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The Role of Nuclear Positioning in Muscle Function

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The Role of Nuclear Positioning in Muscle Function UNIVERSIDADE DE LISBOA Faculdade de Medicina The role of nuclear positioning in muscle function Mafalda Ramos de Melo Pimentel Orientador: Prof. Doutor Edgar Rodrigues Almeida Gomes Tese especialmente elaborada para obtenção do grau de Doutor em Ciências Biomédicas especialidade em Biologia Celular e Molecular 2019 UNIVERSIDADE DE LISBOA Faculdade de Medicina The role of nuclear positioning in muscle function Mafalda Ramos de Melo Pimentel Orientador: Prof. Doutor Edgar Rodrigues Almeida Gomes Tese especialmente elaborada para obtenção do grau de Doutor em Ciências Biomédicas especialidade em Biologia Celular e Molecular Júri: Presidente: Doutor João Eurico Cortez Cabral da Fonseca, Professor Catedrático e Vice-Presidente do Conselho Cientifico da Faculdade de Medicina da Universidade de Lisboa Vogais: Doctor Antoine Guichet, Group Leader and Principal Investigator, Institut Jacques Monod, Université Paris Diderot; Doutor Reto Gassmann, Group Leader and Investigador do Instituto de Biologia Molecular e Celular da Universidade do Porto; Doutor Ramiro Daniel Carvalho de Almeida, Professor Auxiliar do Departamento de Ciências Médicas da Universidade de Aveiro; Doutora Solveig Thorsteinsdottir, Professora Associada com Agregação da Faculdade de Ciências da Universidade de Lisboa; Doutora Maria do Carmo Salazar Velez Roque da Fonseca, Professora Catedrática da Faculdade de Medicina da Universidade de Lisboa; Doutor Edgar Rodrigues Almeida Gomes, Professor Associado Convidado da Faculdade de Medicina da Universidade de Lisboa; Instituição Financiadora: Fundação para a Ciência e Tecnologia SFRH/BD/52227/2013 2019 A impressão desta tese foi aprovada pelo Conselho Científico da Faculdade de Medicina de Lisboa em reunião de 16 de Outubro de 2018. As opiniões expressas nesta publicação são da exclusiva respondabilidade do seu autor. Acknowledgments I would like to express my gratitude to my supervisor Dr. Edgar Gomes, for giving me the opportunity to perform my thesis in his new lab. I am very grateful for his support throughout my PhD and for his sharing of knowledge, experience and out of the box ideas. After this challenging but gratifying project, I know now that science can be “cool”, exciting and curiosity driven. I would like to thank all former and present members of the Edgar Gomes group, for the productive and friendly working atmosphere and for all the group activities inside and outside the lab. In particular, I am grateful to Bruno Cadot and Valerie Vilmont, for receiving me in Paris, making me feel welcomed and helping me with all the initial technicalities and bureaucracies. I would like to thank Judite Costa for all the support and advice, and for always making me see the better side of things. I am thankful to Sara Ferreira for taking care of our lab and making our lives infinite times easier. Many thanks to the muscle team, for all the hours spent doing primary cells (or trying), for all the troubleshooting and shared frustrations. I am especially thankful to Graciano Leal, who became my mRNP mentor/ encyclopedia, and to Helena Pinheiro, who is my science soulmate and makes me enjoy every little detail in our joint scientific lives. I would like to thank my front neighbor, Francisco Calero, for all the molecular biology teachings no matter how hopeless they were. Most importantly, I am grateful to William Roman for changing my paradigms in science and beyond. I would like to thank our neighbor lab, especially to Claudio Franco, for all the meaningful input in our meetings and to Pedro Barbacena for being always there for me. I would like to thank the Figueirencios, for being simply the best ex-lab ever and never stopping taking care of me. I acknowledge the IMM community for being so friendly and helpful all the way. Finally, I would like to express my gratitude to my family and friends for their constant support and immense belief in me. You remind me every day why it is worth it. Thank you! i ii Table of contents List of figures ........................................................................................................... v List of tables ........................................................................................................... vi List of abbreviations .............................................................................................. vii Summary ................................................................................................................ ix Resumo .................................................................................................................. xi 1 Introduction .......................................................................................................... 1 1.1 Skeletal muscle biology ............................................................................. 1 1.1.1 Skeletal muscle structure .................................................................... 1 1.1.2 Skeletal myogenesis ........................................................................... 5 1.1.3 Muscle function ................................................................................... 6 1.1.4 Muscle disorders ................................................................................. 9 1.2 Nuclear positioning and nuclear domains ................................................ 11 1.2.1 Nuclear positioning in skeletal muscle .............................................. 11 1.2.2 Nuclear domain theory ...................................................................... 13 1.2.3 Skeletal muscle research models ..................................................... 15 1.3 Subcellular mRNA localization ................................................................ 18 1.3.1 Relevance of mRNA localization....................................................... 18 1.3.2 Sequence determination and RBPs .................................................. 22 1.3.3 mRNP transport by cytoskeleton motors .......................................... 25 1.3.4 mRNP anchoring and hitchhiking...................................................... 29 1.3.5 mRNA localization in muscle ............................................................ 30 2 Objectives ..................................................................................................... 35 3 Materials and Methods .................................................................................. 37 3.1 Myoblast isolation and in vitro myofiber differentiation ............................ 37 3.2 Immortalized human myoblast culture and co-culture ............................. 38 3.3 Whole muscle isolation and cryosectioning ............................................. 39 iii 3.4 Transfection of plasmids and siRNAs ...................................................... 39 3.5 RNA extraction and RT-qPCR ................................................................. 41 3.6 Drug treatments ...................................................................................... 41 3.7 Immunofluorescence staining and image acquisition .............................. 42 3.8 Active ribosome labelling (Puromycilation) .............................................. 43 3.9 smFISH and total mRNA FISH ................................................................ 43 3.10 SYTO14 live imaging ........................................................................... 45 3.11 Nuclear movement imaging ................................................................. 45 3.12 Light-induced contraction ..................................................................... 45 3.13 Image analysis and quantification ........................................................ 46 3.14 Statistics ............................................................................................... 46 3.15 Protein size and GO term analysis ....................................................... 47 4 Results .............................................................................................................. 49 4.1 Nuclear positioning in myofibers differentiated in vitro ............................ 49 4.2 Perinuclear mRNA localization in mature myofibers ............................... 51 4.3 Giant muscle mRNAs have a particular distribution ................................ 57 4.4 mRNA localization is cytoskeleton dependent ......................................... 59 4.5 Translation correlates with regular mRNA distribution............................. 67 4.6 mRNA localization correlates with muscle function ................................. 70 4.7 Contributions ........................................................................................... 72 5 Discussion ..................................................................................................... 73 6 Appendix ....................................................................................................... 81 6.1 smFISH probes ....................................................................................... 81 6.2 MATLAB script for spatial analysis of smFISH ........................................ 87 6.3 GO term analysis of top10 biggest CDSs in the genome ........................ 91 6.4 Publications ............................................................................................. 94 7 Bibliography ................................................................................................ 101 iv List of figures Figure 1 – Structural organization of skeletal muscle. ...........................................
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