Author: Bridget Mooney
Mooney, Bridget, 2025 Environment-dependent regulation of mitosis and cytokinesis by AMP-Activated Protein Kinase, Flinders University, College of Medicine and Public Health
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The cell cycle is a strictly coordinated process, which sees cells undergo phases of growth and division when environmental conditions are favourable. Upon environmental stress, growth and division is ceased, to prevent the production of damaged daughter cells. Remarkably, cancer cells are able to adapt to their nutrient-poor environment and continue to proliferate under environmental stress, aided by proteins that promote tumorigenesis, such as AMP-Activated Protein Kinase (AMPK). The tumour microenvironment is known to have significantly elevated potassium levels, subsequently forcing cancer cells to proliferate under salt stress. Although AMPK is implicated in promoting cancer cell survival under environmental stress, the mechanisms as to how are poorly understood.
This study utilised microscopy, genetic and biochemical approaches to decipher how AMPK regulates cell division in response to environmental stress and in particular, salt stress. Fission yeast, along with mammalian cell lines were used to gain insight into both in vivo and in vitro regulation of mitosis and cytokinesis.
In response to salt stress, this work established that AMPK regulates two independent checkpoints to promote division. The first, sees a delay to the metaphase to anaphase transition by the well-characterised spindle assembly checkpoint (SAC), where loss of AMPK resulted in a significant delay to SAC silencing, thus delaying anaphase. Opposingly, mutants with elevated AMPK activity were accelerated in silencing the SAC following salt stress, promoting anaphase and advancing mitotic exit. This was demonstrated in both yeast and human cells, displaying evolutionary conservation of AMPK-dependent regulation of the SAC. The latter post-stress checkpoint elicits a delay to actomyosin ring constriction, which provides the mechanical force necessary to divide daughter cells in two. Ring constriction is largely driven by phosphorylation of the myosin regulatory light chain (Rlc1), which increases the motility of myosin motor proteins. Yeast cells without AMPK (Ssp2) lack the ability to constrict the actomyosin ring following salt stress, and biochemical analysis revealed that ssp2∆ cells fail to maintain Rlc1 phosphorylation in this condition. Overexpression of the known Rlc1 kinase Pak2 rescued the inability for ssp2∆ cells to divide following salt stress, and genetic manipulation of Rlc1 revealed that Ssp2 is essential to regulate Rlc1 Ser35 phosphorylation (through Pak2), to promote post-stress cytokinesis.
While AMPK is primarily activated by T-loop phosphorylation in response to salt stress, this study established that PP1(Dis2)-mediated dephosphorylation of the inhibitory phosphorylation on Ssp2 Ser367 contributes to stress-induced AMPK activation. This subsequently drives AMPK-dependent SAC silencing and cytokinesis.
To exploit AMPK signalling in cancer, colorectal cancer (CRC) cells were utilised, as recent work demonstrated that high expression of AMPK was associated with poor survival in CRC patients. Here, mutants with elevated AMPK activity efficiently silenced the stress-induced SAC and were also able to evade mitotic arrest induced by spindle poison, thus supporting the evidence that AMPK promotes cancer cell proliferation.
Together, this study contributes to understanding how AMPK aids cell survival under stress. Targeting the AMPK-dependent regulation of mitosis and cytokinesis established in this work has potential to improve therapeutic outcomes for those living with cancer.
Keywords: AMPK, mitosis, cytokinesis, spindle checkpoint, S. pombe, HCT116, contractile actomyosin ring, Ssp2
Subject: Medical Science thesis
Thesis type: Doctor of Philosophy
Completed: 2025
School: College of Medicine and Public Health
Supervisor: Professor Janni Petersen