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Understanding the Dynamic Regulation of SOCS3 – Nikoletta Kalenderoglou – November 2019

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Understanding the dynamic regulation of SOCS3 Nikoletta Kalenderoglou This dissertation is submitted for the degree of Doctor of Philosophy November 2019 School of Biomedical and Life Sciences “ἓν οἶδα ὅτι οὐδὲν οἶδα” Σωκράτης “All I know is that I know nothing” Socrates ACKNOWLEDGEMENT First and foremost, I would like to acknowledge Dr Rachael Rigby and Dr Karen Wright for giving me the chance to carry out my PhD thesis in their group. I am also grateful that they gave me the freedom to develop my own ideas and for supporting me in their realization. Next, I would like to express my deepest gratitude to Dr Richard Mort, for the donation of tFT vector, for his excellent guidance, immense knowledge and patience. I would never be able to finish my dissertation without his help and support. I would also like to thank the external examiner Dr Pawel Paszek and the internal examiner Dr David Clancy for the valuable suggestions and comments for my thesis. The guidance and support of Dr David Clancy’s, especially in the statistical analysis I am immensely grateful for. My sincerest thanks goes to Dr John Worthington for taking my CRISPR engineered cell clones for cell sorting in Manchester University, Dr Leonie Unterholzner for helping me improve the quality of my CRISPR chapter as well as Dr Christopher Jewell and Dr James Hensman for analysing my data in Chapter 2. One more person to thank is Dr Elisabeth Shaw, not only for helping me improve my Confocal imaging, but also for her resourcefulness and answering my seemingly obvious science questions. I would also like to take this time to thank Biomedical and Life Sciences department for helping me to develop my background skills in Molecular Biology, Cell Biology and Imaging as well as all of the financial lab support, they were able to provide to me in order to make this dissertation possible. I would also like to thank my fellow lab members, past and present, for their help and words of advice, in and outside the lab. ii Special thanks go to Sarita, my long-lasting friend and made of honour, for help, support and always being there for a cigarette break during long lab incubation times. To my friends that I left at home, Sevi, Christina, Georgia, Eirini and Amalia for always having time for coffee and endless PhD ranting. Thank you for being there, in spite of the distance. Your encouragement and friendship led me to where I am today. Yeia Mas! Most of all, I am extremely grateful for my wife Jacqui. Thank you for your love and companionship, you are my rock. Your faith in my abilities gave me the confidence and determination to get me through the dark times. Your car rides to University and back, last minute cell movie analysis advice, bringing me coffee and my favourite snacks, I will be forever in your debt. For last I reserve my deepest expression of gratitude to my parents and my sister. Especially to my mum and dad, who always encouraged me to pursue my dreams and never doubted my abilities. It is for my family that I have completed this thesis. Declaration This thesis has not been submitted in support of an application for another degree at this or any other university. It is the result of my own work and includes nothing that is the outcome of work done in collaboration except where specifically indicated. Many of the ideas in this thesis were the product of discussion with Dr Richard Mort (chapter 4) and my supervisors Dr Karen Wright and Dr Rachael Rigby (chapters 1-3). iv Understanding the dynamic regulation of SOCS3 – Nikoletta Kalenderoglou – November 2019 Abstract In the past two decades, it has become evident that signal transduction pathways are more than two dimensional pathways consisted of proteins that are just activated or supressed in response to distinct cues. Instead, the dynamic nature of key proteins regulates the strength and quality of the signal. Several key signal transduction pathways are controlled by negative feedback loops that are highly dynamic and demonstrate oscillatory behaviours. Negative feedback regulation of the JAK/STAT pathway by Suppressors of Cytokine Signalling (SOCS) is an example of oscillatory signalling. We sought to investigate the oscillatory capacity of the tumour suppressor protein SOCS3 and its role in important cellular functions using whole-cell population and single-cell analysis. An important aspect of cell biology using experimental cell-population techniques is to produce a synchronized cell culture. Serum starvation and subsequent shock is able to capture the oscillatory behaviour of SOCS3 protein to some extent. However, the average response in whole-cell population systems demonstrated to be ‘noisy’ leading to establishment of a single-cell analysis system. To investigate SOCS3 oscillation at the single cell level, we first attempted to generate cell clones stably expressing SOCS3 C-terminal GFPSpark fusion protein from its respective endogenous promoter to monitor its expression in real time with confocal microscopy. Despite careful optimization of each step of CRISPR/Cas9 strategy, the generation of GFPSpark knockin cell line was not successful. Finally, we utilised the tandem fluorescent protein timer (tFT) strategy to investigate localisation and trafficking of SOCS3 protein and monitor its promoter activity in response to different stimuli. The use of tFT provided us the ability to analyse SOCS3 dynamics across spatial and temporal dimensions under either normal culture conditions or different treatments that are known to influence on SOCS3 half-life and degradation rates. vi Contents 1 LITERATURE REVIEW .................................................................................... 17 1.1 Inflammatory Bowel Disease (IBD) ................................................................ 17 1.2 Colorectal Cancer (CRC) ................................................................................ 22 1.3 IBD-associated colorectal cancer (IBD-CRC) ................................................. 24 1.4 Cytokine Signalling in Intestinal Epithelial Cells (IECs) ................................. 27 1.5 Suppressor of cytokine signalling (SOCS) ....................................................... 31 1.6 SOCS3 in IBD and IBD-CRC ......................................................................... 35 1.7 AIMS .............................................................................................................. 38 2 WHOLE-CULTURE SYNCHRONIZATION METHOD: CAVEAT EMPTOR 40 2.1 Introduction .................................................................................................... 40 2.1.1 Dynamics of JAK-STAT signaling pathway ............................................. 45 2.1.2 Serum shock as method to synchronize ultradian oscillations through the circadian clock .................................................................................................. 48 2.1.3 Aims ........................................................................................................ 52 2.2 Material and Methods ..................................................................................... 54 2.2.1 Cell Culture .............................................................................................. 54 2.2.2 Cell-line maintenance and subculturing protocol ...................................... 55 2.2.3 Alkaline Phosphatase (alkP) cell staining ................................................. 56 2.2.4 Cell differentiation protocol...................................................................... 57 2.2.5 Cell cycle synchronization procedure ....................................................... 57 2.2.6 Western Immunoblot ................................................................................ 58 2.2.7 Mathematical Modeling ............................................................................ 61 2.3 Results ............................................................................................................ 63 2.3.1 Effects of serum deprivation on SOCS3 dynamic behaviour ..................... 63 2.3.2 Dynamic behavior of SOCS3 protein in Differentiated Caco2 .................. 70 2.3.3 Serum shock as method to achieve whole population synchrony ............... 78 2.4 Discussion....................................................................................................... 99 2.4.1 Experimental design of horse serum shock ............................................. 104 2.4.2 Limitations of Western Blotting methodology ........................................ 106 2.4.3 Complexity of JAK-STAT pathway ....................................................... 108 2.4.4 Whole cell population versus single cell analysis .................................... 110 2.5 Conclusion and Future Steps ......................................................................... 113 3 CRISPR/CAS9-MEDIATED GENERATION OF KNOCKIN CELL LINE EXPRESSING ENDOGENOUS FLUORESCENTLY TAGGED SOCS3 PROTEIN ............................................................................................................. 115 3.1 Introduction .................................................................................................. 115 3.1.1 Single cell analysis ................................................................................. 115 3.1.2 CRISPR-Cas9 system ............................................................................. 118 3.1.3 Genomic Engineering by Homologous Recombination and CRISPR/Cas9- Induced DSBs ................................................................................................
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