University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2017-12-14 Membrane Proteins Take Different Trafficking Pathways to the Primary Cilium William Joseph Monis University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Cell Biology Commons, and the Developmental Biology Commons Repository Citation Monis WJ. (2017). Membrane Proteins Take Different Trafficking Pathways to the Primary Cilium. GSBS Dissertations and Theses. https://doi.org/10.13028/M2GX0S. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/946 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. MEMBRANE PROTEINS TAKE DIFFERENT TRAFFICKING PATHWAYS TO THE PRIMARY CILIUM A Dissertation Presented By William Joseph Monis Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (DECEMBER, 14, 2017) INTERDISCIPLINARY GRADUATE PROGRAM MEMBRANE PROTEINS TAKE DIFFERENT TRAFFICKING PATHWAYS TO THE PRIMARY CILIUM A Dissertation Presented By William Joseph Monis This work was undertaken in the Graduate School of Biomedical Sciences Interdisciplinary Graduate Program The signature of the Thesis Advisor signifies validation of Dissertation content ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Gregory J. Pazour, Ph.D., Thesis Advisor The signatures of the Dissertation Defense Committee signify completion and approval as to style and content of the Dissertation ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Julie A. Jonassen, Ph.D., Member of Committee ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Mary Munson, Ph.D., Member of Committee ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Silvia Corvera, M.D., Member of Committee ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Jagesh V. Shah, Ph.D., External Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ George B. Witman, Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the School ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences December 14, 2017 iii Dedication For My Mother “The untold want by life and land ne’er granted, Now voyager sail thou forth to seek and find.” - Walt Whitman, Leaves of Grass, 1855 iv Acknowledgments I thank my thesis advisor Gregory Pazour for his mentorship and for the opportunity to conduct research in his lab. I express my gratitude to all the members of my thesis/dissertation committee for their interest, enthusiasm, and valuable advice that helped me move my thesis project forward. Additionally, I thank all the past and present members of the Pazour Lab along with all my friends at UMass Medical School for their support. v Abstract Cilia are conserved organelles that extend from the surface of most eukaryotic cells. During development cilia play key roles in force generation and perception of the extracellular environment. Ciliary defects cause a broad class of human diseases called ciliopathies characterized by pleiotropic symptoms including cystic kidneys, retinal degeneration, cardiac malformations and skeletal deformations. Perception of the environment relies on specific proteins being localized to the ciliary membrane compartment. The mechanism for sorting and trafficking membrane proteins to the cilium is poorly understood. To address this question, I developed a fluorescence-based pulse-chase assay to measure the transport kinetics of ciliary membrane proteins. This assay was used to determine the importance of candidate proteins to the delivery of fibrocystin, polycystin-2, and smoothened to cilia. Using this assay, I found that ciliary delivery of fibrocystin and polycystin-2 requires IFT20, GMAP210, and the exocyst while smoothened delivery is largely independent of these proteins. In addition, I determined that polycystin-2, but not smoothened or fibrocystin require the biogenesis of lysosome related organelles complex-1 (BLOC-1) for ciliary delivery. Consistent with a requirement for BLOC-1 in ciliary transport of polycystin-2, BLOC-1 mutant mice have cystic kidney disease. BLOC-1 functions in endosomal sorting and I find that disrupting the recycling endosome also reduced ciliary polycystin-2 and causes its accumulation in the recycling vi endosome. This is the first demonstration of a role for BLOC-1 in ciliary biogenesis and highlights the complexity of trafficking pathways to the cilium. vii Table of Contents Signature Page.....................................................................................................ii Dedication ...........................................................................................................iii Acknowledgments ..............................................................................................iv Abstract ................................................................................................................v Table of Contents ..............................................................................................vii List of Tables.......................................................................................................ix List of Figures ......................................................................................................x List of Copyrighted Material .............................................................................xii Preface...............................................................................................................xiv Chapter I : Introduction .......................................................................................1 Primary Cilia Structure and Trafficking Barriers...................................................... 1 The Primary Cilium.................................................................................................... 2 Photoreceptors Have A Modified Primary Cilium ...................................................... 2 The Ciliary Gate ........................................................................................................ 5 Vesicle Docking and Fusion Sites ..................................................................................... 5 Transition Fibers ................................................................................................................ 6 Transition Zone.................................................................................................................. 7 Ciliary Targeting Sequences...................................................................................... 8 Apical and Basolateral Membrane Protein Sorting ................................................... 8 Cystoproteins ............................................................................................................ 9 G Protein Coupled Receptors ................................................................................. 15 Trafficking Pathways to the Cilium ......................................................................... 20 Lateral Trafficking Pathway..................................................................................... 21 Direct Trafficking Pathway ...................................................................................... 26 Recycling Trafficking Pathway ................................................................................ 28 Proteins Implicated in Ciliary Trafficking ............................................................... 30 IFT Complex A and B.............................................................................................. 31 Golgi-IFT Complex .................................................................................................. 34 BBSome .................................................................................................................. 37 Molecular Motors..................................................................................................... 39 Small GTPases ....................................................................................................... 40 Arf Small GTPases .......................................................................................................... 41 Arl Small GTPases .......................................................................................................... 43 Rab Small GTPases ........................................................................................................ 46 Exocyst.................................................................................................................... 49 SNAREs .................................................................................................................. 51 BLOC-1 ................................................................................................................... 52 Conclusion................................................................................................................. 53 Chapter II : BLOC-1 is required for selective membrane protein trafficking from endosomes