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Resolving Membrane Receptor Multimerization in Live Cells

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Resolving Membrane Receptor Multimerization in Live Cells RESOLVING MEMBRANE RECEPTOR MULTIMERIZATION IN LIVE CELLS USING TIME RESOLVED FLUORESCENCE METHODS A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Megan J. Kaliszewski Klufas December, 2017 RESOLVING MEMBRANE RECEPTOR MULTIMERIZATION IN LIVE CELLS USING TIME RESOLVED FLUORESCENCE METHODS Megan J. Kaliszewski Klufas Dissertation Approved: Accepted: _____________________________ _____________________________ Advisor Department Interim Chair Dr. Adam W. Smith Dr. Christopher J. Ziegler _____________________________ _____________________________ Committee Member Dean of the College Dr. Chrys Wesdemiotis Dr. John C. Green _____________________________ _____________________________ Committee Member Interim Dean of the Graduate School Dr. David S. Perry Dr. Chand K. Midha _____________________________ _____________________________ Committee Member Date Dr. Michael Konopka ______________________________ Committee Member Dr. Mesfin Tsige ii ABSTRACT The cell membrane is a complex environment made up of thousands of molecular components. The dynamic assembly of these components regulates a myriad of cellular functions, but it is difficult to measure in a biologically relevant context. Pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) is a time resolved fluorescence technique that was used to obtain concentration, mobility and co-diffusion (fc) of membrane proteins in live cells. Several lines of evidence support the hypothesis that homo-dimerization (or even oligomerization) facilitates the function of membrane proteins. The goal of this research was to elucidate the dynamic organization and relative affinity of membrane protein-protein interactions. In order to accomplish this goal, a mathematical model was developed to interpret the cross-correlation value obtained from PIE-FCCS and to quantify the dynamic interactions of membrane receptors in a more rigorous way. This thesis describes three main projects. The first project focused on determining the homo-dimerization of the neuronal membrane protein PlexinA4 before and after ligand stimulation. PIE-FCCS measurements of protein controls with varying degrees of homo-multimerization were used to determine that PlexinA4 receptors assemble into preformed dimers. In the next project, a mathematical model was developed to interpret the PIE-FCCS figure of merit, fc. Several controls systems with varying dimerization iii affinity and degrees of oligomerization were measured and analyzed to verify the accuracy of the model. Lastly, the organization of two class A G protein-coupled receptors (GPCRs) was investigated using PIE-FCCS and a new labeling strategy. The organization of these membrane proteins showed a high degree of cell to cell variability. A simple monomer-dimer equilibrium model failed to describe the range of single cell data, so the mathematical model was altered to describe a cluster model that agreed with the experimental data. This thesis lays the groundwork for application of the mathematical model to quantify the organization of other membrane receptors using PIE-FCCS. iv DEDICATION This work is dedicated to my brother, Michael, whose life was cut short during the completion of this degree. His memory gave me the strength to persevere. Michael, I wish you were here to see this and I miss you. v ACKNOWLEDGEMENTS First, I want to thank my research advisor, Dr. Adam W. Smith, for challenging me to grow outside of my comfort zone and for his continued faith in my abilities. I would like to acknowledge all those who have aided me in my research, specifically my fellow group members: Xiaojun “Roger” Shi, Xiaosi Li, William D. Comar, Shaun Christie, D. Paul Mallory, and So Yeon “Stephanie” Kim. Our group has been very fortune to have a group of very dedicated undergraduate researchers. I want to acknowledge specifically Anthony S. Esway, Margaret Pinkevitch, and Laura Smith who assisted in this research. I want to thank all my friends at Akron who have given me console and much needed relief during this academic adventure: Jeffrey McCausland, Carolyn Scherger, Mena Klittich, Alex Taraboletti, Abegel Freedman, and all the graduate students in my cohort. My family has been instrumental in encouraging me to persevere. I want to thank my husband Dan who has supported me throughout my graduate career. Thank you to my parents who have always believed that I can do anything I put my mind to do. I would also like to express my gratitude to my aunt and uncle with whom I had many meaningful conversations that kept me centered. I have been so blessed to have the support of my entire family (those still living and those lost during these past five years), who I am lucky to have close by whenever I need them. vi TABLE OF CONTENTS Page ABSTRACT ....................................................................................................................... iii DEDICATION .................................................................................................................... v ACKNOWLEDGEMENTS ............................................................................................... vi TABLE OF CONTENTS .................................................................................................. vii LIST OF TABLES .............................................................................................................. x LIST OF FIGURES ........................................................................................................... xi CHAPTER I RESEARCH INTRODUCTION ...................................................................................... 1 II MATERIALS AND METHODS .................................................................................... 5 2.1 Fluorescence Fluctuations Spectroscopy ............................................................. 5 2.2 Instrumental Setup.............................................................................................. 15 2.2.1 Optical Path ................................................................................................. 18 2.2.2 Procedure for Data Acquisition .................................................................. 21 2.2.3 Procedure for Data Analysis ....................................................................... 22 2.3 Snap Tag Labeling ............................................................................................. 24 2.3.1 Materials Needed ........................................................................................ 24 2.3.2 Chinese Hamster Ovary Culture ................................................................. 25 2.3.3 Labeling Protocol ........................................................................................ 26 vii III CLASS A PLEXINS ARE ORGANIZED AS PREFORMED INACTIVE DIMERS ON THE CELL SURFACE .............................................................................................. 28 3.1 Preface ................................................................................................................ 28 3.2 Introduction ........................................................................................................ 29 3.3 Materials and Methods ....................................................................................... 32 3.3.1 Cloning ........................................................................................................ 32 3.3.2 PIE-FCCS instrument ................................................................................. 33 3.3.3 PIE-FCCS data collection ........................................................................... 34 3.4 Results and Discussion ....................................................................................... 39 3.5 Conclusions ........................................................................................................ 59 3.6 Postface .............................................................................................................. 60 IV FLUORESCENCE CROSS-CORREALTION SPECTROSCOPY FOR THE STUDY OF MEMBRANE PROTEIN OLIGOMERS ................................................................... 62 4.1 Introduction ........................................................................................................ 62 4.2 Material and Methods......................................................................................... 65 4.2.1 PIE-FCCS Instrument ................................................................................. 65 4.2.2 Data Collection and Analysis...................................................................... 68 4.2.3 Cell Culture and Transient Transfection ..................................................... 69 4.3 Data Analysis ..................................................................................................... 70 4.4 Theoretical Framework ...................................................................................... 75 4.4.1 Combinatorial Dilution of fc with two Fluorescent Tags ............................ 75 4.4.2 Inclusion of Non-Fluorescent Protein Fusions ........................................... 79 4.4.3 Incomplete Dimerization ............................................................................ 80 4.5 Experimental Comparisons ................................................................................ 85 4.5.1 Experimental PIE-FCCS ............................................................................
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