Development and Characterization of Novel Bioluminescent Reporters of Cellular Activity By Derrick C. Cumberbatch 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 Biological Sciences May 10, 2019 Nashville, Tennessee Approved: C. David Weaver, Ph.D. Douglas McMahon, Ph.D. Qi Zhang, Ph.D. Carl Johnson, Ph.D. To my beloved and supportive wife Alicia, and to my parents Cameron and Marcia Cumberbatch. ii ACKNOWLEDGEMENTS This work was made possible by financial support from the NIMH grants MH107713 and MH116150 awarded to Carl Johnson, Ph.D. as well as funds provided by the Vanderbilt University Dissertation Enhancement Grant, graciously awarded to me by the Graduate School. I appreciate Dr. Carl Johnson for taking me into his lab and providing me with ample tools that aided in the successful completion of my Ph.D. I would like to express gratitude to my committee members Drs. David Weaver, Douglas McMahon, Qi Zhang, and the late Dr. Donna Webb for guiding me through the process of becoming a competent researcher. I would also like to make special mention of Jie Yang, Ph.D. whose persistent efforts and sage advice were an ever-present help during my graduate studies. His one-on-one training provided me with many skills that will serve me well as a molecular biologist. Meaningful contributions from the other current and past members of the Johnson lab group, Yao Xu, Ph.D., Tetsuya Mori, Ph.D., Shuqun Shi, Ph.D., Chi Zhao, Ph.D., Peijun Ma, Ph.D., He Huang, Ph.D., Kathryn Campbell, Briana Wyzinski, Kevin Kelly, Maria Luisa Jabbur, Carla O’Neale and Ian Dew deserve to be highlighted here as well. I am thankful for the many professors, fellow graduate students and undergraduate students who have given me helpful insights and suggestions on my Ph.D. journey, making special mention of Hu Lan, Ph.D., Jeff Jones Ph.D., Siwei He, Ph.D., Xiaohan Wang, Ph.D., Sam Centanni, Ph.D., Kim Kwangho, Ph.D., Gary Sulikowski, Ph.D., Paige Vinson, Ph.D., and Emily Days. iii TABLE OF CONTENTS Page DEDICATION ................................................................................................................................ ii ACKNOWLEDGEMENTS ........................................................................................................... iii LIST OF FIGURES ...................................................................................................................... vii LIST OF SUPPLEMENTARY FIGURES .................................................................................... ix LIST OF ABBREVIATIONS ........................................................................................................ xi Chapter I. GENERAL INTRODUCTION ...................................................................................................1 Bioluminescence .........................................................................................................................1 Illuminating the cell through microscopy ...................................................................................3 Contributions of the humble jellyfish Aequorea victoria ............................................................3 First glimpses of intracellular Ca2+ neural activity using Aequorin bioluminescence ................5 The rise of GFP and fluorescence-based imaging .......................................................................7 Drawbacks and limitations of optical methods .........................................................................10 Optogenetics ..............................................................................................................................11 The fluorescence-optogenetics disadvantage and the bioluminescence advantage ..................14 Bioluminescence reporters continue to shine ............................................................................14 Improvements in bioluminescence imaging ..............................................................................17 Disparity of sensor abundance between fluorescence and bioluminescence-based reporters of cellular activity ......................................................................................................19 Return of bioluminescence imaging to circumvent the drawbacks of fluorescence imaging ......................................................................................................................................21 II. DEVELOPMENT OF CALFLUXVTN, A NOVEL RATIOMETRIC, BIOLUMINESCENCE CALCIUM SENSOR FOR LIVE-CELL IMAGING .............................23 Abstract .....................................................................................................................................23 Introduction ...............................................................................................................................23 Materials and Methods ..............................................................................................................26 Construction of Plasmids ........................................................................................................26 Protein Purification and in vitro experiments .........................................................................27 Cellular expression of Ca2+-sensors and recording ................................................................28 iv Compatibility with optogenetic rhodopsins............................................................................30 Hippocampal viral injection and brain slice preparation ........................................................30 Flow-through microscopic imaging experiments of acute brain slices ..................................31 Microscopic imaging and data analysis ..................................................................................32 Results .......................................................................................................................................35 In vitro characterization of BRET Ca2+ sensor .......................................................................35 BRET Ca2+ sensor expressed in cell cultures .........................................................................39 CalfluxVTN is an optimal optogenetic partner with melanopsin ...........................................41 Calflux reports Ca2+ flux in hippocampal neurons and slices ................................................43 Neuronal responses to optogenetically induced depolarization .............................................47 Discussion .................................................................................................................................49 III. NOVEL BIOLUMINESCENT CALCIUM ION REPORTERS ALLOW FOR GREATER UTILIZATION OF SMALL MOLECULE LIBRARIES IN HIGH THROUGHPUT SCREENS ..........................................................................................................60 Abstract ....................................................................................................................................60 Introduction ..............................................................................................................................60 Materials and Methods ..............................................................................................................63 DNA plasmid construction .....................................................................................................63 In vitro Ca2+ assays .................................................................................................................64 Mammalian cell expression and selection of stably-expressing cell lines ............................64 Optical data acquisition for in vivo experiments ....................................................................65 Coelenterazine analogs ...........................................................................................................66 Carbachol checkerboard .........................................................................................................66 BRET ratio measurements under the influence of fluorescein ...............................................67 Carbachol concentration response curves (CRCs) .................................................................67 Scopolamine concentration response curves (CRCs) .............................................................67 Impact of fluorescein on recordings of antagonist activity ....................................................68 Data analysis ...........................................................................................................................68 Results .......................................................................................................................................69 In vitro characterization of CalfluxCTN ................................................................................69 Effects of different luciferase substrates in cells ....................................................................72 Application of Calflux for HTS ..............................................................................................75 Fidelity of BRET signals in highly fluorescent backgrounds ................................................77 BRET sensor reveals antagonist action undetected by fluorescent sensors ...........................79 Discussion .................................................................................................................................81