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Identification and Characterization of Moesin-, PIP2-Mediated Solid Particle Phagocytosis

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Identification and Characterization of Moesin-, PIP2-Mediated Solid Particle Phagocytosis University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2018-08-09 Identification and Characterization of Moesin-, PIP2-mediated Solid Particle Phagocytosis Tu, Zhongyuan Tu. Z. (2018). Identification and Characterization of Moesin-, PIP2-mediated Solid Particle Phagocytosis (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/32804 http://hdl.handle.net/1880/107622 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Identification and Characterization of Moesin-, PIP2-mediated Solid Particle Phagocytosis by Zhongyuan Tu A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN MICROBIOLOGY AND INFECTIOUS DISEASES CALGARY, ALBERTA AUGUST, 2018 © Zhongyuan Tu 2018 Abstract Phagocytosis is the defining feature of professional phagocytes of the innate immune system. This function is typically carried out by phagocytic receptors on the cell surface. These receptors can mediate binding and engulfment of solid particles. However, these phagocytic receptors have evolved very recently in history comparing to phagocytosis as a conserved cellular function. This suggests a primordial form of phagocytosis might exist. Years ago, our laboratory uncovered an expected phagocytic mechanism that solid particle can bind to membrane lipids on phagocytes to trigger lipid sorting. Consequently, this can lead to phagocytosis akin to FcγR-based phagocytosis regarding its dependence on Immunoreceptor Tyrosine-based Activation Motif (ITAM), Src-family kinases, Syk, and phosphoinositide 3- kinase (PI3K). Based on these findings, we proposed a hypothetical mechanism for solid particle phagocytosis termed “Signaling Equivalent Platform” (SEP). In short, membrane engagement with solid structures, either via ligand/receptor binding or merely being stabilized by an approaching solid surface will lead to a shared downstream pathway with the same dependence on ITAM and Syk. Both modes of phagocytosis are equivalent for its activation by solid structures. However, the identity of the ITAM-containing molecule and the exact involvement of lipid during solid particle phagocytosis under SEP is still unclear. This thesis serves to strengthen the idea of SEP by identifying the ITAM-containing molecule and further characterizing the involvement of the ITAM-containing molecule and lipids during solid particle phagocytosis. We used a generic ITAM sequence as a probe and identified moesin as the ITAM- containing molecule from the mouse genome. We further demonstrated that a solid structure ii binding to the cell surface leads to autonomous accumulation of phosphatidylinositol 4, 5- bisphosphate (PIP2) to the site of contact, which attracts moesin, a conserved structural linker, to the plasma membrane. Moreover, Moesin, via its ITAM, is sufficient to activate phagocytic programming including Syk and downstream signaling that is virtually identical to that initiated by Fcγ receptors. Bioinformatic analysis suggested that this moesin-mediated signaling predates modern Fcγ and immune receptors. This thesis, therefore, reveals an evolutionarily conserved moesin-, PIP2-mediated signaling platform for the evolutionarily conserved phagocytosis that provides essential components for modern ITAM-based signaling cascades. iii Preface This thesis is original, unpublished, independent work by the author, Zhongyuan Tu. The author would like to take this opportunity to lay out how this thesis is written. Chapter 1 provides the background for the entire thesis. It is structured in a funnel shape such that the broadest topic is introduced first. The topics discussed will progressively get narrower and eventually the topic at issue is introduced and discussed at last. As the topics narrows, descriptions become more detailed. This technique was first introduced to me by Dr. Mark McDermott, Professor Emeritus and a top-notch graduate educator at McMaster University, and has been useful ever since. Chapter 2 concerns the material and methods used in this thesis. I structured the methods sections according to their purpose. Specific to this thesis, section 2.4.2 presents the topic of microscopy. This section can be read independent of other parts of the thesis and intends to serve as a practical guide on how to perform good microscopy for junior graduate students in need. This part is written in consultation with Dr. Tie Xia. Chapter 3 to 5 contains all results of this thesis. There are two major flows when writing these chapters. The vertical flow is the results section. In it, results are presented in logical order and conclusions are made. Results are from the interpretation of data. Therefore, prudence and appropriate caution should be exercised. The horizontal flow is the discussion section. It is like a replay of a football game in which each move are highlighted and analyzed. In it, vertically flowed results are horizontally discussed, and critically examined. Also, new topics are introduced if necessary to facilitate discussion. For example, in Section 4.3, membrane domains were introduced to provide context for results discussion. Also, in section 5.3, the idea of “effective engagement” was put forward in an attempt to explain the results further. The iv discussion points were identified from, but not limited to, the previous discussion with committee members, discussions among colleagues and comments from reviewers of the manuscript submitted. Critical thinking and the defense elements of a thesis should be reflected in these sections. The entire writing process on the discussion sections can be described as playing a chess game against the most critical version of oneself because one must contemplate all possibilities and weigh strengths and weakness of alternative scenarios all the time. In other words, one must think hard. It is both a taxing and intellectually stimulating experience. Of special note, it is my personal view that weakness of results should be acknowledged upfront instead of looking the other way in the hope of forcing specific narratives, especially with thesis writing. The truth may be uncomfortable, inconvenient, but it shall set you free. The Chief Justice of the Supreme Court of United States, John G Roberts Jr, stated on multiple occasions that the Court appreciates when an attorney acknowledges the weak points of their argument because the Court understands the topics brought before the Supreme Court are hard, and there are merits for each side of the argument. This is true for law, and this is true for science. Because the questions investigated in a Ph.D. thesis usually involves many unknowns and are limited by current techniques, it is helpful to exercise candor and then discuss possibilities and solutions. Chapter 6 is the final chapter serving as the grand finale of the thesis. Results are summarized, and a model is proposed. This model is further examined in the Future Directions section for possible ways to improve this model. For example, in Section 6.2, how phagocytic signaling occurs is discussed fundamentally, and previously discussed concepts of membrane domain, “effective engagement”, checks and balances between ITAM and ITIM are fitted into this grand discussion to help better understand our work and to improve the model in the future. In a way, this chapter can serve as a prequel to a future thesis. v Acknowledgements The official completion of this thesis will mark the end of the early part of my life as a scientist. Looking back, this is a long and winding road with success and much more failures as the natural progression in science. I am privileged to meet and sometimes travel alongside some of the kindest, wittiest human beings I know on this road. This section of the thesis serves to express my deepest gratitude towards these people. First and foremost, I thank my supervisor Dr. Yan Shi for giving me such a unique opportunity to develop my graduate research and to mature as a human being. It is my firm belief that the critical thinking and interpersonal skills I have developed during my graduate studies will benefit me for the rest of my life. Next, I thank my committee members Drs Robin Yates and Matthias Amrein for carefully guiding me through my graduate studies. It is evident to me that I gained experience points and leveled up every time coming out of a committee meeting or a candidacy exam. I believe any student is fortunate to have you as their supervisors. I would also like to thank Dr. Tie Xia for being a personal friend and my go-to guy for imaging-related problems. Being nice is sometimes a very overrated and murky virtue as it can be misjudged. However, I would still want to make it to the record that Tie Xia is a genuinely nice person. One can always count on him for personal advice, imaging expertise, and recommendation for vegan restaurants. The newly-minted Dr. Libing Mu is critical for developing and finishing this thesis. The fact that we have gone through this together means everything. Therefore, nothing more can be said. vi I would also like to acknowledge Jiahua Chen, Hua Rong, Yifei Zhang, Xiaoting Wang for their companionship while they are in Canada. I enjoyed every meal they shared with me and every conversion we had. I would like to thank the administrative staffs. Those include our lab manager Melanie Stenner and administrative assistant Florence Yang. They made my life as a graduate student much easier by doing their job exceptionally well. I would also like to acknowledge all the other lab members at Tsinghua University. I would like to thank Ning Kang for her quick wit.
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