This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Electron tomography of muscle cross‑ bridge by regulatory light chain labelling with APEX2 Mufeeda, Changaramvally Madathummal 2019 Mufeeda, C. M. (2019). Electron tomography of muscle cross‑ bridge by regulatory light chain labelling with APEX2. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/85724 https://doi.org/10.32657/10356/85724 Downloaded on 09 Oct 2021 08:25:28 SGT ELECTRON TOMOGRAPHY OF MUSCLE CROSS- BRIDGE BY REGULATORY LIGHT CHAIN LABELLING WITH APEX2 MUFEEDA CHANGARAMVALLY MADATHUMMAL SCHOOL OF BIOLOGICAL SCIENCES 2019 ELECTRON TOMOGRAPHY OF MUSCLE CROSS- BRIDGE BY REGULATORY LIGHT CHAIN LABELLING WITH APEX2 MUFEEDA CHANGARAMVALLY MADATHUMMAL SCHOOL OF BIOLOGICAL SCIENCES A thesis submitted to the Nanyang Technological University in partial fulfilment of the requirement for the degree of Doctor of Philosophy 2019 2 4 ACKNOWLEDGMENTS First and foremost, I wish to express my sincere gratitude to my advisor, Professor Michael Alan Ferenczi for his expert guidance and warm encouragement. I have always adored his passion for science and expert knowledge about the Muscle biology field. Without Mike extensive knowledge in muscle biology, I could not have learned as much as I have. I wish to express my sincere thanks to my mentor Assistant Professor Alexander Ludwig for his mentoring, encouragement, support and guidance. I am deeply grateful to him for the long discussions that helped me sort out the technical details of my work. My sincere thanks for his great help, guidance throughout my work and for giving me a wonderful opportunity. This thesis has been possible only because of Mike and Alex help, constant support and patience. I feel fortunate to be a part of their lab and shall remain ever grateful to them for providing a conducive environment to grow as a student of science. I would like to extend my gratitude to Assistant Professor Sara Sandin for agreeing to be my co-supervisor and letting me use her laboratory and research facility for my experiments. Special thanks to Meiling Wee, Pei yin Tan and Kayen Low (FEI) for their practical advice in ultramicrotomy and TEM. I am very thankful to each and every present and past member of Sara lab for all the help and support. I wish to express my thanks to my fellow lab mates Dr. Haiyang Yu, Dr. Song Weihua and for technical advice in muscle dissection. My appreciation goes to all other Mike’s lab members for providing me with a pleasant work environment. I will be always grateful to Ben and Kasturi for helping me and spending their time for helping me finish this thesis. Special thanks to Malini and Archita for their help during thesis writing. Special thanks to my besties Remya and Divya for their cheering conversation during thesis writing. Sincere thanks to Rahul and Rebu for their support and standing with me as best friends. Special thanks to Remya and Parjanya for their all help. They stand as a family through their love and I am thankful for all the happy times that we have shared and those are yet to come. i Most importantly thanks for my mother and father for all their support. Unless they believed me I would not have reached this position. Thanks for my sisters for their love, patience, understanding and endless support. Special thanks to my Koya uncle for guiding me and advising me to take Biotechnology field. I think he might be looking at me from another world and being proud of me. Last but not the least; I would like to thanks my love Nitin for his love, care and support throughout my life. He was always there cheering me up and stood by me through the good times and bad. Without him, I would not have achieved this PhD. I am truly thankful for having you in my life. I wish to take this opportunity to express my most sincere gratitude to all my teachers and people who guided me all through my life to pursue this dream. Special thanks to Dr. Anil Kumar Gopala for all his scientific teaching and guidance to pursue my PhD. I gratefully acknowledge the financial support rendered by the Nanyang Technological University of Singapore and LKC school of Medicine Singapore. I would like to thank my thesis committee: Asst. Prof. Dr. Zhang Li-feng and Asst. Prof. Wang Xiaomeng for their insightful comments and encouragement during our meetings. ii TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................... i TABLE OF CONTENTS .................................................................................................... iii LIST OF FIGURES ........................................................................................................... vii LIST OF TABLES .............................................................................................................. ix ABBREVIATIONS ............................................................................................................. x ABSTRACT ...................................................................................................................... xiii 1. INTRODUCTION .................................................................................................. 1 1.1. Skeletal muscle structure ........................................................................................ 3 1.2. Sarcomere structure and organisation ..................................................................... 6 1.3. Sarcomeric proteins ................................................................................................ 9 1.3.1. Thin filament ....................................................................................................... 9 1.3.1.1. Actin ............................................................................................. 9 1.3.1.2. Troponin and tropomyosin ......................................................... 10 1.3.2. Thick filament ................................................................................................... 12 1.3.2.1. Myosin light chains .................................................................... 16 1.3.3. Other filament proteins...................................................................................... 18 1.4. Regulation of muscle contraction ......................................................................... 19 1.5. Historical perspectives on muscle contraction mechanism studies. ..................... 20 1.5.1. Biochemical studies for establishing actin-myosin interaction ......................... 21 1.5.1.1. Swinging cross-bridge cycle ...................................................... 21 1.5.2. Early structural studies ...................................................................................... 25 1.5.2.1. Sliding filament theory ............................................................... 25 1.5.3. Impact of high-resolution EM and X-ray studies on building the structural model for muscle contraction ............................................................................ 27 1.5.3.1. Swinging lever arm hypothesis .................................................. 29 1.5.4. Spectroscopical structural studies ..................................................................... 32 1.6. Novel labelling techniques for Transmission electron microscopy (TEM) and electron tomography (ET) ..................................................................................... 32 1.6.1. Electron tomography ......................................................................................... 33 1.6.2. APEX2, a protein tag to visualise proteins by EM ........................................... 36 1.7. Aim of the thesis and significance of RLC labelled EM structure ....................... 38 1.8. Research gaps and hypotheses .............................................................................. 39 1.9. Myosin Regulatory light chain exchange process ................................................ 41 2. MATERIALS AND METHODS ........................................................................ 42 iii 2.1. Materials… ........................................................................................................... 43 2.2. Methods…............................................................................................................. 46 2.2.1. Molecular biology methods ............................................................................... 46 2.2.1.1. DNA analytical methods ............................................................ 46 2.2.1.2. Competent E. coli cells for the transformation of pET-3D-RLC- APEX2 and pET-3D-APEX2 plasmids ................................................................. 46 2.2.1.3. E. coli Transformation for introducing recombinant plasmid into bacteria………… ................................................................................................... 47 2.2.1.4. Plasmid and oligonucleotides to generate recombinant vector: pET-3D-RLC-APEX2 and pET-3D-APEX2 ......................................................... 48 2.2.1.5. Polymerase chain reaction (PCR) for amplification of RLC and APEX2 cDNAs……… .......................................................................................... 49 2.2.1.6. Vector preparation ...................................................................... 51 2.2.1.7. Small-scale plasmid isolation from bacteria for RLC-APEX2 and APEX2 gene cloning.............................................................................................