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How do healthy cells age - a worm’s tale of exploration into the decline of DNA replication and the structure of the nuclear membrane
Secondary Supervisor(s): Dr Zita Balklava
University of Registration: Aston University
BBSRC Research Themes: Integrated Understanding of Health (Ageing)
Project Outline
Staged within the nucleus of the cell, DNA replication is the fundamental process for cell division and occurs within the majority of the trillions of cells we have in the body. As healthy cells age, DNA replication fidelity and efficiency decreases, while the integrity of the nuclear membrane also deteriorates [1-3]. Such changes have never been studied together however and so the aim of this project is to characterise both events in the ageing process, to determine which occurs first - defects in DNA replication or breakdown of the nuclear membrane? The knowledge gained from this project will help us to better understand the ageing process and the connections that exist between DNA replication machinery and the nuclear membrane, which are important in many ageing-related diseases such as cancer and Progeria.
In order to ensure that all of the DNA is fully duplicated in a precise manner, DNA replication is performed through a number of highly regulated steps [4]. In higher eukaryotic cells, onset of DNA replication requires formation of an intact nuclear membrane, which surrounds the chromatin and concentrates replication factors within the nucleus. Components of the nuclear membrane have also been found to directly interact with DNA replication machinery however, and to affect many aspects of the process.
In order to study and characterise mechanisms of ageing within the lab, we need a model system, which ages in a relatively short time and which mimics humans as much as possible. This project will thus make use of the C. elegans (nematode worm) model organism, which ages within two-three weeks and which comprises complex organ systems.
This project will combine my expertise in working on DNA replication (>10 years) with that of Dr Zita Balklava, who has >10 years’ worth of experience in working with C. elegans. The first aim of the project is to monitor the health of DNA replication and the structure of the nuclear membrane over the life span of the worms. For this, we will employ western blotting and immunofluorescence microscopy techniques with antibodies against known essential components, such as the MCM2-7 helicase complex (DNA replication) and Lamin 1 (nuclear membrane).
Secondly, we will make use of a commercially available C. elegans strain, which expresses GFP-tagged Lamin 1 (LMN-1) and determine the localisation and strength of signal for this protein within cells as the worms age, as well as interactors of this protein throughout the life span using mass spectrometry.
Finally, with support from the AIME cohort, we will extract nuclei from cells at different stages of the life span and perform biochemical and biophysical assays to determine the size, shape and composition of the nucleus and nuclear membrane. These results will help us to characterise how and when fundamental aspects of cell biology decline with age, thus deepening our understanding of the ageing process.
References
1. Pathak, R.U., M. Soujanya, and R.K. Mishra, Deterioration of nuclear morphology and architecture: A hallmark of senescence and aging. Ageing Research Reviews, 2021. 67: p. 101264.
2. D'Angelo, M.A., et al., Age-Dependent Deterioration of Nuclear Pore Complexes Causes a Loss of Nuclear Integrity in Postmitotic Cells. Cell, 2009. 136(2): p. 284-295.
3. Flach, J., et al., Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells. Nature, 2014. 512(7513): p. 198-202.
4. Jones, R.M. and E. Petermann, Replication fork dynamics and the DNA damage response. Biochem J, 2012. 443(1): p. 13-26.