Preparation and Properties of Isolated Z-disks Thesis by Mara Camelia Rusu Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Biological Sciences School of Molecular and Cellular Biology August 2014 i The candidate confirms that the work submitted is her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2014 The University of Leeds and Mara Camelia Rusu ii Acknowledgements I would like to thank my supervisor, Professor John Trinick, for the unique opportunity to work on this exciting and challenging project. Throughout the four years his advice, patience and enthusiasm proved to be invaluable resources, especially during stressful periods. I would like to thank my co-supervisor, Dr Larissa Tskhovrebova, for her contributions to this project. My thanks to all the members of the MUZIC consortium with which I have spent great moments during our meetings, workshops and conferences. Lucian Barbu- Tudoran has sparked my interested in electron microscopy and I would like to thank him and Dr. Peiyi Wang, Dr. Stephen Muench, Martin Fuller, Dr. Kyle Dent and Rebecca Thompson for the technical help and discussion, especially during the steep learning curve of cryo-electron microscopy. I would like to extend my sincere gratitude to Dr. Sonja Welsch, Dr. Sacha de Carlo (FEI), Dr. Shaoxia Chen, Dr. Greg McMullan and Dr. Cristos Savva (MRC LMB) for their help with data collection on the Titan Krios. I am truly grateful to Dr. Kenneth Taylor for tomogram reconstruction and subtomogram averaging and to Dr. Peiyi Wang for the 2D crystal unbending. My thanks to Dr. Kathryn White for her help with fluorescence microscopy and to Dr. Marcin Wolny for his assistance during protein purification. Also I would like to thank the members of the Molecular Contractility Group at Leeds University. I am grateful to my good friends Emanuel Fertig, Marcin Wolny, Sarika Khasnis, Ania Wroblewska-Wolna, Kasia Makowska, Marta and Marcin Kurzawa, Kat White, Kieran Lee, Fran Parker, Ruth Hughes, Ada Klyszejko-Kupinska, Adam Kupinski, Matt Batchelor, Derek Revill, Anna Lopata and Charlie Green for the moral support they have given me throughout my stay in Leeds and for making me feel right at home. My deepest gratitude extends to my friends and family who have supported me emotionally during this time of learning Ovidiu Hosu, Ioana Mihalache, Georgiana Delea, Ioana Cilean, Sabin Bogdan, Cristian Iacob, Lorena Popa and Eugen Gheorghiasa. I am truly grateful to my mother who raised me to be the person I am today. Her strength during times of adversity motivates me to become better each day. My late iii father fueled my passion in biology and my parents were always supportive and understood my interest in science, encouraging me every step of the way. The work presented has been funded by the European Seventh Framework Programme through the Marie Curie Initial training Network – MUZIC. I am truly grateful for the funding and support given by the network and the European Research Council. iv Abstract Z-disks form the boundaries of the sarcomeres, the basic contractile units of muscle cells. Within the Z-line thin filaments containing mainly actin interdigitate and are crosslinked by α-actinin. Ends of the giant proteins titin and nebulin are also anchored in the Z-disk. The Z-line was originally thought to have the purely mechanical function of transmitting contractile force along the myofibrils. However, more recently, the Z- disk has emerged as a highly dynamic structure involved in stress sensing and important signaling pathways that govern muscle homeostasis. In order to fully understand how the Z-disk functions a detailed description of its molecular organization is essential. Even though the structure the structure of the Z-disk has been studied by electron microscopy techniques its molecular organization is known only in outline to a resolution of about 5 nm, whereas at least 3 nm is required to begin distinguishing protein shapes and to accurately dock crystal structure. Reports describing the isolation of intact Z-disks from insect indirect flight muscle date from 30-40 years ago, but these preparations have not been subjected to modern electron microscopy techniques. We improved the existing methods for the isolation of the Z-disk from honeybee flight muscle and investigated its structure using cryo- electron tomography and subtomogram averaging. The preliminary data indicate that the resolution was improved when compared with past studies of plastic sectioned muscle. We have also investigated the protein composition of the preparations to monitor the components that are washed away during preparation. Methods for the isolation of intact Z-disks from vertebrate muscle are not available. We explored strategies for isolating Z-disks from skeletal and cardiac muscle. Even though such a preparation has not been achieved we present promising approaches that, with optimization, should enable isolation of Z-disks from vertebrate muscle. v Table of Contents Acknowledgements ........................................................................................................ iii Abstract ............................................................................................................................ v Table of Contents ........................................................................................................... vi List of Figures ................................................................................................................. ix List of Tables .................................................................................................................. xi List of Abbreviations .................................................................................................... xii 1. Introduction ................................................................................................................. 1 1.1 Striated muscle: structure and function ......................................................................... 1 1.1.1 Overview of vertebrate muscle structure ..................................................................... 2 1.1.1.1 Vertebrate A-band and M-line ........................................................................................... 5 1.1.1.2 Vertebrate I-Band ............................................................................................................... 6 1.1.2 Overview of insect flight muscle (IFM) structure ....................................................... 9 1.1.2.1 IFM A-band ....................................................................................................................... 9 1.1.2.2 IFM I-Band ........................................................................................................................ 9 1.1.3 Mechanism of contraction in all muscle types .......................................................... 10 1.1.4 The Z-disk ................................................................................................................. 12 1.1.2.1 Proteins in the Z-disk ....................................................................................................... 12 1.1.2.2 Structure of the Z-disk ..................................................................................................... 22 1.1.3 The Z-disk as a stretch sensor ................................................................................... 26 1.2 Electron microscopy and electron tomography of biological specimens ................... 27 1.2.1 Development of electron microscopy ........................................................................ 27 1.2.2 Overview of the transmission electron microscope................................................... 28 1.2.3 Image recording in transmission electron microscopy .............................................. 31 1.2.4 Sample preparation for biological electron microscopy ............................................ 32 1.2.4.1 Electron microscopy preparation of isolated particles ..................................................... 33 1.2.4.2 Electron microscopy preparation of tissues and cells ...................................................... 36 1.2.5 Biological electron microscopy techniques ............................................................... 39 1.2.5.1 Electron crystallography .................................................................................................. 39 1.2.5.2 Single particle electron microscopy ................................................................................. 40 1.2.5.3 Electron tomography (ET) ............................................................................................... 42 1.3 Mass spectrometry (MS) ................................................................................................ 45 1.4 Thesis aims ...................................................................................................................... 46 2. General materials and methods ............................................................................... 47 vi 2.1 Materials .......................................................................................................................... 47 2.2 Protein handling and analysis