A cellular understanding of shift work

Background

Having evolved on a rotating planet, organisms on Earth have developed an internal biological clock to allow them to anticipate the 24hr day-night cycle. These ‘circadian rhythms’ regulate almost all aspects of biology, from sleep to wound healing. Most importantly, circadian rhythms are cell-autonomous – every single cell in your body keeps time. They do this using a handful of proteins that interact to form a series of macromolecular complexes that drive daily rhythms in transcription of ~40% of the genome. For a circadian rhythm to be useful, it must align the timing of these transcriptional complexes with the external day-night cycle. This alignment becomes disrupted in shift work, where external timing cues such a light and food occur at unexpected times with regards to the cellular circadian ‘clock’. This mistiming of cues results in ‘circadian disruption’, which is associated with increased disease risk, most notably type-2 diabetes and cancer: the World Health Organisation defines shift work as a type-2 carcinogen. 1 in 5 of the UK working population engage in shift work. Despite this clear biomedical need to explore the mechanisms underlying the circadian disruption, it is not currently understood how mistimed circadian cues results in disease. Previous work from our group suggests that major aspects of circadian disruption can be recapitulated at the cellular level (Crosby et al. 2019). Furthermore, recent work from the scientific team of the Crosby and van Ooijen labs uncovered conserved mechanistic links between the cell and circadian cycles (Gil*, Crosby* et al. 2024). This project uses a combination of cell biology, protein biochemistry, and bioinformatics approaches build on these prior findings to uncover how shift work alters the proteins of the mammalian circadian clock under shift work conditions to drive increased cancer risk at the cellular level.

Aims

1. Optimise cellular model of shiftwork We will optimise an existing cellular microfluidic system to mimic shift work at the cellular level (Crosby et al. 2019). Optimising this system, which was pioneered and established in the lab, will enable higher throughput, resolution and fidelity than current approaches. This will syngerise with other projects in the lab, as well as providing an improved tool for this project.
2. Characterize changes in circadian protein complexes under circadian disruption Using cell lines expressing fluorescently-tagged circadian proteins in static culture and the microfluidic system, we will use Fluorescence Cross-Correlation Spectroscopy (FCCS) and bioinformatic image analysis of cells under shift work conditions to identify changes in the localization, kinetics and composition of the core circadian protein complexes. We will subsequently make structurally-informed mutations in these proteins to test the role of specific complex constituents in regulating circadian changes in response to shift work cues.
3. Identify changes in cellular function under circadian disruption The mechanistic links between circadian disruption and cancer remain unknown. Using the microfluidics developed in Aim 1, combined with fluorescence microscopy and molecular biology approaches, we will investigate oncogenic propensity in cells under circadian disruption using a number of markers, including the cell cycle reporters previously used in collaboration between the van Ooijen and Crosby labs (Gil*, Crosby* et al. 2024). We will subsequently test potential behavioural and pharmacological therapeutic interventions that have the potential to reduce the oncogenic risk of cells under shift work conditions.

Training outcomes

1. Gain experience in a wide range of techniques, including mammalian cell culture, genome editing, bioluminescence and fluorescence imaging, live-cell microscopy, pharmacology, image analysis and bioinformatics.
2. Gain skills in the generation of new methodologies, including high throughput microfluidics.
3. Gain competence in academic skills of data analysis and interpretation, experimental design and presentation of scientific findings to expert and non-expert audiences.

References

1. Crosby, P. et al. Insulin/IGF-1 Drives PERIOD Synthesis to Entrain Circadian Rhythms with Feeding Time. Cell, (2019)
2. Gil Rodríguez, S.*, Crosby, P.*, et al. Potassium rhythms couple the circadian clock to the cell cycle. bioRxiv (2024)