Investigating the effect of emetic and aversive compounds on Dictyostelium identifies a novel non-sentient model for bitter tastant research Steven Jann Robery Research thesis submitted for the degree of Doctor of Philosophy at Royal Holloway University of London in July 2013 Declaration of Authorship I, Steven Robery, hereby declare that this thesis and the work presented in it is entirely my own. Where I have consulted the work of others, this is always clearly stated. Signed: Date: 2 Acknowledgements First and foremost, I would like to thank my supervisors, Professor Robin Williams and Professor Paul Andrews. I would like to thank Robin for providing me with the support and direction he has given me over the past few years, as well as teaching me to critique my own work to make me a better scientist. I would like to thank Paul for providing me with support, advice and passion throughout my PhD. Your meetings were always inspirational. I would also like to thank my laboratory colleagues for all of the help, support and banter whilst I have been at Royal Holloway. Secondly, I would like to thank my funding body, Universities Federation for Animal Welfare. Without you I would not have been given a PhD and been able to undertake this exciting project, nor would I have been able to contribute to the world of science in the way that I have. I would also like to thank my friends and family for all of the support you have given me over the years. In particular I would like to thank my girlfriend Annabelle, for putting up with me and for providing me with laughs and generally being there for me when I needed her to be. I would also like to thank my parents, Peter and Elisabeth, as well as my sister, Nina for providing me with all the support and encouragement I needed. I would also like to thank my all my friends, providing me with the banter and encouraging me to “get a move on” and complete my PhD. Finally, I can’t forget my dog, Plato, who let me know time and again whilst I was writing that it is time to clear my mind and go for a much needed walk. 3 Abstract Nausea and vomiting are common but serious side effects associated with many therapeutic drugs. Whilst the physiological mechanisms behind the generation of the vomiting response are well characterised, the range of emetic stimuli that can generate the response are poorly understood. The potential of using Dictyostelium discoideum, a eukaryotic amoeba, as a model for predicting emetic liability was examined in this thesis. The effects of a range of known emetic and aversive compounds on Dictyostelium cell behaviour was investigated, resulting in the identification of a small number that strongly inhibited cell migration in a concentration-dependent and reversible manner. These active compounds included a range of bitter compounds and the pungent tastant, capsaicin. A Dictyostelium mutagenesis screen was then used to identify genes controlling sensitivity to bitter tastants. This screen identified a mutant containing a disrupted grlJ gene as showing partial resistance to phenylthiourea in growth and behavioural changes in movement. GrlJ is a G- protein coupled receptor that regulates a phenylthiourea-dependent effect by inhibition of a phosphatidylinositol (PIP3) signalling pathway. A search for proteins sharing homology to GrlJ identified an uncharacterised GABAB-like receptor, Q8NHA5, involved in the detection of phenylthiourea in Dictyostelium. This thesis has therefore identified Dictyostelium as a potentially useful model for the identification of bitter and pungent tastants. In addition, this thesis has identified the Dictyostelium protein, GrlJ, as well as an uncharacterised human protein, Q8NHA5, involved in the detection of the bitter tastant, phenylthiourea. 4 Table of Contents Acknowledgements.......................................................................................... 3 Abstract ............................................................................................................... 4 List of Figures and Tables ................................................................................. 10 Abbreviations .................................................................................................... 14 Chapter 1 .......................................................................................................... 17 1.1. Nausea and Vomiting........................................................................... 18 1.2. Physiology of Nausea and Vomiting .................................................... 19 1.2.1. Pre-ejection phase ........................................................................ 19 1.2.2. Ejection phase ............................................................................... 20 1.2.3. Post-ejection phase ....................................................................... 20 1.3. Pharmacology of Nausea and Vomiting ............................................... 20 1.4. Emetic Compounds ............................................................................. 23 1.4.1. Ligand gated ion channels ........................................................... 24 1.4.2. Ion Channels ................................................................................. 27 1.4.3. G Protein-Coupled Receptors ....................................................... 28 1.4.4. Transporters and Enzymes ........................................................... 34 1.5. Animal Models in Emetic Research ..................................................... 35 1.6. Dictyostelium discoideum .................................................................... 38 1.7. Dictyostelium as a Biomedical Model .................................................. 40 1.7.1. Channels and Receptors in Dictyostelium ........................................... 42 1.7.2. GABAB-like Receptors ................................................................... 43 1.7.3. cAMP Receptors ........................................................................... 43 1.8. Dictyostelium movement ...................................................................... 44 1.8.1. Detection of cAMP ......................................................................... 44 1.8.2. F-actin polymerisation ................................................................... 46 1.8.3. The Phosphatydylinositol-3-kinase Pathway ................................. 48 1.8.4. Target of Rapamycin complex 2 .................................................... 52 1.8.5. The Phospholipase A2 Pathway .................................................... 53 1.8.6. Alternative Amplification Pathways................................................ 54 1.9. Aims of this work .................................................................................. 56 Chapter 2 .......................................................................................................... 57 2.1. Materials ................................................................................................. 58 2.1.1. General reagents ............................................................................. 58 5 2.1.1.1. Reagents purchased from Sigma-Aldrich Co. Ltd (Dorset, England) 58 2.1.1.2. Reagents purchased from other suppliers ........................................ 58 2.1.1.3. Antibiotics ......................................................................................... 59 2.1.1.4. Molecular weight standards .............................................................. 59 2.1.1.5. Restriction enzymes ......................................................................... 59 2.1.1.6. Other enzymes ................................................................................. 60 2.1.1.7. Antibodies ......................................................................................... 60 2.1.1.8. Kits .................................................................................................... 60 2.1.1.9. Escherichia coli (E.coli) strains ......................................................... 61 2.1.1.10. Primers ........................................................................................... 61 2.1.1.11. Equipment....................................................................................... 61 2.2. Methods .................................................................................................. 62 2.2.1. Dictyostelium methods ................................................................... 62 2.2.1.1. Cell culture ........................................................................................ 62 2.2.1.2. Growth curve assays ........................................................................ 62 2.2.1.3. Colony formation assays .................................................................. 63 2.2.1.4. Development assays ........................................................................ 63 2.2.1.5. Spore shape ..................................................................................... 63 2.2.1.6. Motility assays .................................................................................. 64 2.2.1.7. Random cell movement assays ........................................................ 64 2.2.1.8. Cell viability assays ........................................................................... 65 2.2.1.9. PHCRACGFP global stimulation experiments...................................... 65 2.2.1.10. PIP3 mass ELISA ...........................................................................