REGULATION OF THE CENTRIOLE LIFE CYCLE BY MDM1 A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOLOGY AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Daniel Patrick Van de Mark August 2014 © 2014 by Daniel Patrick Van de Mark. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/gf463wx1210 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Tim Stearns, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Martha Cyert I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. William Nelson I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Rajat Rohatgi Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost for Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii Abstract Centrosomes are composed of microtubule based structures called centrioles surrounded by a proteinaceous pericentriolar material. They serve as the primary microtubule organizing center (MTOC) in mammalian cells, and have important roles in intercellular trafficking, cellular organization and motility, and the formation of mitotic spindles. In addition to these roles, centrioles can also nucleate the formation of cilia. Mutations in centrosome and cilium protein coding genes are implicated in human diseases such as cancer and a group of disorders characterized by cilia-related phenotypes, collectively called ciliopathies. Understanding the functions of centrosome and cilium proteins is useful for understanding the causes behind the development of these diseases. Here we study the gene Mdm1, which we previously identified as being transcriptionally upregulated in multiciliated mouse tracheal epithelial cells (MTECs). We show that MDM1 is a core centriole protein with microtubule binding and stabilizing properties that localizes to newly replicated procentrioles beginning in mitosis, coincident with the end of centriole duplication. Furthermore, we show that depletion and overexpression of MDM1 results in phenotypes consistent with the protein acting as a negative regulator of centriole duplication. Lastly, we study the function of MDM1 in MTECs, demonstrating that MDM1 does not appear to be associated with the cellular machinery responsible for driving centriole amplification in these cells, but localizes to centrioles upon their formation. We further show that MDM1 disperses from centrioles upon ciliation in mature multiciliated cells. Depletion of MDM1 in this system did not block entry into iv the multiciliated cell fate program, but we did observe a ciliation defect, suggesting MDM1 depletion may cause a delay in the development pathway of these cells. v Acknowledgements This work was supported by funding from the Stanford Genomics Training Program (SGTP), the Stanford Department of Biology, and by NIH grant R01GM52022 (T.S.). The 3D-SIM super-resolution experiments were supported, in apart, by Award Number 1S10OD01227601 from the National Center for Research Resources (NCRR). Thank you to my advisor, Tim Stearns, for his guidance, support, and ideas over the last several years. He has had a tremendously positive effect on both how I think about, and how I communicate science. Thank you to all of the current and former Stearns lab members whom I overlapped with during my time here. I learned a lot from everyone in lab over the years, and it was a pleasure to work with them all. In particular, thanks to Joanna Lee and Yin Loon Lee who worked at the benches to either side of mine throughout the majority of my graduate school experience, and who both became very good friends of mine. Additionally, the MTEC experiments in this thesis really got off the ground because of guidance that I received early on from Yin Loon in working with the system. Thank you also to Miranda Stratton, whom I had the great fortune of mentoring during her time rotating in the lab. She was a fantastic student to work with, and I ended up learning a lot from the experience as well. Also thanks to Ramona Hoh who was both my mentor when I rotated in the Stearns lab and who first identified MDM1, the topic of this thesis, as a centrosome protein of interest. And thank you to Eszter Vladar, a former Stearns lab member, who was incredibly helpful with the MTEC studies, providing reagents and extremely useful comments and guidance along the way. Thank you to my committee members, Martha Cyert, James Nelson, and Raj Rohatgi, and my defense chair, Lucy O’Brien, for all of the help they have given me over the past 5 years. And lastly, thanks to my family and to all of the friends I have made during my time at Stanford. They all have been so supportive, and made the times when experiments were not working so much better. I am very grateful to have them all in my life. vi Table of Contents Chapter 1: Introduction 1 Abstract 2 Introduction 2 The centriole life cycle 4 Proteins involved in the licensing and initiation of centriole duplication 6 Negative regulation of centriole duplication 14 Centriole duplication in multiciliated cells: an example of altered regulation of the centriole duplication pathway 21 Areas of future study 22 Figures 23 References 25 Chapter 2: MDM1, a microtubule binding protein that negatively regulates centriole duplication 34 Abstract 35 Introduction 35 Results 39 Discussion 51 Materials and methods 56 Figures 65 References 86 Chapter 3: The Role of MDM1 in multiciliation of tracheal epithelial cells 92 Abstract 93 Introduction 93 Results 96 Discussion 103 Materials and methods 107 Figures 113 References 124 Chapter 4. Concluding remarks and future directions 127 References 134 vii List of Figures Chapter 1: Introduction Figure 1. Asymmetries in centriole structures 23 Figure 2. Positive regulation of the centriole duplication pathway 24 Chapter 2: MDM1, a microtubule binding protein that negatively regulates centriole duplication Figure 1. Rabbit and mouse anti-MDM1 antibodies recognize N- and C-terminal regions of the protein 65 Figure 2. MDM1 localizes to centrioles 67 Figure 3. Overexpressed MDM1 colocalizes with microtubules in RPE-1 cells 69 Figure 4. MDM1 is a microtubule binding protein 71 Figure 5. MDM1 stabilizes microtubules 73 Figure 6. MDM1 overexpression inhibits centriole duplication in cycling RPE-1 cells 75 Figure 7. MDM1 overexpression blocks centriole reduplication in S-phase arrested U2OS cells 76 Figure 8. MDM1 depletion controls 77 Figure 9. MDM1 depletion causes an accumulation of supernumerary centrin foci 79 Figure 10. Characterization of supernumerary centrin foci 81 Figure 11. Fluorescence intensity analysis of duplication pathway proteins upon MDM1 depletion 83 Figure 12. Model of MDM1 as a negative regulator of centriole duplication 85 Chapter 3: The Role of MDM1 in multiciliation of tracheal epithelial cells Figure 1. Stages of multiciliated cell development in MTECs 113 Figure 2. MDM1 is expressed at all stages of early MTEC development 114 Figure 3. Additional colocalizations of MDM1 with known centriole/basal body markers during MTEC development 115 Figure 4. The rabbit anti-MDM1 antibody we produced stains non-specifically in mouse cells 116 Figure 5. MDM1 is not associated with deuterosomes 118 Figure 6. MDM1 transiently localizes to centrioles in MTECs 119 Figure 7. MDM1 depletion does not block entry into the multiciliated cell fate program 120 Figure 8. MDM1 depletion decreases the percentage of MTEC cells that reach a multiciliated state 122 viii Chapter 1: Introduction 1 Abstract Centrosomes, the primary microtubule organizing center (MTOC) in mammalian cells, are composed of two microtubule-based structures called centrioles that are surrounded by a proteinaceous matrix called the pericentriolar material. As MTOCs, centrosomes nucleate and anchor microtubules that play roles in intercellular trafficking, providing cell shape, cell motility, and organizing the mitotic spindle. However an additional function of centrosomes is to nucleate the formation of cilia, organelles appreciated to be important for a wide diversity of chemical and mechanical signal transduction pathways. Centriole number is tightly controlled as aberrations in centriole and cilium numbers can contribute to cell death and disease [1]. This chapter reviews the processes and proteins involved in forming new centrioles, with an emphasis given to positive and negative regulation of the duplication pathway. Introduction Centrioles are microtubule based, barrel shaped structures with nine-fold symmetry. In most organisms, the barrel is made up of triplet microtubules and is approximately 250 nm wide and 500 nm long [2]. However, deviations in this structure are observed across different organisms. Notably, the C. elegans centriole is composed of doublet microtubules, and the D. melanogaster centriole is composed of singlet microtubules [3]. Throughout this chapter, I focus primarily on the centriole structure and life cycle as it relates to they typical proliferating vertebrate cell, but I will occasionally point out evolutionary and cell type deviations along the way (see [3] 2 and [4] for a detailed discussions of evolutionary differences in centriole and cilia structures). Most cells contain two parental centrioles at any given time, which are capable of nucleating two procentrioles during centriole duplication.