Investigating the Functional Consequence of Pik3c2b Ablation in a Skeletal Muscle Model by Kamran Rezai A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Kamran Rezai 2019 Investigating the Functional Consequence of Pik3c2b Ablation in a Skeletal Muscle Model Kamran Rezai Master of Science Molecular Genetics University of Toronto 2019 Abstract Phosphoinositide 3-kinases (PI3Ks) and its three distinct classes are involved in a myriad of cellular processes, including: cell growth, survival and intracellular trafficking. The Class II PI3Ks remain one of the least studied classes of lipid kinases. There are three isoforms of class II kinases, PIK3C2α, PIK3C2β and PIK3C2γ. Our laboratory identified PIK3C2β as a modifier of X-linked myotubular myopathy (XLMTM) caused by mutations in MTM1. However, PIK3C2β’s role in muscle and the consequences PIK3C2β ablation has not been elucidated. To answer this question. I generated a skeletal-muscle specific PIK3C2β KO mouse and found lower fasting glucose levels and increased AKT activation in muscle in an age-dependent manner. Next, I created and characterized PIK3C2β KO C2C12 myoblasts and discovered increased surface GLUT4 levels upon insulin stimulation. These new insights into the consequences of PIK3C2β ablation represent an opportunity to develop novel therapeutic strategies in XLMTM and metabolic disorders. ii Acknowledgements I would like to acknowledge my supervisor Dr. James Dowling for providing his leadership and guidance throughout my graduate degree. The opportunity to learn, grow and do research in his laboratory within the Hospital for Sick Children was a valuable experience I can take with me towards my future career in science. I would also like to thank my committee members Dr. Mikko Taipale and Dr. John Brumell for their advice, critiques, and encouragement at our meetings. To everyone in the laboratory who made themselves available to bounce ideas around and troubleshoot experiments, thank you for making the laboratory a fun and collaborative place to work. I was very lucky to join a great group of fellow graduate students who provided leadership, positivity, friendship and fun outside of the lab; a special thanks to all. I would like to thank my family and close friends for their support throughout this journey. Making the move to a new city to take on this challenge could not be done without each of you. iii Table of Contents Contents Acknowledgements .......................................................................................................... iii Table of Contents ............................................................................................................ iv List of Figures .................................................................................................................. vi Chapter 1 Introduction ...................................................................................................... 1 1.1 Phosphoinositides ................................................................................................. 1 1.2 Phosphatidylinositol 3-Kinases .............................................................................. 2 1.3 Class II PI3Ks ........................................................................................................ 3 1.4 PIK3C2B, a Class II PI3K ...................................................................................... 5 1.5 PIK3C2B, An X-linked Myotubular Myopathy Disease Modifier ............................ 7 1.6 Insulin Regulation of GLUT4 Translocation ........................................................... 9 1.7 Endosomal Recycling of GLUT4 ......................................................................... 11 1.8 Summary ............................................................................................................. 11 Chapter 2 In-vivo Characterization of the Loss of PIK3C2B in a Skeletal-Muscle Specific Knockout Model ............................................................................................................. 14 2.1 Acta Driven Expression of Cre-recombinase Generates Pik3c2b KOs ............... 14 2.2 Metabolic Analysis of Pik3c2b KO Mice .............................................................. 15 Chapter 3 In-vitro Characterization of the Loss of PIK3C2B in C2C12 Myoblasts ......... 20 3.1 Creating CRISPR-Cas9 Mediated Pik3c2b KOs in C2C12 Myoblasts ................ 20 3.2 Characterizing Pik3c2b KO C2C12 Myoblasts .................................................... 22 Chapter 4 Discussion ..................................................................................................... 29 Chapter 5 Future Directions ........................................................................................... 33 5.1 Specific Aim 1: Determine the cause of increased surface GLUT4 in Pik3c2b KO myoblasts ................................................................................................................... 33 iv References ..................................................................................................................... 35 Appendix I: Materials and Methods ................................................................................ 44 v List of Figures Figure 1. Specifics of the Pik3c2b mouse model ......................................................................... 15 Figure 2. GTT of Pik3c2b KO mice compared to WT show a decreased fasting blood glucose level ............................................................................................................................................... 17 Figure 3. Insulin tolerance test (ITT) of Pik3c2b KO mice compared to WT .............................. 18 Figure 4. Insulin stimulation in vivo displays a trend towards enhanced phospho-AKT (S473) activation ....................................................................................................................................... 19 Figure 5. Experimental Design of CRISPR-CAS9 Editing of Pik3c2b and Sequence Analysis. 21 Figure 6. Western Blot Analysis of CRISPR-CAS9 Pik3c2b KO Clones ................................... 22 Figure 7. Insulin time-course in Pik3c2b KO myoblasts exhibit increased AKT activation compared to WT .......................................................................................................................... 23 Figure 8. Analysis of mTORC1 activation in Pik3c2b KO myoblasts ......................................... 24 Figure 9. GLUT4 translocation assay (OPD) performed on WT and Pik3c2b KO C2C12 myoblasts. ...................................................................................................................................... 26 Figure 10. Total GLUT4 protein expression in WT and Pik3c2b KO C2C12 myoblasts ............ 27 Figure 11. GLUT4 endocytosis assay performed on WT and Pik3c2b KO C2C12 myoblasts .... 28 vi Chapter 1 Introduction 1.1 Phosphoinositides Phosphoinositides (PIPs) are important cellular signaling molecules generated and regulated by PI kinases and phosphatases. PIPs have been extensively studied due to their involvement in a myriad of cellular processes, such as: endosomal trafficking, exocytosis, autophagy and signal transduction1. PIPs belong to a family of membrane lipids characterized by the phosphorylation state of its base structure: phosphatidylinositol (PtdIns). These lipid molecules are composed of two fatty acid chains and a glycerol backbone tethered to an inositol ring with a phosphate linker2. Reversible phosphorylation of positions D3, D4 and D5 of the inositol ring results in seven distinct species of PIPs. Regulation of these different PIPs is vital for cellular function as each PIP varies in its expression, localization and interactions with effector proteins. Additionally, these molecules are short lived and are present at low concentrations predominantly at cytosolic surface of membranes. PtdIns comprises 80% of the total cellular levels of PIPs3,4. PtdIns4P and PdtIns(4,5)P2 represent the second most abundant PIPs at approximately 10% of total PIPs. The least abundant PIPs, PtdIns3P, PtdIns(3,4)P2, PtdIns5P, PtdIns(3,5)P2 and PtdIns(3,4,5)P3 each represent less than 1.5% of total PIP levels3. The seven PIPs vary widely in their subcellular localization, function and their ability to recruit specific effector proteins that mediate cellular signaling events. Generally, PI 3-phosphates are involved in endosomal trafficking while PI 4- phosphates are found at the plasma membrane and within the exocytic pathway1. PIPs mediate their function through direct binding to effector proteins altering their enzymatic activity and/or localization leading to a cellular response. PIPs are able to direct and target binding partners via several specific lipid-binding domains: pleckstrin homology (PH), FYVe (Fab-1, YGL023, Vps27 and EeA1), phox (PX) and Epsin N-Terminal Homology (ENTH)5. These lipid-binding domains offer PIPs another level of complexity. For example, the PH domain was first shown to bind PtdIns(3,4)P2 but later some PH domains were demonstrated to have high affinity for PIPs with adjacent phosphates5,6. Further experimental approaches to define PH domain specificity revealed low affinity for PtdIns3P, PtdIns4P and PtdIns(3,5)P2. 1 Additionally, there is cross-specificity of PX and FYVe domains that are able to bind PtdIns3P with high affinity7. Several PIPs are generated