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Mechanisms of Ciliary Targeting of the Olfactory Cyclic Nucleotide- Gated Channel

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Mechanisms of Ciliary Targeting of the Olfactory Cyclic Nucleotide- Gated Channel Mechanisms of Ciliary Targeting of the Olfactory Cyclic Nucleotide- Gated Channel by Paul Michael Jenkins A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmacology) in The University of Michigan 2010 Doctoral Committee: Associate Professor Jeffrey Randall Martens, Chair Professor Lori L. Isom Professor Benjamin L. Margolis Associate Professor Kristen J. Verhey © Paul Michael Jenkins All Rights Reserved 2010 To my family ii ACKNOWLEDGEMENTS I would like to first extend my sincerest gratitude to my research mentor, Dr. Jeffrey R. Martens. The time I have spent in his laboratory has shaped me both professionally and personally. His drive and dedication to science serve as a model that I strive towards every day. The past few years have been extremely rewarding for me due to his friendship, patience, and continual mentoring. I would not be where I am today without his guidance. I would also like to acknowledge my thesis committee members, Dr. Lori Isom, Dr. Ben Margolis and Dr. Kristen Verhey. I have been extremely lucky to have a committee that not only acted as thesis advisors, but also as collaborators, mentors, and friends. I would like to thank the numerous members of the Martens laboratory, present and former: Kristin Arendt, Dave Dudek, Nikhil Iyer, Sajida Jackson, Qiuju Li, Dyke McEwen, Jeremy McIntyre, Sarah Schumacher, Laurie Svoboda, Kristin van Genderen, Eileen Vesely, Tiffney Widner, Liz Williams, Kendra Yum, and Lian Zhang. Your antics in lab have kept me alternating between sanity and insanity and have left me with many goo d memories. Special thanks to the many collaborators I have worked with in my time spent in graduate school: the Benjamin Margolis laboratory, including Albert Liu and especially iii Toby Hurd for advice and technical help; the Donna Martin laboratory, including Elizabeth Hurd, Wanda Layman, and Jennifer Skidmore for assistance with the in situ hybridization experiments; the Yehoash Raphael laboratory, especially Lisa Beyer, for help with the electron microscopy; and the Margaret Gnegy laboratory, including Bipasha Guptaroy, Cheryse Furman, Rong Chen and Kathryn Luderman for help with the radiolabeling experiments. I would also like to acknowledge my funding from the Pharmacological Sciences Training Program, Hearing Balance and Chemical Senses Training Grant, and the National Institute on Deafness and Other Communication Disorders. I would like to show appreciation to my parents, Beverly and Richard, as well as my siblings, Ken and Katie, for their support through the years. Finally I would like to thank my wife, Jackie, and our daughter, Molly, for all of the great times. Your love and unconditional support have been so important to me throughout graduate school. I could not have done it without you. Thank you all so much. iv TABLE OF CONTENTS Dedication ........................................................................................................... ii Acknowledgements ............................................................................................ iii List of Figures .................................................................................................... ix List of Abbreviations .......................................................................................... xi Chapter 1 ............................................................................................................. 1 Olfactory Cilia: Linking Sensory Cilia Function and Human Disease .................. 1 Summary .................................................................................................... 1 Introduction ................................................................................................ 2 Anatomy of the Olfactory Epithelium ......................................................... 3 Olfactory Cilia Structure ............................................................................. 3 Axoneme ............................................................................................ 5 Lipid Composition ............................................................................. 7 Ciliary Necklace ................................................................................. 8 Basal Body ......................................................................................... 9 Ciliary Rootlet .................................................................................. 10 Ciliogenesis ...................................................................................... 10 Intraflagellar Transport (IFT) .................................................................... 13 Regulation of Ciliary Protein Entry .................................................. 16 Dynamics of Protein Movement within Olfactory Cilia .................... 19 Fate of Mistargeted Ciliary Cargo .................................................... 20 Ciliary Genomics and Proteomics ............................................................. 20 Olfactory Cilia and Human Disease .......................................................... 21 Olfactory Ciliopathies ...................................................................... 22 The OSN as a Site for Pathogen Entry .............................................. 25 v Conclusions .............................................................................................. 26 Acknowledgements ................................................................................... 26 Chapter 2 ........................................................................................................... 30 Ciliary targeting of olfactory CNG channels requires the CNGB1b subunit and the kinesin-2 motor protein, KIF17 .................................................................... 30 Summary .................................................................................................. 30 Results ...................................................................................................... 31 Discussion ................................................................................................ 36 Experimental Procedures .......................................................................... 37 Antibodies ........................................................................................ 37 Olfactory Epithelium Preparation ..................................................... 37 Mutagenesis ..................................................................................... 38 Immunoprecipitation ........................................................................ 40 Cell culture, transfection, and immunocytochemistry ....................... 42 Confocal Imaging ............................................................................. 43 Quantification of Ciliary Targeting ................................................... 44 Fluorescence Recovery After Photobleaching (FRAP) ..................... 45 Kinesin Constructs ........................................................................... 45 Acknowledgements ................................................................................... 46 Chapter 3 ........................................................................................................... 59 PACS-1 Mediates Phosphorylation-Dependent Ciliary Trafficking of the CNG Channel in Olfactory Sensory Neurons .............................................................. 59 Summary .................................................................................................. 59 Introduction .............................................................................................. 60 Results ...................................................................................................... 61 PACS-1 is expressed in Olfactory Sensory Neurons ......................... 61 The CNGB1b Subunit can Interact with PACS-1 and Serve as a Substrate for CK2 Phosphorylation .................................................. 62 Mutation of the CK2 Phosphorylation Sites on CNGB1b Inhibits Ciliary Delivery of the CNG Channel ............................................... 64 Loss of PACS-1 Function Impairs CNG Channel Ciliary Transport .. 64 vi Inhibition of CK2 Alters CNG Channel Localization ........................ 65 CK2 Phosphorylation is Necessary for the Ciliary Localization of CNG Channel In Vivo and Proper Olfactory Function...................... 66 Adenoviral Expression of Non-Phosphorylatable PACS-1 in Native OSNs Impairs Ciliary Localization of the Endogenous CNG Channel ............................................................................................ 67 Discussion ................................................................................................ 68 Experimental Procedures .......................................................................... 71 Antibodies ........................................................................................ 71 Cell culture....................................................................................... 71 Adenovirus Preparation .................................................................... 71 Intranasal injection ........................................................................... 72 Tissue preparation ............................................................................ 72 Immunostaining ............................................................................... 73 Confocal Imaging ............................................................................
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