University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 5-2014 Mechanisms of action and co-optive evolution for hypervariable courtship pheromones in plethodontid salamanders. Wilburn Beau Wilburn University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Wilburn, Wilburn Beau, "Mechanisms of action and co-optive evolution for hypervariable courtship pheromones in plethodontid salamanders." (2014). Electronic Theses and Dissertations. Paper 1569. https://doi.org/10.18297/etd/1569 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The nivU ersity of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The nivU ersity of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. MECHANISMS OF ACTION AND CO-OPTIVE EVOLUTION FOR HYPERVARIABLE COURTSHIP PHEROMONES IN PLETHODONTID SALAMANDERS By Damien Beau Wilburn B.S., University of Louisville, 2005 A Dissertation Submitted to the Faculty of the School of Medicine of the University of Louisville in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Department of Biochemistry and Molecular Biology University of Louisville Louisville, KY May 2014 MECHANISMS OF ACTION AND CO-OPTIVE EVOLUTION FOR HYPERVARIABLE COURTSHIP PHEROMONES IN PLETHODONTID SALAMANDERS By Damien Beau Wilburn B.S., University of Louisville, 2005 A Dissertation Approved on March 31, 2014 by the following Dissertation Committee: ___________________________________________ Richard C. Feldhoff ___________________________________________ Pamela W. Feldhoff ___________________________________________ Ronald G. Gregg ___________________________________________ Thomas E. Geoghegan ___________________________________________ Michael H. Perlin ___________________________________________ S. Randal Voss ii DEDICATION This dissertation is dedicated to my teacher, mentor, and friend Kirk Barnett for instilling in me a love and passion for biology. iii ACKNOWLEDGEMENTS First and foremost, I must thank Dr. Richard Feldhoff for seven years of outstanding mentorship. I am not sure who or where I would be today without him, and I won’t ever be able to thank him enough for everything he has done to help me grow as a scientist, a person, and a lover of wine. I’ll never forget the demonstrations in the lab, the chats in his office, the many road trips, the nights of salamander collecting, the dinners in Valentine, and the life lessons on the porch of the Duplex – I could not have asked for a better adviser. Next I have to thank Kathleen Bowen. In countless ways, this dissertation would be a fraction of the quality without her extensive efforts to support me both scientifically and personally; her friendship has been invaluable. I must thank Kari Doty for her dedication and patience, always allowing me to use her as a sounding board as needed. And a great many thanks to Dr. Pamela Feldhoff for the support, enthusiasm, and keeping her friend in line. My friends and family have kept me motivated during the best and worst of times, and I could not have made it this far without their constant encouragement and support. I thank my parents, Cliff and Judy Wilburn, my sister Tiffany, and brother-in-law Steve. And I owe a huge debt of gratitude to my three best friends: Tyler Kilgore, my oldest friend and brother, for always being by my side; Tom Lucas for the inside jokes, continued companionship, and many, many shenanigans; and Tyler “Tweek” Adams for the games, the drinks, and the laughs. And I thank my “wife” Lindsey Becker for being my friend and constant Biochemistry support. iv I have been extraordinarily fortunate to have talented, motivated collaborators who have shown me tremendous kindness and support on many levels, and I sincerely thank all of them: Drs. Lynne Houck and Stevan Arnold for their guidance and assistance on all things salamander-related, and hosting me during my stay in Oregon; Dr. Sarah Eddy for her amazing friendship and academic support; Dr. Sarah Woodley and Chris Fonner for hosting me in Pittsburgh and their assistance on the neurophysiology project; Dr. William Pierce, Dr. Jian Cai, and Ned Smith for assistance in mass spectroscopy; Drs. Andrew Lane and Sam Arumugam for their patience and support on the NMR project; Christy Baggett, Jess Thomas, Sean Darrow, and Adam Chouinard for their outstanding field assistance, and keeping me awake on the late night collecting trips. I also thank my dissertation committee for their invaluable expertise and continued support: Drs. Ronald Gregg, Thomas Geoghegan, Michael Perlin, and Randal Voss. A special thanks goes to Dr. William Dean for always lending an ear and providing feedback whenever I needed it. None of this work would have been possible without the support of Highlands Biological Station and its fabulous staff, particularly Dr. Jim Costa, Dr. Anya Hinkle, Michelle Ruigrok, and Cynthia Soderstrom. I thank my funding sources: the National Science Foundation (IOS-0416834, IOS-0818649, IOS-1147271, the Graduate Research Fellowship Program, and Doctoral Dissertation Improvement Grant IOS-1311309), the UofL IRIG program, and the Highlands Biological Station Grant-in-Aid program. Finally, I thank Heine Brothers coffee for – often literally – giving me the stamina and energy to survive the last 5 years of graduate school. v ABSTRACT MECHANISMS OF ACTION AND CO-OPTIVE EVOLUTION FOR HYPERVARIABLE COURTSHIP PHEROMONES IN PLETHODONTID SALAMANDERS Damien Beau Wilburn March 31, 2014 Pheromones are an important type of chemical cue used by most animals to convey information between individuals. For more than 100 million years, male plethodontid salamanders have utilized a system of non-volatile, proteinaceous pheromones to regulate female mating behavior and receptivity. One of these pheromone components, Plethodontid Modulating Factor (PMF), is a hypervariable protein related to the three-finger protein (TFP) superfamily. Previous studies revealed that PMF persists as a rapidly evolving multi-isoform mixture. However, many characteristics of PMF as a pheromone remained undetermined, including gene structure and transcriptional regulation, translational regulation, protein structure, evolutionary mechanisms, and the isoform effects on female behavior and neurophysiology. Therefore, the broad aim of this dissertation was to characterize the mechanisms of action and evolution for PMF using the red-legged salamander, Plethodon shermani. The molecular and proteomic diversity of PMF was determined by RT-PCR and mass spectroscopy. The PMF complex is the product of at least 13 gene duplications in 3 gene classes containing highly conserved 5’ and 3’ untranslated regions (UTRs). These UTRs are bound by cold inducible RNA binding protein, which likely plays a key role in coordinating expression of the many vi diverse PMF isoforms during gland development. Using mass spectroscopy and multidimensional NMR, the 3D structure of the most abundant PMF isoform was determined to have a novel structure compared to all other TFPs. In particular, an altered disulfide bonding pattern promoted greater backbone flexibility in the most rapidly evolving segments of PMF to possibly enhance male pheromone and female receptor interactions. Functional assays testing different mixtures of PMF isoforms revealed that isoform diversity is a key requirement for increasing female receptivity, likely through synergistic interactions in the vomeronasal organ and/or the brain. Examination of pheromones in a different plethodontid species (P. cinereus) revealed that the majority of PMF duplications occurred within the last ~20 million years. In summary, in response to female sexual selection, the PMF gene complex has evolved through an unusual disjunctive evolutionary process as part of a birth-and-death model of gene evolution to permit coordinated expression of dozens of flexible proteins that synergistically function to regulate female behavior. vii TABLE OF CONTENTS PAGE DEDICATION iii ACKNOWLEDGEMENTS iv ABSTRACT vi LIST OF TABLES x LIST OF FIGURES xi CHAPTER I. INTRODUCTION 1 II. PROTEOMIC AND UTR ANALYSES OF A RAPIDLY EVOLVING HYPERVARIABLE FAMILY OF VERTEBRATE PHEROMONES Chapter Overview 19 Introduction 21 Methods 25 Results 29 Discussion 43 Conclusions 49 III. STRUCTURAL INSIGHTS INTO THE EVOLUTION OF A SEXY PROTEIN: NOVEL TOPOLOGY AND RESTRICTED BACKBONE FLEXIBILITY IN A HYPERVARIABLE PHEROMONE FROM THE RED-LEGGED SALAMANDER, PLETHODON SHERMANI Chapter Overview 50 Introduction 52 Materials and Methods 56 Results 64 Discussion 85 Conclusions 89 IV. DIFFERENTIAL FEMALE BEHAVIOR FROM A HYPERVARIABLE COURTSHIP PHEROMONE IN THE RED-LEGGED SALAMANDER, PLETHODON SHERMANI Chapter Overview 90 Introduction 92 Materials and Methods 98 Results 103 Discussion 105 viii V. OLFACTORY EFFECTS OF PMF ISOFORM VARIABILITY IN THE RED-LEGGED SALAMANDER, PLETHODON SHERMANI Chapter Overview 109 Introduction 111 Results 115 Discussion 120 Experimental procedures 125 VI. GENE EXPRESSION DURING SEASONAL ORGANOGENESIS: PROFILING TRANSCRIPTOME CHANGES IN AN ANNUAL CYCLE OF GLAND DEVELOPMENT AND HYPERVARIABLE PHEROMONE SYNTHESIS Chapter Overview 129