Analysing the switches that govern mitotic control in mammalian cells

Suarez Peredo Rodriguez, Maria Fernanda (2020) Analysing the switches that govern mitotic control in mammalian cells. Doctoral thesis (PhD), University of Sussex.

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Abstract

Entry into mitosis is mediated by the switch-like activation of CDK/Cyclin B. This needs to be tightly regulated to ensure that mitosis occurs at the right time in the cell cycle after completion of DNA replication. Mathematical models of this feedback system predict that mitotic entry is bistable, meaning that cells can only exist in either an Interphase or mitotic state, but not arrest in intermediate stages. A positive feedback in the Cdk1 activation loop that results in the removal of inhibitory phosphorylation sites on Cdk1, T14 and Y15, has been proposed to generate different thresholds for transitions between Interphase and M phase.

Recent work on mitotic control also suggests that the major Cdk1 counteracting phosphatase, PP2A-B55, needs to be inhibited as cells progress into mitosis. This requires Gwl kinase generating an additional feedback loop. In vitro experiments suggest that the regulation of PP2A-B55 By Gwl also constitutes a bistable switch. How Cdk1 activation and PP2A inactivation are interlinked in the G2/M switch and how either pathway contributes to bistability remains to be explored in vivo.

We have addressed these questions by combining quantitative cell biology assays and mathematical modelling that includes both kinase and phosphatase regulation using quantitative assays in human cell lines to estimate numerical parameters for the model. Our results show that the mitotic switch displays hysteresis and bistability and that phosphatase inhibition and kinase activation are each suficient to maintain it; only abrogation of both mechanisms eliminates a stable barrier between interphase and mitosis. Loss of bistability has as consequences for mitotic progression and cell proliferation, as they fail to stabilize the metaphase state and are also unable to initiate progression through mitosis toward cell division and G1. Our model predicted a third stable steady state that represents a prophase arrest in between mitosis and interphase. We verified experimentally this prediction of the model which supports the predictive value of it.

Further work explored the contribution of other phosphatases to the G2/M switch system and a synergistic effect between Gwl kinase and inhibition of Cdk4. Overall, this work presents a comprehensive quantitative model for the mammalian mitotic entry switch and sheds new light on its regulation.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Subjects: Q Science > QH Natural history > QH0301 Biology > QH0573 Cytology > QH0605 Cell division > QH0605.2 Mitosis
Q Science > QH Natural history > QH0301 Biology > QH0426 Genetics > QH0438.4 Special aspects of the subject as a whole, A-Z > QH0438.4.M3 Mathematical models
Depositing User: Library Cataloguing
Date Deposited: 02 Dec 2020 12:37
Last Modified: 03 Dec 2020 06:56
URI: http://sro.sussex.ac.uk/id/eprint/95219

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