Letton, William; (2019) Studying time and disease using two different cell line models. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The scientific approach of modelling uses manipulation of simpler systems in order to understand aspects of more complex ones. This method is extensively used to understand the biology of complex organisms through studying representative cells artificially cultured in vitro. The work presented here uses in vitro cell line model systems to investigate two aspects of cell biology: the coupling between the circadian clock and cell cycle, and the role BEST1 protein localisation in Autosomal Dominant Vitreoretinochoroidopathy (ADVIRC) disease. 1. The circadian clock is an internal time-keeping mechanism that allows organisms to anticipate daily environmental changes. This mechanism operates at an organismal level as well as being intrinsic to each cell through oscillating genetic negative feedback loops. One of the many cellular functions that has been found to be regulated by the circadian clock is cell cycle progression. However, it is still an outstanding question as to whether the circadian clock drives cell cycle progression actively or whether it passively gates particular cell cycle transitions to certain times of day. In the first experimental chapter zebrafish (Danio rerio) cell lines are used to investigate the relationship between the circadian clock mechanism and cell cycle behaviour. This relationship is examined at a population and single cell level to address the question as to the effect of varying the circadian clock period on the timing and average length of the cell cycle output. It is demonstrated that altering the period length of the circadian oscillation affects the timing of cell cycle progression, but not the length of the cell cycle or its phases. This provides strong evidence in support of the gating theory of circadian regulation of the cell cycle. In the second experimental chapter the effects of cell density on this coupling between the circadian clock and cell cycle are considered. It is shown that this coupling is not observed in cell populations that are at low density and that are highly proliferative, providing insight into how the assay conditions used in previously published work on this coupling could be contributing to conflicting results. 2. Bestrophinopathies are a group of retinal dystrophic diseases that share a common cause: mutations in the Bestrophin-1 (BEST1) gene, expressed in the retinal pigmented epithelium (RPE). Epithelial cell models, particularly derived from the kidney, have been used as models to study the function and dysfunction of the BEST1 protein in RPE cells through overexpression of the BEST1 gene. The results of these studies have suggested that in ADVIRC disease mutant BEST1 shows mis-splicing defects. However, this has not been supported by work using induced 5 pluripotent stem cell-derived RPE (iPS-RPE) cell models, raising the question as to whether the model cells used are providing accurate insights into the actual properties of native RPE. In this section human epithelial cell lines are used to investigate the relationship between specific point mutations in the gene Bestrophin1 and the localisation within the cell of the resulting protein product. It is shown that the localisation of an ADVIRC mutant BEST1 protein depends partially on the cell line used to express it, as well as the cell culture conditions used. This suggests that the use of non-RPE model cell lines for investigating Bestrophin-1 may be leading to conclusions that do not apply in vivo.

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