DOPAMINERGIC REGULATION OF INSULIN SECRETION INVESTIGATED BY FLUORESCENCE FLUCTUATION SPECTROSCOPY by Brittany Catherine Caldwell Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biomedical Engineering May, 2016 Nashville, Tennessee Approved by: Professor David Piston Professor Melissa Skala Professor John Gore Professor Hassane Mchaourab Professor Anne Kenworthy ACKNOWLEDGEMENTS I am very thankful to my advisor, Dave Piston, who provided me with challenging problems to solve and the methods with which to solve them. Going to the lab even on a challenging day was exciting with the lab environment Dave created. My committee members, Melissa Skala, John Gore, Hassane Mchaourab, and Anne Kenworthy provided me with excellent feedback that helped develop and strengthen my project. I am grateful for their time and patience. Working in the Piston Lab was a wonderful experience. I am thankful for all the current and previous lab members who made the lab a fun place to go each day. I owe most of my molecular biology knowledge to Alessandro Ustione who is a fantastic mentor and wonderful friend. I also want to thank Amy Elliott, Chris Reissaus, Troy Hutchens, and Zeno Lavagnino who provided helpful feedback and many laughs during my time in the Piston Lab. I am blessed with four parents who have supported me on this journey. Most importantly though, I must thank my mother who has always encouraged education, not just by words, but also by example. One of my earliest memories is dragging my children’s books over to the dining table where I watched her study so that I could be like her. Mom, without your influence, I would most certainly would not be where I am today. Thank you. Last, I want to thank my husband who has given me infinite support and encouragement on this journey. There are no words to describe how thankful I am for ii your support as we lived apart so that I could achieve my goals. This accomplishment would not have been possible without you. Thank you. iii Table of Contents LIST OF FIGURES ....................................................................................................... vii LIST OF TABLES........................................................................................................... ix Chapter 1. PHYSIOLOGY INTRODUCTION ............................................................................... 1 Introductory Comments ........................................................................................... 1 Physiology of Endocrine Pancreas .......................................................................... 2 Pancreas Physiology ........................................................................................ 2 Islets of Langerhans ......................................................................................... 3 Blood Glucose Regulation ................................................................................ 6 Diabetes Mellitus ..................................................................................................... 8 Type 1 .............................................................................................................. 8 Type 2 .............................................................................................................. 8 G Protein Coupled Receptor Regulation of Insulin ................................................ 10 Introduction ..................................................................................................... 10 G Protein-Coupled Receptor Structure ........................................................... 11 Heterotrimeric G-proteins ............................................................................... 12 GPCR in Islets (β-cell) .................................................................................... 14 Dopamine .............................................................................................................. 14 Introduction ..................................................................................................... 14 Dopamine Receptors ...................................................................................... 16 Dopamine Mediated Negative Feedback Pathway ................................................ 18 Glucose Stimulated Insulin Secretion ............................................................. 18 Dopamine in Islets .......................................................................................... 19 Proposed Dopamine Mediated Negative Feedback Pathway.......................... 21 2. FLUORESCENCE FLUCTUATION SPECTROSCOPY ........................................... 23 Tools for Understanding Protein–protein Interactions ............................................ 23 Historical Background ........................................................................................... 25 FFS Theory ........................................................................................................... 29 FCS Theory .................................................................................................... 31 PCH Theory .................................................................................................... 35 FFS Measurements ............................................................................................... 37 Instrumentation ............................................................................................... 37 Single Component Measurements .................................................................. 40 iv Two-Color Cross-Correlation Measurements .................................................. 43 Photon Counting Histogram .................................................................................. 44 Single Component PCH .................................................................................. 44 Two Component PCH ..................................................................................... 46 3. METHODOLOGY ..................................................................................................... 49 Introduction ........................................................................................................... 49 Cell Preparation .................................................................................................... 50 MIN6 Cell Line ................................................................................................ 50 Cell Media and Culturing ................................................................................ 51 Cell Transfection Background ......................................................................... 51 Cell Electroporation Transfection .................................................................... 53 Cell Imaging Buffer ......................................................................................... 54 Instrumentation and Calibration ............................................................................. 54 General Overview ........................................................................................... 54 Alignment ....................................................................................................... 56 Data Acquisition .............................................................................................. 56 Data Analysis ........................................................................................................ 58 Constructs ............................................................................................................. 61 EGFP and mApple Bacterial Expression Plasmids ......................................... 62 EGFP and mApple Mammalian Expression Plasmids ..................................... 62 EGFP DRD3 ................................................................................................... 63 EGFP DRD2 ................................................................................................... 63 mApple Gγ2 Subunit ........................................................................................ 63 mApple Linked to EGFP construct .................................................................. 64 mCerulean Gβ1 Subunit................................................................................... 65 EGFP G-protein Inwardly Rectifying Potassium Channel 3 ............................. 65 mApple G-protein Inwardly Rectifying Potassium Channel 3 .......................... 65 Protein Characterization ........................................................................................ 66 Summary ............................................................................................................... 69 4. DOPAMINE RECEPTOR SIGNALING THROUGH Gβγ COMPLEX.......................... 70 Introduction ........................................................................................................... 70 Materials and Methods .......................................................................................... 71 Constructs and Cells....................................................................................... 71 Instrumentation ............................................................................................... 72 v Theory and Data Analysis ............................................................................... 72 Two-color FFS of Separate and Linked Fluorescent Proteins ...............................