i PFF1320C an Essential but Presumptive Plasmodium Myosin Light Chain By Melody Robinson Wright March 2017 A Dissertation Presented to the Faculty of Drexel University College of Medicine In partial fulfillment of the Requirements for the Degree of Doctor of Philosophy ii DEDICATION This dissertation is dedicated to my grandmother, Dorothy Ann Gilmore Robinson and to my parents Colonel Ronald Robinson and Reverend Mary C. Sloan. iii ACKNOWLEDGEMENTS The completion of this dissertation would not have been possible without the unwavering support of my family, friends, and many within the Drexel Community. Throughout this scientific journey I have been blessed with many amazing mentors. I would like to thank Dr. Lawrence W. Bergman for allowing me the pleasure to learn and grow in his lab over the past seven years. His strong passion for science and overall enthusiasm for life created the perfect lab environment for which I was able to discover confidence in myself as an independent scientist. His support for my project has always been steadfast even at times when I begin to question it all. For this I am truly grateful. To the remaining members of my dissertation committee Dr. James Burns, Dr. Theodore Taraschi, Dr. Anand Mehta, and Dr. Patrick Loll I appreciate all your support, helpful suggestions, advice, and constant motivation through the more troubling times of this endeavor. I would especially like to thank Dr. Burns his help with troubleshooting experiments. More importantly I would like to thank him to taking the time out to meaningfully ask me how I was doing. Simple gestures such as this can go a long way in the life of graduate student. To great lab environment is only as great as the people who are in it. To Tom Daly, words could never express my gratitude for everything you’ve taught iv me as well as your contribution to my research. You taught me how to look at things differently and appreciate the beauty in the techniques in ways that I will never forget. To Dr. Bethany Jenkins, thank you for helping me transition into the lab, for being an amazing lab mate, and more importantly a great friend. It was a pleasure to grow into a scientist along side you. Thank you to Aaron Tocker for his assistance with different aspects of the project. I would like to also thank Dr. Sumit Kumar for helping me in the early days of starting my project. I would like to thank the Vaidya lab for all their collaborations and support throughout the years. I would like to give a special thanks to Joann Morrisey, for her wisdom and helpful advice when it came to culturing parasites. I would like to thank all colleagues within the Micro/Immuno department along with my batch mates for making my time here at Drexel memorable, all the collaborative talk and friendship. Thank you to Andrea Partridge, Dr. Ming Yang, Dr. April Pershing, Liz Parzych, Sezin Patel, Lindsay Kleinwaks, and Jacqueline Schneider. To my parents, sisters, grandmothers, and extended family. Thank you so much for your enormous support and for being my biggest cheerleaders. I could not have made it to the end without you. To my husband Marcus Wright, thank you for being a solid rock while these crazy winds were blowing. To my dogs Jada and Jujubee, thank you for always giving me a reason to smile whenever I came home. I love you all! v Table of Contents Chapter 1 .................................................................................................................... 1 Introduction ................................................................................................................ 1 History Of Myosins ..................................................................................................................... 6 Actin And Unique Actin Binding Proteins ................................................................................. 10 Apicomplexan Myosins ............................................................................................................ 12 History Of Myosin Light Chains ................................................................................................ 20 Conditional Knockdown Strategies ........................................................................................... 34 Proximity Dependent Labeling ................................................................................................. 38 Thesis Aims...................................................................................................................39 CHAPTER 2 ................................................................................................................ 41 MATERIALS AND METHODS ....................................................................................... 41 Plasmodium Cell Culture And Transfection .............................................................................. 42 Western blot ............................................................................................................................. 42 Microscopy ............................................................................................................................... 43 Generation of parasite expression vectors .............................................................................. 43 Genomic DNA Isolation From Parasite Pellet ........................................................................... 46 vi Growth Assay For Flow Cytometry ........................................................................................... 47 Isolation Of Biotinylated Proteins ............................................................................................ 47 ATc Treatment .......................................................................................................................... 48 Mass Spectrometry .................................................................................................................. 48 CHAPTER 3 ................................................................................................................ 51 RESULTS .................................................................................................................... 51 Potential Essential Nature Of PFF1320c ................................................................................... 52 Generation Of Epitope Tagged Transgenic PFF1320c Parasites And Expression In The Blood Stages. ...................................................................................................................................... 52 Cellular Distribution Of PFF1320c ............................................................................................ 57 PFF1320c Co-Localize With PfMyoC In a Inconsistent Manner. ............................................... 58 Knockdown Of PFF1320c Does Not Show An Effect On Growth Of The Parasite. .................... 59 The TetR-Dozi Aptamer System Enhances Regulation of PFF1320C. ....................................... 63 Biotinylation In 1320-BirA* Parasites Is Distinct From Cytoplasmic BirA* And Localizes To The Cytoplasm. ................................................................................................................................ 69 The Identification Of Presumed Myosin Complex Proteins Through Mass Spectrometry of MyoB-BirA* and MTIPB-BirA* Pulldowns. ............................................................................... 75 CHAPTER 4 ................................................................................................................ 97 DICUSSION/CONCLUSION .......................................................................................... 97 REFERENCES ............................................................................................................111 APPENDENCIES.........................................................................................................120 vii LIST OF TABLES Table 1.1 Apicomplexan myosins naming convention................................................14 Table 1.2 Grouping of Apicomplexan myosin sequences............................................19 Table 3.1 Proteins identified by mass spectrometry in 1320-BirA* samples..............85 Table 3.2 PfMyoC proteins found in biotinylation.......................................................88 Table 3.3 Proteins found similar in data sets from 1320-BirA*-Aptamer....................90 Table 3.4 PfMyoB and PfMTIPB associated proteins identified by mass spectrometry...............................................................................................94 viii LIST OF FIGURES Figure 1.1 The Plasmodium Life Cycle.............................................................................4 Figure 1.2 Myosin Class XIV Phylogeny ........................................................................17 Figure 1.3 Repertoire of myosin light chain-like proteins in Apicomplexa....................31 Figure 1.4 Amino Acid Sequence Alignment of Plasmodium Myosin Light Chains (EF- Hands)............................................................................................................32 Figure 1.5 PFF1320c conserved only in Primate Plasmodia..........................................33 Figure 3.1 Generation of transgenic PFF1320c using the PLN System..........................53 Figure 3.2 Succesfful expression of tagged PFF1320c in blood stage parasite ............. 54 Figure 3.3 PFF1320c is essential for blood stage growth. .............................................. 55 Figure 3.4 PFF1320c is expressed during