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Control of Ciliogenesis by an Evolutionarily Ancient Peptide Amidating Enzyme Dhivya Kumar [email protected]

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Control of Ciliogenesis by an Evolutionarily Ancient Peptide Amidating Enzyme Dhivya Kumar Dhivkmr@Gmail.Com University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 3-13-2017 Control of Ciliogenesis by an Evolutionarily Ancient Peptide Amidating Enzyme Dhivya Kumar [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Kumar, Dhivya, "Control of Ciliogenesis by an Evolutionarily Ancient Peptide Amidating Enzyme" (2017). Doctoral Dissertations. 1466. https://opencommons.uconn.edu/dissertations/1466 Control of Ciliogenesis by an Evolutionarily Ancient Peptide Amidating Enzyme Dhivya Kumar, PhD University of Connecticut, 2017 Cilia are highly complex microtubule-based cellular extensions present on most eukaryotic cells that play key roles in sensing the extracellular environment, processing developmental signals and generating propulsive force. Although many details of the ciliary assembly process are well understood, there are numerous aspects of this overall process that remain very unclear. Accumulating evidence points to an ancient connection between peptidergic signaling and cilia. A crucial last step in generating active neuropeptides capable of intercellular signaling involves modification of the C-terminus to a protease-resistant and uncharged amide group in the lumen of the secretory pathway. This modification is mediated by peptidylglycine alpha-amidating monooxygenase (PAM) and is generally essential for neuropeptide bioactivity and indeed in mammals is required for life. Phylogenetics revealed that metazoans lacking nervous systems contain PAM, and that PAM-like genes are present in unicellular green algae. In this thesis, I describe the identification of active PAM enzyme in the green alga Chlamydomonas reinhardtii, and demonstrate that it localizes to both Golgi and punctate structures within cilia; PAM is also present in both motile and immotile (primary) cilia in mammals. The presence of this novel enzymatic activity in cilia of organisms across phylogeny suggests these organelles may have a previously unappreciated role in peptide-based signaling that emerged early in the history of eukaryotes. Furthermore, I demonstrate that this enzyme is absolutely required for normal ciliary assembly in Chlamydomonas, planaria and mice; in the absence of PAM, these organelles either do not assemble past the transition zone (Chlamydomonas), fail during Dhivya Kumar – University of Connecticut, 2017 remodeling (planaria) or are much shorter than controls (mouse). In Chlamydomonas, loss of PAM alters Golgi structure and function, and affects levels of specific ciliary assembly proteins. Biochemical analysis reveals that PAM is released in extracellular vesicles (ectosomes) that are shed from the surface of cilia in Chlamydomonas, which may have important implications for cell-cell communication. Finally, I also describe connections between PAM and the actin cytoskeleton, and demonstrate that both cilia and actin-based microvilli are lost from the pronephros of PAM-null zebrafish. This raises the possibility that PAM plays a general role in the formation and/or maintenance of membrane-bound cellular extensions. Control of Ciliogenesis by an Evolutionarily Ancient Peptide Amidating Enzyme Dhivya Kumar B.Tech. Indraprastha University, India, 2008 M.S., University of Tennessee, Knoxville, USA 2012 Ph.D., University of Connecticut, USA 2017 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2017 i Copyright by Dhivya Kumar 2017 ii APPROVAL PAGE Doctor of Philosophy Dissertation Control of Ciliogenesis by an Evolutionarily Ancient Peptide Amidating Enzyme Presented by Dhivya Kumar, B.Tech., M.S., Ph.D. Major Advisor ___________________________________________________________________ Betty A. Eipper Major Co-Advisor ___________________________________________________________________ Stephen M. King Associate Advisor ___________________________________________________________________ Richard E. Mains Associate Advisor ___________________________________________________________________ Peter Setlow Associate Advisor ___________________________________________________________________ Bing Hao University of Connecticut 2017 iii Acknowledgements First and foremost, I would like to thank my advisors, Drs. Betty Eipper, Richard Mains and Stephen King for their support and guidance throughout this journey. My PhD journey has been a transformative experience and I feel extremely lucky to have worked with outstanding advisors. I walk away from this experience a more confident and better scientist then I thought was ever possible. The “We can do it!” poster that hangs outside Betty’s office is very fitting, for she is an incredible role model for women in science. I will forever be in awe of her dedication to science and mentorship to trainees. I appreciate the freedom she gave me to develop and pursue my own ideas and all the sound advice over the years that have kept me sane. Perhaps the most valuable lesson I learnt from Dick and Betty is to not be afraid to learn new things. I thank them for always leaving their doors open for questions, no matter how big or small. Steve’s enthusiasm and love for science is contagious. I feel very fortunate to have walked- both literally and figuratively- along the “tetsugaku no michi” (path of philosophy) with him. I enjoyed every minute I spent interacting with him over the years- both at work and during our wonderful adventures in Japan and Amsterdam. Thank you for being my constant cheerleader- I am very appreciative and I hope I make you proud. I have to thank all the present and past members of the Neuropeptide and Molecular Motors laboratories for their support and comradery. It was a pleasure working with such a talented group of people. Dr. K.S. Vishwanatha deserves a special thank you for always patiently answering my innumerable questions over the years. Thanks to Darlene D’Amato, Yanping Wang and Andrew Yanik for outstanding technical assistance. A big thanks to Taylor LaRese for help with the animal experiments and conversations about coffee and life. I would also like to acknowledge Ramila Patel-King for help with the planarian experiments and Maya Yankova for iv her expertise with electron microscopy. I enjoyed working with Myah Bartolotta, a very sharp undergraduate from Wheaton College for two consecutive summers and I appreciate her hard work and efforts on generating extracellular vesicles from Chlamydomonas. This work would not have come together without the continued support of many collaborators. I would like to thank Dr. Sabeeha Merchant, Dr. Daniela Strenkert and Dr. Crysten Blaby-Haas for helping us navigate through the tricky world of Chlamydomonas biology. The zebrafish experiments presented in this dissertation would not have been possible without the efforts of Dr. Jonathan Gitlin and Dr. Rebecca Thomason at Marine Biological Laboratory. I would like to thank the entire Molecular Biology and Biophysics department for fostering a fantastic environment for graduate students to learn and grow in. Special thanks to Drs. Peter Setlow and Bing Hao for serving on my thesis committee and their interest, advice and helpful comments over the years. I also thank Drs. Kimberly Dodge-Kafka and Larry Klobutcher for their willingness to serve on my general exam committee. I appreciate the efforts and dedication of Dr. Barbara Kream, Jody Gridley, Anna Sagan, Bridget Clancy-Tenan and Patricia Schultz in the Neuroscience, Molecular Biology and Biomedical Science graduate programs. Last, but certainly not least, I am indebted to my family and friends for their unconditional love and endless patience throughout these years. I am very grateful to my partner, Arvind for his unwavering support, even during countless late nights spent in the lab. Thanks to my brother, Bharani for helping me see the lighter side of life. These past years would have been very dreary without my friends- Sudershana, Nozomi, Kristen, Swati and Pragya. Finally, I thank my parents for believing in me and instilling in me the value of hard work: I owe all my success to you. v Table of Contents Outline ……………………………………………………………………………………………… 1 Chapter 1 Cilia: Form, Function and Genesis …………………………………………………………… 3 Abstract …………………………………………………………………………………………….. 3 Introduction ………………………………………………………………………………………… 4 One organelle, many functions ……………………………………………………………… 4 Form follows function …………………………………………………………………………. 8 How do cells build a cilium? …………………………………………………………………. 10 When cilia go awry ……………………………………………………………………………. 14 Birth of an organelle ………………………………………………………………………….. 15 More questions than answers ……………………………………………………………….. 17 References …………………………………………………………………………………………. 20 Chapter 2 60 Years of POMC: From POMC and alpha-MSH to PAM, Molecular Oxygen, Copper, and Vitamin C …………………………………………………………………………………….. 23 Abstract …………………………………………………………………………………………….. 23 POMC and PAM: Where it all began ……………………………………………………………. 25 What have we learned about PAM since its identification? …………………………………... 28 Structure of the PAM gene …………………………………………………………………… 28 A PAL for PAM ………………………………………………………………………………… 30 Cellular requirements for PAM activity ……………………………………………………… 31 Crystal structures and insights into the mechanism of peptide amidation ……………… 32 More than just an enzyme – a multi-tasking protein …………………………………………… 35 Evolutionary distribution of PAM ……………………………………………………………… 35 vi The cytosolic domain of PAM ………………………………………………………………...
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