The Role of Ion Channel TRPM7 in B Cell Development and Function By Mithunah Krishnamoorthy A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy (Ph.D.) Cell and Systems Biology University of Toronto © Copyright by Mithunah Krishnamoorthy, 2018 The Role Ion Channel TRPM7 in B Cell Development and Function Mithunah Krishnamoorthy Doctor of Philosophy (Ph.D.) Cell and Systems Biology University of Toronto 2018 Abstract The channel-kinase Transient Receptor Potential Subfamily M7 (TRPM7) is known to regulate magnesium homeostasis and was the first channel implicated in the survival of a B cell line. Our study is the first to show that B cells require TRPM7 for development in a murine model. By using a mouse model where TRPM7 is specifically deleted in B cells under the control of the mb1 promotor, we show that B cells are absent in all peripheral lymphoid tissues due to apoptosis of Pre B cells. By using an in vitro stromal cell line system, we demonstrate that B cell development can be partially rescued by high levels of extracellular magnesium. Interestingly, the lack of B cells is accompanied by an expanded granulocyte population in the spleen. In addition to identifying TRPM7 as an essential factor for B cell development, we show that TRPM7 is also an important regulator of B cell activation. DT40 B cells lacking TRPM7 fail to contract and gather antigen when activated. To investigate the role of the kinase domain of TRPM7 we made use of B cells expressing a kinase dead point mutant. These cells were also unable to gather antigen, showing that the kinase domain is an important regulator of this process. ii We also show that the kinase domain may potentially interact with another important regulator of B cell activation, PLCγ2 to mediate antigen collection and cell contraction. Importantly, primary murine B cells expressing only one allele of TRPM7 or treated with a TRPM7 inhibitor both displayed defects in antigen gathering, confirming our results in the DT40 cell line. Lastly, we show that TRPM7 is essential for antigen internalization, a process that is important for the recruitment of T cell help and ultimately, antibody production. iii Acknowledgements I could not have finished this degree without the support of several individuals. While I knew that on this journey I would meet new people, I had no idea of the lasting impact that these individuals would have on my life. First and foremost, I would like to thank Dr. Bebhinn Treanor. This work would have been entirely impossible without her. From editing what feels like a million figures, reports and presentations to providing sound advice, she is a tour de force that has always supported me. Thank you Dr. Treanor, for being an excellent mentor and for teaching me how to be a better scientist every day. I could not have asked for a better supervisor. I would also like to thank the members of my supervisory committee, Drs. Rene Harrison and Blake Richards, for their advice and guidance over the years. I would also like to thank Dr. Mauricio Terebiznik for his kindness and help. Huge thanks goes to my Mom, who has supported me in all my endeavors, even though she did not entirely understand what I did or why I had to be in the lab all the time. Mom, I am so glad that I can always come to you for advice regarding any aspect of my life and for being the one person I can always count on no matter what. No amount of thanks can even begin to acknowledge what you have done for me. I also want to thank my Dad for being my personal chauffeur from day 1. To my dearest lab mates, you have made coming into the lab everyday a treat. I will miss our impromptu Timmy’s runs, going out for all you can eat (until you die) sushi, Pump it up exercise sessions and random office conversations. I will always cherish those moments; I know they will put a smile on my face for years to come. Thank you, Tina Zhao, Anh Cao, Josephine Ho, Laabiah Wasim, Trisha Mahtani, Hifza Buhari, Nouf Alluqmani and Logan Smith for your friendship and support. Special thanks go to Tina, for her unwavering emotional support and for iv being the shoulder I can always go to cry on. She has never failed to cheer me up when I was feeling down and discouraged. For that, I will be forever grateful. Thank you, Tina, for spending almost a year troubleshooting those terrible total ERK blots. Honestly, I have no idea how you kept going. Your level of persistence is inhuman and is something I strive to achieve going forward. I will never forget how important your friendship has been to my success. v Declaration Chapters 2 and 3 have been submitted to the journal of Science Signalling under the following titles: M. Krishnamoorthy, F.H.M Buhari, T. Zhao, P. Brauer, K. M. Burrows, E. Cao, V. Moxley-Paquette, A. Mortha, J. C. Zúñiga-Pflücker, and B. Treanor. The ion channel TRPM7 is required for B cell lymphopoiesis. Science Signalling, accepted. M. Krishnamoorthy, L. Wasim, F.H.M Buhari, T. Zhao, T. Mahtani, J. Ho, S. Kang, A- L. Perraud, C. Schmitz, and B. Treanor. The channel-kinase TRPM7 regulates antigen gathering and internalization in B cells, Science Signalling, accepted. I performed all the experiments contained within this thesis with the exceptions of those performed by Laabiah Wasim and Josephine Ho (Figures 3.2A-C, 3.4B-D, and 3.6C-D), Hifza Buhari (Figure 2.3, 3.5C-D), Tiantian Zhao (Figure 2.5, Figures 3.8 H-I), and Trisha Mahtani (3.8A) vi Table of Contents Abstract ........................................................................................................................................ ii Acknowledgements .................................................................................................................... iv Declaration.................................................................................................................................. vi List of Figures .............................................................................................................................. x List of Tables .............................................................................................................................. xi List of Abbreviations ................................................................................................................ xii CHAPTER 1: Introduction ........................................................................................................ 1 1.1 B Cell Biology .................................................................................................................... 1 1.1.1 Overview ............................................................................................................... 1 1.1.2 B cell development ............................................................................................... 2 1.1.3 B cell activation .................................................................................................... 5 1.1.4 Microclusters in the spreading and contraction response ..................................... 8 1.1.5 Antigen internalization and presentation ............................................................ 14 1.2 Secondary Lymphoid Tissues .......................................................................................... 15 1.2.1 Overview ............................................................................................................. 15 1.2.2 Spleen .................................................................................................................. 15 1.2.3 Lymph nodes ....................................................................................................... 17 1.2.4 Peyer’s patches.................................................................................................... 18 1.3 The Transient Receptor Potential Channel Superfamily .................................................. 19 1.3.1 Overview ............................................................................................................. 19 1.3.2 TRPM subfamily ................................................................................................. 20 1.4 TRPM7 Structure and Function ....................................................................................... 24 1.4.1 Overview ............................................................................................................. 24 1.4.2 Structure of TRPM7 ............................................................................................ 25 1.4.3 Function of TRPM7 ............................................................................................ 28 1.5 Magnesium Function ........................................................................................................ 31 1.5.1 Overview ............................................................................................................. 31 1.5.2 Importance of magnesium in the immune system .............................................. 33 1.6 Thesis Rational and Hypothesis ....................................................................................... 34 vii CHAPTER 2: TRPM7 is Essential for B Cell Development ................................................. 36 2.1 Introduction ...................................................................................................................... 36 2.2 Results .............................................................................................................................. 38 2.2.1