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Signature Redacted. Amy E A genetic platform for the study of protein perturbation and prion-based inheritance by Gregory A. Newby B.S., Biological Sciences Carnegie Mellon University (2009) SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY June 2017 Massachusetts Institute of Technology 2017. All rights reserved. Signature redacted Signature of Author................................. Department of Biology redacted C e rtifie d B y .. Signature ............... I.R..... F..... / Gerald R. Fink Professor of Biology Acting Thesis Supervisor in Place of Susan Lipdquist (deceased) Accepted By............ Signature redacted. Amy E. Keating MASSACHUSETTS INSTITUTE Professor of Biology OF TECHNOLOGY Chair, Graduate Student Committee MAY 2 3 2017 LIBRARIES I 1 2 A genetic platform for the study of protein perturbation and prion-based inheritance By Gregory Arthur Newby Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology SUMMARY Proteins mediate every cellular process. In order for life to exist, each protein must be finely tuned to carry out its function at the proper time and place. Because the environment is dynamic and often unpredictable, the regulation of proteins must be responsive to environmental stimuli. Mutations, age, and severe or repeated insults can decrease the quality of protein regulation, leading to disease. The study of protein regulation and its dysfunction in disease are of vital importance. Regulatory and disease phenomena involving protein assembly or aggregation are common but currently understudied on account of their intractability with existing techniques. In order to equip scientists with better tools to tackle these difficult phenomena, my collaborators Ahmad Khalil and Szilvia Kiriakov (Boston University) and I developed the yTRAP platform (standing for: yeast transcriptional reporters of aggregating proteins). yTRAP couples the activity of a synthetic transcriptional activator to a protein's solubility. It enables sensitive measurement of a protein's state within a eukaryotic cellular context, preserving complex interactions that may be lost using in vitro techniques. yTRAP can be measured in high throughput to enable large studies, screens, and selections on aggregation phenomena. The reporter output is modular and can be customized to desired purposes and measurement modalities. Using a fluorescent output, the signal from yTRAP is readily quantifiable. The combination of these desirable properties enables many kinds of previously-impossible studies. Furthermore, because of its exquisite sensitivity, yTRAP can be used to broadly screen for protein perturbation beyond the context of aggregation. For example, it can report on alterations in protein localization, binding partners, or degradation. I applied yTRAP to track yeast prions, which have previously been difficult to study due to a lack of simple and reliable reporters. Prions are protein-based elements of inheritance that have profound implications for the evolution of single-celled organisms. I first utilized yTRAP to identify factors that faithfully switch prion states on and off, thus proving that prion states can be deterministically regulated. I used these factors to create new cellular tools out of prions: 1) a memory device that records elevated temperatures experienced by a yeast population, and 2) an anti-prion drive that eliminates prions from mating partners and progeny. Separately, I conducted an ecological study into the yeast prion [SW*]. I found that [SWI*] confers a 'pioneering' cellular program that encourages migration and diverse mating partners. Loss of the prion confers a protective 'settled' cellular program with growth and survivability advantages. yTRAP greatly facilitated this study through reliable tracking of the prion state. Prion-like phenomena are now ripe for study with yTRAP. Thesis Supervisor: Susan Lindquist, Professor of Biology 3 4 Acknowledgments I will be forever grateful for the time I spent in Susan Lindquist's laboratory. Susan was an amazing scientist, mentor, and person who gave so much of herself to everyone she worked with. In our many meetings throughout the years, I never failed to leave revitalized and excited about the work to come. She had enormous vision for the future and a never-ending stream of ideas. She was also a highly skilled and thoughtful manager who assembled a top-notch team to help run the lab - together they brought in and supported an amazing set of people with whom it was a pleasure to come to work every day. In large part that pleasure was due to the friendly and collaborative environment fostered directly by Sue. Her death was felt deeply by all of us; we will always miss the days we shared together when our laboratory was still whole. I hope that in this thesis and in my future work, I can live up to Susan's high expectations. I would like to thank all of the members of the Lindquist lab, past and present, for their friendship and scientific contribution to my experience and training. In countless ways each person added to the unique and happy environment, where unlimited help was never more than a question away. I would particularly like to thank members of the prion subgroup, Peter Tsvetkov, Can Kayatekin, Erinc Hallacli, Kendra Frederick, Sohini Chakrabortee, Lauren Pepper, Bill Hesse, Dan Jarosz, Manoshi Datta, my co-leader of chocolate subgroup Georgios Karras, my rotation mentor Mikko Taipale, the administrative staff Brooke Bevis, Linda Clayton, Bob Burger, Audrey Madden, Rosemary Benson, and the materials assistants Ndubuisi Azubuine and Tsering Yangchen. I could fill the page with the names of my many labmates, who all contributed to the wonderful experience I had has a graduate student. I would like to thank my collaborator Ahmad "Mo" Khalil, his graduate student Szilvia Kiriakov, and the other members of his lab. Mo and Szilvia have been my closest collaborators since very early on in my thesis work. It has been a pleasure working closely with them, and I have come to think of Mo as a second mentor to me. Together, the three of us co-authored the manuscript that I have included in this thesis as chapter 2. A great deal of credit for the ideas, text, and figures is owed to them. I hope that our collaborative work together is only just beginning. I would like to thank the members of my thesis committee, Gerry Fink, Dane Wittrup, Tim Lu, Graham Walker, and lain Cheeseman, for their advice and encouragement provided at each meeting, and for their input as I consider future steps. I have valued their guidance greatly. My graduate work was made possible by the many wonderful scientists who trained me before. I would like to thank my mentors and from Carnegie Mellon, Peter Berget, Beth Jones, and Jim Burnette, and also my many labmates and teachers. I thank Andreas PlOckthun who hosted and mentored me during my Fulbright project, and his laboratory. I would also like to thank the wonderful friends I have made in graduate school and those I met long before. We have had a lot of fun through all manner of good times, and 5 stuck together even through much sadness. I thank The Ten Groomsmen, Rohit Ramnath, Fowler Brown, Jon Chastek, Andy Echenique, Kevin Knockenhauer, Rob Mathis, Isaac Oderberg, Ethan Sokol, Bradely Yates, and Timmy Zhu. I would also like to thank the Ramnath and Zhu families, who on several occasions hosted me in their homes and treated me as a son. I thank all of my classmates for making the transition to graduate school a fun and easy one. I especially thank all members of the Biomansion community, who lived together with me, hosted, and/or attended many fun events every single week. Finally, I would like to thank my dear family - the many cousins and relatives scattered throughout the globe, my sisters Kristin, Laura, Colette, and Alexandra, my parents Richard and Hilary, and my wife Hee Yeon. Over the years they have all shown me so much love and support that I am overwhelmed to think of it. I would especially like to thank my parents for the incredible amount of effort it took on their parts to get me to where I am today, and for inspiring me through their strong work ethic, inquisitive spirits, and commitment to reason. And to Hee Yeon I owe a very special debt of gratitude - she willingly signed up for this partnership despite not knowing what trials lay ahead! Each shared day with her is a new adventure, and her steadfastness is a constant comfort and source of strength for the journey. 6 Table of Contents Chapter 1: Introduction.............................................................................................. 9 S u m m a ry ........................................................................................................ 1 0 References ...................................................................................................... 29 Chapter 2: A genetic sensor for protein aggregation enables synthetic memory and anti-prion drives................................................................................................ 37 S u m m a ry ........................................................................................................ 3 8 Introduction.................................................................................................... 38 R e s u lts ..............................................................................................................
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