. This process is facilitated by several protein complexes, such as SMC complexes, topoisomerases, and barrier elements. It also coincides with structural changes to the underlying chromatin and activation of protein complexes that facilitate the chromosome division. My research has also delved into studying other forms of chromatin folding and division via micromanipulation. The four projects I have undertaken during my thesis research all use micromanipulation of chromosomes extracted from live cells using micropipettes to stretch the isolated chromosome. This has included the development of a new strategy of isolating the chromosomes from the cell. The first project is the study of protein depletion of condensin I, condensin II, both isoforms, cohesin, and Ki-67 using auxin-induced degradation to study the morphology and mechanics of mitotic chromosome bundles and isolated chromosomes. The next project observed how altering histone post-translational modifications and other controllers of chromatin state affect the stiffness of mitotic chromosomes to study the relationship between the underlying chromatin and the stiffness of the whole mitotic chromosome. The other project on mitotic chromosomes involved the study of the centromere’s stretch in the chromosome in relation to other parts of the kinetochore. The other project studied meiotic chromosomes and the stiffness and structural relationship with the synaptonemal complex. Our results show that there are several morphological effects from depleting protein complexes, but their rapid degradation does not weaken mitotic chromosomes. By contrast, the underlying chromatin was shown to affect mitotic chromosomes through control of heterochromatin, where increasing heterochromatin by histone post-translational modifications stiffened mitotic chromosomes but removing heterochromatin proteins weakened mitotic chromosomes. We demonstrated that the centromere was very stiff compared to the chromosome arms but was not affected by depleting members of the inner kinetochore. Finally, we demonstrated that meiotic I prophase chromosomes were structurally like mitotic chromosomes, but much stiffer. The increase in meiotic chromosome stiffness was not dependent on the presence of the central synaptonemal complex 1 protein. I conclude this thesis by presenting a synthesis of a theme for general findings of chromosomes and future projects, both related and unrelated to the projects described in this document.

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