Analysing the mechanisms that govern centrosome separation

Echegaray Iturra, Fabio Rodrigo (2021) Analysing the mechanisms that govern centrosome separation. Doctoral thesis (PhD), University of Sussex.

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Abstract

Centrosomes form the poles of the mitotic spindle in animal cells. Their separation in late G2/early prophase requires precise spatial coordination. Failure in this process can cause an increase in sister chromatid attachment and segregation errors that may contribute to aneuploidy and tumorigenesis. As cells enter mitosis, centrosome positioning is determined by a balance of forces that support and antagonise centrosome separation. Although the major effector of centrosome separation is kinesin Eg5, the antagonising forces that regulate Eg5 are not as well characterised. In this Thesis, we analyse the balance of forces that counteract Eg5 in centrosome separation by the use of a chemical-genetical assay in mammalian cell lines. By manipulating candidate components we found that microtubule polymerisation against a stiff actin cortex acts as the major Eg5 antagonising positioning mechanism. Surprisingly, the system directs centrosomes towards the centre of the nucleus rather than the cell centre. Accordingly, we identified perinuclear actin structures that form in late G2/early prophase that directly interact with microtubules emanating from the centrosomes. These structures cause microtubule bending at the nuclear boundary further supporting the notion of perinuclear actin/microtubule interactions in prophase. Data fitting of microtubule images against an Euler-Bernoulli rod model allowed us to estimate this interaction force in the range of tens of pN. Interestingly, the microtubule (MT)-network branches at points of high curvature, suggesting a load regulation mechanism. Disrupting these structures by breaking the interactions of the LINC complex with perinuclear actin filaments abrogates the nuclear centring mechanism of the centrosomes and causes a dramatic increase in subsequent chromosome segregation errors. Overall, our results provide a systematic analysis of the major components that coordinate centrosome position and separation before nuclear envelope breakdown to ensure accurate chromosome segregation.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QH Natural history > QH0301 Biology > QH0573 Cytology > QH0597 Centrosome. Centriole
Q Science > QH Natural history > QH0301 Biology > QH0573 Cytology > QH0605 Cell division > QH0605.2 Mitosis
Depositing User: Library Cataloguing
Date Deposited: 13 Apr 2021 13:50
Last Modified: 13 Apr 2021 13:50
URI: http://sro.sussex.ac.uk/id/eprint/98435

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