Understanding the role of LC4-like in the regulation of the wave initation point of flagellar beats in trypanosomatids Lore Baert Student number: 01509541 Promoter Ghent University: Prof Dirk de Graaf Erasmus/Opinno Promoter: Dr Richard Wheeler Scientific supervisor: Dr Richard Wheeler Master’s dissertation submitted to Ghent University to obtain the degree of Master of Science in Biochemistry and Biotechnology. Major Microbioloby Academic year: 2019 – 2020 Ghent University - Department of Biochemistry and Microbiology - Laboratory of Molecular Entomology and Bee pathology University of Oxford, Oxford, United Kingdom - Nuffield Department of Medicine Preface The past year has been unforgettable, to say the least While the world was affected by a virus I am sure we are all tired of hearing about, I spent my time writing this thesis, a work which marks a huge turning point in my life. I cannot express how grateful I am for all the chances I got along the way. Nevertheless, I would like to take this opportunity to thank some people for making this thesis a reality. First of all, I want to express my gratitude to Richard Wheeler and Jack Sunter for giving me the opportunity to write my thesis in their labs and introducing me to the marvellous world of parasites. Although my time there was cut short, this international experience has helped me grow, both as a person and a scientist. Thank you to Prof. Dirk de Graaf as well, for being my Belgian promoter. In particular, I want to thank Richard, for his time and guidance, and especially for managing to come up with a new project when the coronavirus interfered with the original one. Also thanks for the weekly skype meetings, the support and the constructive feedback. Moreover, I want to thank all the wonderful people in the Wheeler, Gluenz and Sunter labs for immediately making me feel welcome and making my stay in Oxford the amazing experience it was. Thank you in particular to Lara, Andreia and Sophia, for letting me stay in their homes. I also want to thank my family. My mum, for the unconditional support and love, and for always making me coffee. My sister, for taking over my responsibilities in my youth movement, so I could focus on writing this thesis. Dobby, for forcing me to take a break from my computer screen every day, to take her on a much needed walk. And finally, I want to thank my Bro’z form the Boekenclub: Gloe, Naomi, Bosper, Julie, Cilinder, Marie and Eva. As I am writing this on my GamePC, I cannot help but look back at all the wonderful times we have had together in the past five years. Talking people under the table, crying on the floor while eating bread, flying heads and collecting garden material… So thanks for all these moments, I cannot imagine it any other way. In short, thank you to everyone who believed in me and supported me along the way. Lore I Table of contents Preface ..................................................................................................................................................... I Table of contents .................................................................................................................................... II List of abbreviations .............................................................................................................................. IV Preamble ................................................................................................................................................. V Summary ................................................................................................................................................ VI 1. Introduction ........................................................................................................................................ 1 1.1 The Trypanosomatids ................................................................................................................... 1 1.2 The role of the flagellum .............................................................................................................. 2 1.3 Introduction of the different flagellar beat types of trypanosomatids ...................................... 3 1.4 Overview of the flagellar structures ............................................................................................ 4 1.4.1 Axoneme ................................................................................................................................. 6 1.4.2 Paraflagellar rod (PFR) .......................................................................................................... 10 1.5 Role of calcium in flagellar regulation ....................................................................................... 11 1.6 The regulatory role of the ODA and IDAs in trypanosomes: Determination of wave initation point .................................................................................................................................................. 11 1.7 ODA asymmetries in other organisms ....................................................................................... 13 2. Aim .................................................................................................................................................... 14 3. Results ............................................................................................................................................... 16 3.1 Understanding the calcium binding properties of EF-hand containing proteins ..................... 16 3.1.1 Selection and mapping the localisation of the EF-hand containing proteins ....................... 16 3.1.2 Description of the canonical binding loop sequence ........................................................... 18 3.1.3 The EF-hands in axonemal proteins have a lower predicted calcium affinity than PFR proteins ......................................................................................................................................... 19 3.1.4 The EF-hand containing proteins in the distal flagellar end show different properties ...... 21 3.2 Determination of distal flagellar proteins complexes based on localisation profiles .............. 22 3.2.1 Calculation of intensity profiles of proteins located to the distal flagellum ........................ 22 3.2.2 There are four possible functional protein complexes with a distal flagellar localisation ... 26 3.2.3 Paralogues with a different flagellar localisation show differences in either the N- or C- terminal end .................................................................................................................................. 28 3.3 Understanding how the LC4-like and the dDCs are targeted to the distal flagellum ............... 29 3.4 Evolutionary analysis of the outer dynein arm light chains ...................................................... 33 4. Discussion .......................................................................................................................................... 37 4.1 The evolution of the outer dynein arm light chains .................................................................. 37 4.2 Unravelling the complexity of the distal asymmetry along the flagellum ............................... 38 II 4.3 The distal flagellar proteins are likely not regulated by calcium-binding ................................ 40 4.4 The distal flagellar localisation profile is not mediated by a single localisation signal ........... 42 5. Material and methods ...................................................................................................................... 45 5.1 Comparison of EF-hand domain properties between different flagellar localisations ............ 45 5.1.1 Selection and mapping of EF-hand containing proteins ...................................................... 45 5.2.2 Scoring of binding loop sequences ....................................................................................... 45 5.2.3 Statistical tests ...................................................................................................................... 45 5.2 Construction and comparison of intensity profiles of the distal flagellar proteins ................. 46 5.2.1 Selection of proteins and measurements of intensity along flagellum ................................ 46 5.2.2 Calculation of intensity profiles ............................................................................................ 46 5.2.3 Clustering of intensity profiles ............................................................................................. 48 5.2.4 Sequence analysis ................................................................................................................. 48 5.3 Evolutionary analysis of the outer dynein arm light chains ...................................................... 48 References............................................................................................................................................. 50 Attachments .......................................................................................................................................... 58 III List of abbreviations HAT Human African Trypanosomiasis DALY Disability-adjusted life years VSG Variable surface glycoprotein ODA Outer dynein arm IDA Inner dynein arm FAZ Flagellar attachment zone PFR Paraflagellar rod CP Central pair N/DRC Nexin-dynein regulatory complex RS Radial spokes AAA ATPases associated with cellular activities