FACTOR ASSOCIATED WITH NEUTRAL SPHINGOMYELINASE ACTIVATION (FAN), A MEMBER OF THE BEACH FAMILY OF PROTEINS, REGULATES LYSOSOME SIZE by Rishna Shrestha A thesis submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Master of Science in Laboratory Medicine and Biomedical Science Department of Pathology The University of Utah August 2015 Copyright © Rishna Shrestha 2015 All Rights Reserved The University of Utah Graduate School STATEMENT OF THESIS APPROVAL The thesis of Rishna Shrestha has been approved by the following supervisory committee members: Diane M. Ward , Chair 05/07/2015 Date Approved Jerry Kaplan , Member 05/07/2015 Date Approved Matthew A. Mulvey , Member 05/07/2015 Date Approved and by Peter E. Jensen , Chair/Dean of the Department/College/School of Pathology and by David B. Kieda, Dean of The Graduate School. ABSTRACT Chediak-Higashi Syndrome (CHS) is a rare, autosomal recessive genetic disorder caused by mutations in the lysosomal trafficking regulator gene (CHS1) that affects vesicle morphology. The CHS1 gene encodes a protein, Lyst, which belongs to a family of proteins containing a conserved BEACH (BEige And Chediak-Higashi) domain and WD40-repeat domain. The beigej mouse has been the best-studied animal model for CHS. Factor associated with neutral sphingomyelinase activation or FAN is the smallest member of the BEACH family. FAN interacts with the Tumor Necrosis Factor Receptor (TNFR1) to activate Neutral SphingoMyelinase2 (NSMase2). The absence of Lyst or FAN gives rise to enlarged lysosomes. Loss of both proteins results in an additive effect, -/- as demonstrated by increased lysosome size in FAN /beigej mouse. An additive phenotype suggests that there are at least two pathways to regulate lysosome size. Overexpression of Lyst or FAN results in smaller than normal lysosomes. Overexpression of FAN in the absence of Lyst does not give rise to small lysosomes. NSMases are present on lysosomes and inhibition of NSMases, using GW4869 or 3-O- Methylsphingomyelin, results in enlarged lysosomes. Further, the decrease in lysosome size seen in FAN-overexpressing cells is blocked by GW4869. These results suggest that FAN activation of NSMase at the lysosome is a crucial step in regulating lysosome size. TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii LIST OF FIGURES ............................................................................................................ vi LIST OF TABLES ........................................................................................................... viii ACKNOWLEDGEMENTS ............................................................................................... ix CHAPTERS 1. INTRODUCTION ......................................................................................................... 1 Lysosomes ............................................................................................................... 2 Functions of Lysosomes .......................................................................................... 3 Diseases Associated with Lysosomal Dysfunction ................................................. 5 Chediak-Higashi Syndrome (CHS) ......................................................................... 6 Lyst Protein ............................................................................................................. 7 Neutral Sphingomyelinase Activation Associated Factor (FAN or NSMAF) ........ 9 FAN-deficient Mouse ............................................................................................ 10 FAN Binding Partners ........................................................................................... 10 Aims ...................................................................................................................... 12 2. MATERIALS AND METHODS ................................................................................ 18 Cells and Media ..................................................................................................... 19 Plasmids ................................................................................................................ 19 Transfections ......................................................................................................... 19 RNA Interference .................................................................................................. 20 mRNA Analysis by RT-PCR ................................................................................ 20 Microscopy ............................................................................................................ 21 Neutral Sphingomyelinase Inhibition .................................................................... 22 Neutral Sphingomyelinase Activity Assay (14C Assay) ........................................ 22 Lipidomic Analysis ............................................................................................... 23 3 RESULTS ..................................................................................................................... 25 -/- Generation of FAN /beigej Mouse ....................................................................... 26 Inhibition of NSMase Activity ............................................................................. 26 Overexpression of NSMase2 ................................................................................. 26 Overexpression of FAN ........................................................................................ 27 RNA Interference of NSMase1 and NSMase3 ..................................................... 28 Overexpression and RNA Interference of FAN Binding Partners ........................ 29 Lipidomic Analysis .............................................................................................. 29 4 DISCUSSION .............................................................................................................. 48 Loss of Lyst and FAN Shows an Additive Effect on Altering Lysosome Size .... 49 Effect of NSMase Activity on the Lysosome Size ................................................ 49 Role of FAN in Regulation of Lysosome Size ...................................................... 50 Overexpression and RNA Interference of FAN Binding Partners ........................ 51 The Absence of FAN Alters the Lipid Profile ..................................................... 52 Summary ............................................................................................................... 53 APPENDIX: CONVENTIONAL AND SECRETORY LYSOSOMES ........................... 55 REFERENCE .................................................................................................................... 77 v LIST OF FIGURES Figure Page 1.1 Enlarged lysosomes in CHS patient fibroblasts ........................................................ 13 1.2 The Lyst protein ........................................................................................................ 14 1.3 Mammalian BEACH family of proteins ................................................................... 15 -/- 1.4 Enlarged lysosomes in beigej and FAN mouse fibroblasts ..................................... 16 1.5 FAN binding partners ................................................................................................ 17 -/- -/- 3.1 Coat color difference between C57BL/6, beigej, FAN , FAN /beigej mice ........... 31 3.2 The absence of FAN and Lyst results in enlarged lysosomes ................................... 32 3.3 NSMase inhibition induces an increase in lysosome size ......................................... 33 3.4 Time-dependent effect of NSMase inhibitor 3-O-Methylsphingomyelin on lysosome size ..................................................................................................................................... 34 3.5 NSMase2 overexpression does not alter lysosome size ............................................ 35 3.6 FAN overexpression does not reduce lysosome size in the absence of Lyst ............ 36 3.7 Inhibition of NSMase blocks the reduction in lysosome size in cells overexpressing FAN ................................................................................................................................... 37 3.8 The absence of NSMase 2 does not affect lysosome size or the FAN-mediated reduction in lysosome size ................................................................................................ 38 3.9 fro/fro cells exhibit decreased NSMase activity in vitro ........................................... 39 3.10 mRNA analysis of NSMase1 and NSMAse3 by RT-PCR ...................................... 40 3.11 RACK1 overexpression does not alter lysosome size ............................................. 41 3.12 EED overexpression does not affect lysosome size ................................................ 42 3.13 mRNA analysis of EED by RT-PCR ...................................................................... 43 3.14 mRNA analysis of RACK1 by RT-PCR ................................................................. 44 -/- 3.15 PCA analysis of C57BL/6, beigej and FAN .......................................................... 46 3.16 Volcano plot of FAN-/- vs. C57BL/6 ....................................................................... 47 A.1 A model of the immunologic synapse in Cytotoxic