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Investigations Into Roles for Endocytosis in LIN-12/Notch

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Investigations Into Roles for Endocytosis in LIN-12/Notch Investigations into roles for endocytosis in LIN-12/Notch signaling and its regulation Jessica Chan Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2020 © 2020 Jessica Chan All Rights Reserved Abstract The LIN-12/Notch signaling pathway is highly conserved in all animals, and is crucial for proper development. It is a key pathway in specifying cell fate in many cellular contexts, and dysregulation of the pathway can have deleterious consequences. Therefore, understanding how LIN-12/Notch signaling is regulated in different contexts has been a main area of interest in the field. Previous studies in different model organisms have identified many modes of regulation of the signaling pathway, one of which is endocytosis of the ligand and receptor. Here, I further investigated the role of endocytosis in LIN-12/Notch signaling in multiple developmental contexts in Caenorhabditis elegans. Work in Drosophila and vertebrates had previously established that ligand-mediated activation of Notch requires ubiquitination of the intracellular domain of the transmembrane ligand and the activity of the endocytic adaptor Epsin in the signaling cell. The consensus in the field is that Epsin-mediated endocytosis of mono-ubiquitinated ligand generates a pulling force that exposes a cleavage site in Notch for an ADAM protease, a critical step in signal transduction. In contrast, in this thesis, I examined two different transmembrane ligands in several different cell contexts and found that activation of LIN-12/Notch and the paralogous GLP-1/Notch in C. elegans does not require either Epsin-mediated endocytosis or ubiquitination of the intracellular domain of the ligand. Results obtained by a collaborator indicate that C. elegans ligand and receptor interactions are tuned to a lower force threshold than are Drosophila ligand and receptor interactions, potentially accounting for these differences. I also looked at the role of endocytosis in regulating LIN-12 signaling in the context of vulval development. The cell fate pattern of six vulval precursor cells (VPCs) is mediated by EGFR and LIN-12/Notch signaling. Previous work using multicopy transgenes in fixed specimens indicated that LIN-12 is post-translationally downregulated via endocytosis in response to EGFR activation in the VPC named P6.p, an event that appeared essential for ligands to activate LIN-12/Notch in neighboring VPCs. In this thesis, I manipulate the endogenous lin- 12 gene and examine live specimens to show that LIN-12 appears to be regulated transcriptionally in P6.p and evidence that there may be additional potential endocytic motifs that may regulate LIN-12 in this context. Table of Contents List of Tables and Figures.............................................................................................................. iii Acknowledgments........................................................................................................................... v Chapter 1: Introduction ................................................................................................................... 1 1.1 Overview of Notch signaling in C. elegans .......................................................................... 2 1.2 Overview of Notch signal transduction ................................................................................ 6 1.3 Notch activation by DSL .................................................................................................... 10 1.4 Role of secreted DSL .......................................................................................................... 13 1.5 Known roles of epn-1/Epsin in C. elegans ......................................................................... 14 Chapter 1 Figures ...................................................................................................................... 17 Chapter 2: Results ......................................................................................................................... 27 Summary ................................................................................................................................... 28 Results ....................................................................................................................................... 29 Chapter 2 Figures ...................................................................................................................... 37 Chapter 3: Discussion ................................................................................................................... 48 Chapter 3 Figures ..................................................................................................................... 58 Chapter 4 Materials and Methods ................................................................................................. 61 References ..................................................................................................................................... 74 Appendix ....................................................................................................................................... 87 Appendix A: Introduction ......................................................................................................... 88 Introduction Figures .............................................................................................................. 97 i Appendix B: Results ............................................................................................................... 102 Results Figures .................................................................................................................... 111 Appendix C: Discussion ......................................................................................................... 126 Appendix D: Materials and Methods ...................................................................................... 132 ii List of Tables and Figures Chapter 1. Figures……………………………………………………………….……….………18 Figure 1. Notch Signaling in C. elegans embryogenesis…….………………………….….……18 Figure 2. Notch Signaling in C. elegans germline development.…………………….……….…19 Figure 3. Notch Signaling in C. elegans AC/VU decision.………………………….….……….20 Figure 4. Notch Signaling in C. elegans VPC specification ………………….…….….………..21 Figure 5. Overview of Notch signaling cascade…………...………………….…….….………..22 Figure 6. DSL in C. elegans, Drosophila and vertebrates…………...…..…….…….…………..23 Figure 7. Domain organization of C. elegans, Drosophila and human Notch………..…………24 Figure 8. Epsin domain structure and function………….……..……………………..………….25 Figure 9. An In vivo force sensor in Drosophila…………………………….………..…………26 Chapter 2. Figures……………………………………………………………….……………….39 Figure 1. epn-1 is not required for GLP-1 signaling at 4 cell stage ……………………………..39 Figure 2. epn-1 is not required for LAG-2 function in specifying excretory cell ……....………40 Figure 3. Ubiquitination is not required for LAG-2 function in embryogenesis and AC/VU decision…………………………………………………………………………………………..41 Figure 4. Membrane anchor is required for ligand function……………………………………..42 Supplemental figure 1. epn-1::zf1::GFP expression in L3……………………………………...43 Supplemental figure 2. A difference in LAG-2 intracellular domain requirement in AC/VU decision……………………………………………………………………………………...…...44 Chapter 3. Figures………………………………………………………………………………..59 Figure 1. Predicted DSL in Bilateria and non-Bilateria………………………………………….59 Figure 2. Alignment of NRR sequence from human, Drosophila, and nematode Notches……...60 iii Appendix Figures……………………………………………………………….….….……….98 Introduction Figure 1. Vulval Precursor Cell specification ……………………………………..98 Introduction Figure 2. LIN-12 expression in VPCs…………….……………………..…...........99 Introduction Figure 3. LIN-12 internalization by Downregulation Targeting Sequence (DTS)………………………………………………………………………..………………….100 Introduction Figure 4. LIN-12 downregulation mediated by alx-1 and wwp-1…..… .……..….101 Results Figure 1. VPC-specific RNAi screen…………………..……………………..….…….112 Results Figure 2. Single copy transgenes of lin-12 and lin-12 mutants… ...………………...…113 Results Figure 3. Summary of lin-12(DTS)::GFP multicopy transgene expression…..…..…...114 Results Figure 4. Endogenous expression of lin-12::GFP and lin-12Δ(DTS)::GFP …….........115 Results Figure 5. Endogenous lin-12 transcriptional reporter.....……………………..………..116 iv Acknowledgments I am writing this amid the COVID-19 pandemic, serving as a stark reminder of the privileges I have and the many things that I am grateful for throughout this journey. First and foremost, I am deeply grateful for my advisor, Dr. Iva Greenwald, for her guidance throughout my time in the lab. There were many times when I encountered obstacles during my research, as well as in personal life, and she has always been patient and provided valuable advice. Her dedication to both teaching and life-long learning, as well as the creativity and rigor she applies to science has been beyond inspiring and certainly worth emulating no matter what career path I end up in. I would also like to thank my thesis committee, Dr. Dan Kalderon and Dr. Julie Canman for the valuable suggestions and input over the years during my committee meetings. I thank Dr. Gary Struhl and Dr. Barth Grant for reading my dissertation and for serving on my thesis committee. I would also like to thank my collaborators
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