SINGLE QUANTUM DOT APPROACH FOR MOLECULAR DISSECTION OF SEROTONIN TRANSPORTER REGULATION IN LIVING CELLS By CHIA HUA CHANG 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 Chemistry May, 2013 Nashville, Tennessee Approved: Professor Sandra J. Rosenthal Professor David W. Wright Professor Ned A. Porter Professor Randy D. Blakely Copyright © 2013 by Chia Hua Chang All Rights Reserved ii Dedicated to my beloved grandfather. May you rest in peace in heaven. iii ACKNOWLEDGEMENTS Thank you to my family for your love and support throughout my life. Thank you to my thesis advisor, Sandy, for your encouragement and investment in my research and my future as a scientist. Thank you to my thesis co-advisor, Randy, for bringing me to the field of neuroscience and consistently pushing me to become a better scientist. Thank you to Dr. Louis De Felice, Dr. Dave Piston, Dr. Sam Wells, Dr. David Wright, and Dr. Ned Porter for your advice and input at numerous points along the way. A good support group is important to surviving in graduate school. I am very fortunate to have been surrounded by sub-group members we like to call "bio-side" and refer to Michael (Warnement), Oleg, Emily and myself. I would have been lost in graduate school without your countless discussions, support, and companionship. Best wishes to you, Oleg, as you finish up. Thank you to other present and past members of the Rosenthal group, including Amy, James, Noah, Sarah, Scott, Joe, Toshia, Melissa, Danielle, Albert, Michael (Schreuder), Michael (Bowers), Ian, Shawn, Teresa, Jessica, Kevin, and Nat. The past seven years have not been an easy ride for me, both academically and personally. Each and everyone of you have supported and/or helped me in many ways. I am very fortunate to join such a loving and down to earth group. Additionally, I also like to thank the members of the Piston and Blakely group, in particular Alessandro Ustione and Dr. Hideki Iwamoto, for helping important part of my experiments. Finally, I would like to thank the Department of Chemistry at the Vanderbilt University, the Vanderbilt Institute of Nanoscale Science and Engineering (VINSE), and the National Institutes of Health (NIH) for funding. iv ABSTRACT The presynaptic serotonin (5-HT) transporter (SERT) is targeted by widely prescribed antidepressant medications. Altered SERT expression or regulation has been implicated in multiple neuropsychiatric disorders, including anxiety, depression and autism. It has been previous reported that SERT is regulated by lipid raft, a cholesterol- enriched subdomain  in the plasma membrane that has been frequently reported a platform to facilitate neuronal signaling. To better understand the membrane diffusion dynamics of SERT, I developed a single quantum dot (QDot) tracking approach that exploits antagonist-conjugated single QDots to monitor, for the first time, single SERT proteins on the surface of serotonergic cells. We document two pools of SERT proteins defined by lateral mobility, one that exhibits relatively free diffusion, and a second, localized to cholesterol and GM1 ganglioside-enriched microdomains, that displays restricted mobility. Receptor-linked signaling pathways that enhance SERT activity mobilize transporters that, nonetheless, remain confined to membrane microdomains. Mobilization of transporters arises from a p38 MAPK-dependent untethering of the SERT C terminus from the juxtamembrane actin cytoskeleton. Our studies establish the utility of single QDot tracking approach for analysis of the behavior of single membrane proteins and reveal a physical basis for signaling-mediated SERT regulation. In line with the single QDot-SERT analysis, single QDot-labeled ganglioside GM1 was incorporated in this dissertation that aimed to quantitatively measure the diffusion dynamics and membrane compartmentalization of lipid raft in living RN46A cells. Diffusion measurements revealed that single QDot-labeled GM1 ganglioside complexes –2 2 undergo slow, confined lateral diffusion with a diffusion coefficient of 7.87 × 10 μm /s v and a confinement domain about 200 nm in size. Further analysis of their trajectories showed lateral confinement persisting on the order of tens of seconds, comparable to the time scales of the majority of cellular signaling and biological reactions. Hence, our results provide further evidence in support of the putative function of lipid rafts as signaling platforms. Finally, I discussed the recent progress of single-QDot techniques, with emphasis on their applications in exploring membrane dynamics and intracellular trafficking. In recent years, single QDot imaging approach has been introduced as a sub- category of single molecule fluorescent techniques for revealing the single protein/vehicle dynamics in real-time. One of the major advantages of using single QDots is the high signal-to-noise ratio, which is beneficial due to the unique photophysical properties of QDot such as extraordinarily high molar extinction coefficients and large Stokes shifts. Although there are some limitations due to the physical nature of the QDots, advances in QDot synthesis and surface chemistry show significant potential to eliminate these pitfalls. Considering the applications of a single QDot approach in the past few years, I am optimistic that the use of single QDots in bioimaging will largely advance our understanding in the biological research field in the near future. vi TABLE OF CONTENTS Page DEDICATION ................................................................................................................. iii ACKNOWLEDGEMENTS .............................................................................................. iv ABSTRACT ……………………………………………………………………………………. v LIST OF TABLES ............................................................................................................ xi LIST OF FIGURES ......................................................................................................... xii Chapter 1 SINGLE-QUANTUM DOT IMAGING FOR MOLECULAR NEUROSCIENCE ….. 2 1.1 Introduction ............................................................................................................ 2 1.2 Water-Soluble Quantum Dot for Biological Labeling ............................................. 4 1.3 Overview of Single-Quantum Dot tracking ............................................................ 9 1.4 Recent Advances of Single-Quantum Dot Tracking in Neuroscience Research .. 13 1.5 Summary ............................................................................................................... 15 1.6 Reference .............................................................................................................. 17 2 SINGLE QUANTUM DOT IMAGING TECHNIQUES (METHODS) ...................... 22 2.1 Microscopy Setup for QDot-Based Single-Molecule Observation ........................ 22 2.2 Imaging Data Processing and Subpixel Localization ........................................... 25 2.3 Diffusion Theory and Calculation ......................................................................... 28 2.4 Imaging System Calibration Using Spin-Cast Single Quantum Dots ................... 32 2.5 Single Quantum Dot Labeling in Living Cells ....................................................... 34 2.6 Tracking Programs for Single-Molecule/Quantum Dot Analysis .......................... 36 2.7 References ........................................................................................................... 42 vii 3 PROBING MEMBRANE DYNAMICS OF LIPID RAFTS WITH SINGLE-QUANTUM DOT IMAGING ..................................................................................................... 45 3.1 Introduction .......................................................................................................... 45 3.2 Material and Methods .......................................................................................... 46 3.2.1 Single QDot Labeling of Ganglioside GM1 in RN46A cells .................................. 46 3.2.2 Microscopy ........................................................................................................... 46 3.2.3 Subpixel Localization and Trajectory Generation ................................................. 47 3.2.4 Instantaneous Velocity ......................................................................................... 48 3.2.5 Diffusion Coefficients and Membrane Confinement ............................................. 48 3.3 Results .................................................................................................................. 50 3.3.1 Instrument response and single-molecule sensitivity inspection .......................... 50 3.3.2 Basal membrane diffusion in living RN46A Cells ................................................. 52 3.3.3 Single quantum dot tracking of lipid raft constituent ganglioside GM1 ................. 54 3.3 Summary .............................................................................................................. 59 3.4 References ........................................................................................................... 62 4 SINGLE MOLECULE ANALYSIS OF SEROTONIN TRANSPORTER REGULATION USING ANTAGONIST-CONJUGATED QUANTUM DOTS …….. 65 4.1 Introduction ........................................................................................................... 65 4.2 Materials and Methods ......................................................................................... 67 4.2.1 Cell Culture, Treatments, and SERT Activity Assay ............................................. 67 4.2.2 Labeling RN46A Cells with Ligand-Conjugated Quantum Dots ........................... 68 4.2.3 Microscopy ........................................................................................................... 69 4.2.4 Data Analysis of Single Quantum Dot Imaging .................................................... 69 4.3 Results ................................................................................................................. 70 4.3.1 Single Molecule Analysis of QDot-labeled SERT Reveals a Membrane Microdomain-Associated Subpopulation of Transporters With Confined Diffusion 70 viii 4.3.2 Single QDot-Labeled SERT Proteins Demonstrate Increased SERT Lateral Mobility, Despite a Confined Distribution, after 8-Br-cGMP Treatment …………. 78 4.3.3 The p38 MAPK Inhibitor SB203580 Attenuates 8-Br-cGMP Induced Enhancements in SERT Lateral Mobility ...................................................................................... 80 4.3.4 IL-1β Activated Single SERT Proteins Reveal p38 MAPK-Dependent Subpopulation ...................................................................................................... 81 4.3.5 Cytoskeletal Disruption Mobilizes SERT Molecules That Remain Confined to Membrane Microdomains .................................................................................... 84 4.4 Discussion ........................................................................................................... 89 4.5 References ......................................................................................................... 94 5 SUMMARY AND FUTURE PERSPECTIVE ....................................................... 98 5.1 Introduction ........................................................................................................ 98 5.2 Mapping Receptor Membrane Diffusion ............................................................ 102 5.3 Endosomal Trafficking and Endocytosis ............................................................ 105 5.4 Dynamic Processes of Intracellular Targets ....................................................... 107 5.5 Single-Quantum Dot FRET ................................................................................ 110 5.6 3-D Single Quantum Dot Tracking ..................................................................... 112 5.7 Conclusion and Future Perspective ................................................................... 114 5.8 References ......................................................................................................... 116 Appendix A QUANTUM DOT DISPLACEMENT ASSAY FOR ANTIDEPRESSANT DRUG SCREENING ...................................................................................................... 120 A.1 Abstract .............................................................................................................. 120 A.2 Introduction ........................................................................................................ 121 A.3 Experimental Section ......................................................................................... 123 A.3.1 IDT318 ligand synthesis .................................................................................... 123 ix A.3.2 hSERT-expressing oocyte prepapation ............................................................. 123 A.3.3 Quantum dot-hSERT labeling and displacement in oocytes .............................. 124 A.3.4 Microscopy ......................................................................................................... 125 A.3.5 Two-electrode voltage-clamp electrophysiological analysis .............................. 126 A.3.6 Data Analysis ..................................................................................................... 126 A.4 Results ............................................................................................................... 129 A.5 Discussion ......................................................................................................... 137 A.6 References ........................................................................................................ 138 x