ASSEMBLY AND DISASSEMLY OF THE WNT BETA-CATENIN DESTRUCTION COMPLEX Kristina Schaefer A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Genetics and Molecular biology in the School of Medicine. Chapel Hill 2018 Approved by: Mark Peifer Robert Duronio Amy Shaub Maddox Kevin Slep Benjamin Major © 2018 Kristina Schaefer ALL RIGHTS RESERVED ii ABSTRACT Kristina N. Schaefer: Assembling and Disassembling the Wnt Beta-catenin destruction complex (Under the direction of Mark Peifer) Wnt signaling provides a key example for cell-cell signaling pathways that regulate embryonic development and stem cell homeostasis and then are frequently inappropriately activated in cancers. Wnt signaling acts by regulating levels of β-catenin (βcat), an essential transcriptional coactivator of Wnt target genes. The tumor suppressors APC and Axin, along with the kinases GSK3 and CK1, form the core of the multiprotein destruction complex (DC), which targets βcat for phosphorylation, ubiquitination and destruction. In the presence of Wnt ligands, DC function is down-regulated, allowing levels of βcat levels to rise, eventually entering the nucleus to activate transcription of Wnt target genes. Based on earlier work, we hypothesize that the DC is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulation of assembly and stability of the DC regulates its function. We tested this hypothesis in Drosophila embryos by combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging, and molecule counting. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these puncta to the membrane, a decrease in the number of Axin molecules, while cytoplasmic Axin levels increase, suggesting altered assembly. GSK3 regulates DC recruitment to the membrane and the release of Armadillo/βcat from the DC. Manipulating Axin or APC2 levels had no effect on DC activity when Wnt signals were absent, but, surprisingly, had opposite effects on the DC when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced iii inactivation. We also found that endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels. Hetero-polymerization between Dishevelled and Axin via their DIX domains is essential for Wnt down regulation of the DC, yet the mechanism of regulation is unknown. Our data suggest both absolute levels and the ratio of these three core components affect destruction complex function, supporting models in which competition among Axin partners determines complex activity. iv PREFACE “One, remember to look up at the stars and not down at your feet. Two, never give up work. Work gives you meaning and purpose and life is empty without it. Three, if you are lucky enough to find love, remember it is there and don't throw it away.” Stephan Hawking We were invited by Developmental Cell to write a review on the current view of Wnt signaling. Therefore, instead of a typical introduction chapter, we wrote a review. This review centered around our hypothesis that the destruction complex is really a biomolecular condensate. We discuss recent research in the field and describe how several recent reports support our hypothesis. This chapter was a collaboration between Mark and I. We have now submitted the manuscript. Chapter 2 describes the majority of my research. This chapter was published in PLoS Genetics in 2018 (PLoS Genet 14(4): e1007339). Here we investigated the mechanistic role of Axin, APC2, GSK3, and Dsh in regulating destruction complex both in the presence and absence of Wnt signaling. We utilized Drosophila genetics, biochemistry, and high-resolution microscopy. This work was completed under the supervision of Mark Peifer. Teresa Bonello analyzed protein levels of Axin, APC, and Dsh and critical reviewed the manuscript. Shiping Zhang aided in visualization of Axin:GFP and analyzing Arm levels, and statistical analyses. v Clara Williams analyzed Engrailed expression, and helped with determining embryonic lethality, terminal cuticle phenotypes, and prepping embryos for imaging analysis. Dave Roberts imaged and analyzed Arm levels in APC2 mutants. Dan McKay analyzed RNAseq data. I helped organize the team of authors on this paper and performed all other experiments. The manuscript was written by me and Mark Peifer with input from the other authors. Chapter 3 describes my work on how beta-catenin is transferred from the destruction complex to the E3 ligase. The groundwork of this project was started when I was a rotation student, and many tools utilized in this project were created by an undergraduate honors research student Lauren Bauer, who I supervised. At the time of this preface, we still finishing up a few experiments and plan to include this data as part of a manuscript we hope to submit later this year. Mira Pronobis conducted and analyzed the FRAP data. I performed the biochemistry experiments, acquired the confocal and super resolution images. vi ACKNOWLEDGEMENTS Before I even entered graduated school, I was given a series of article about how difficult it would be, and then I was questioned whether I really wanted to purse my Ph.D. Throughout my graduate school career I have regretted this decision and have been elated I went back to school. I want to thank those who helped me back on the path of science and discovery. 1) To Dr. Xin Zhang, without him I would have never found my love for research or decided to attend graduate school in the first place. 2) To Mark Peifer who pushed me to be a greater thinker, to make a better argument, and to be healthily skeptical about my own data. 3) To my other lab members and peers - thanks for being my sounding board, my late- night confidant, for helping me stay the course, and for getting me out the lab and doing something fun every once and a while. 4) Thanks to my family who have always supported me in any they could. I would not have made it through without you guys. 5) Lastly to Nathan, thank you for being my practice audience, for believing in me when I was unable, and for supporting my crazy career choice knowing what we would have to sacrifice to accomplish it. vii TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................xii LIST OF TABLES.....................................................................................................................xiv CHAPTER 1: EMERGING IDEAS IN REGULATING WNT SIGNALING: REGULATION BY POLYMERIZATION AND PARALLEL BEHAVIOR WITH BIOMOLECUAR CONDENSATES .. 1 OVERVIEW: ................................................................................................................................................ 1 The textbook model of Wnt signaling ............................................................................................. 6 The Wnt-regulatory destruction complex—is it a biomolecular condensate? .................... 8 The destruction complex is an internally ordered structure that assembles by polymerization .................................................................................................................................... 10 A functional destruction complex contains many more than four proteins ...................... 13 destruction ........................................................................................................................................... 15 .............. 17 Other conserved sequences in APC’s intrinsically disordered region also play key functions............................................................................................................................................... 18 APC may play additional positive and negative roles in Wnt signaling .............................. 19 Regulating a biomolecular condensate: Wnt signaling changes destruction complex localization and assembly ............................................................................................................... 20 Wnt signaling causes a switch in the destruction complex mix, destabilizing it ............. 21 One consequence of supermolecular assembly: the kinase GSK3 plays both positive and negative roles in the destruction complex via its access to many targets ................ 23 Axin post-translational regulation plays complex roles .......................................................... 24 APC mutations in colorectal cancer target specific aspects of destruction complex function ................................................................................................................................................. 27 The destruction complex is a multifunctional machine with other targets including the cytoskeleton ........................................................................................................................................ 28 REFERENCES ......................................................................................................................................... 33 CHAPTER 2: SUPRAMOLECULAR ASSEMBLY OF THE BETA-CATENIN DESTRUCTION COMPLEX AND THE EFFECT OF WNT SIGNALING ON ITS LOCALIZATION,