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Myth4-FERM Myosin Based Filopodia Initiation MyTH4-FERM Myosin based filopodia initiation A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Ashley L. Arthur IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Margaret A. Titus, PhD ADVISOR July 2020 © Ashley L Arthur 2020 ACKNOWLEDGEMENTS I would first and foremost like to advisor my mentor, Dr. Margaret Titus, for her unassailable commitment to training, enthusiasm for science and her sup- port of my career. Meg has been superb advisor, has made me a better scientist and communicator and I genuinely enjoyed working for her. I am grateful for the long list of positive experiences and opportunities I gained while working in the Titus lab. I would like to thank all of my past and present lab mates. Thank you to Hilary Bauer, Sinzi Cornea and Zoe Henrot for welcoming me into the lab when I started and especially to Karl Petersen who share his imaging and analysis ex- pertise. I am so grateful for the help and from my PLA project teammate Livia Songster, you brought such great energy to the project and to the lab. Thanks Casey Eddington, Annika Schroeder for their support, encouragement and help reading and discussing many aspect of this work. Thanks to the University of MN undergraduate students who joined my on research projects over the years espe- cially to Himanshu Jain. I would like to thank Jordan Beach at Loyola, Guillermo Marques, Mark Sanders, for their help with imaging. I thank Ashim Rai for his as- sistance with motor purification and motility assays. Thanks to all the members of the Houdusse Lab for their work on the PLA project and for Anne Houdusse for her critical reading of my work and her help with my manuscripts. Thanks also to Drs. Holly Goodson (Notre Dame), Robert Insall (Beatson), John Cooper (Wash- ington U), Lil Fritz-Laylin and members of the Fritz-Laylin lab (UMass Amherst) for helpful comments and discussion about the work. Thanks to Borris Demeler and Akash Bhattacharya for their help with analytical centrifugation, to Mike Fea- ley for assistance with CD. Thank you to Gaku Ashiba for many insightful conver- sations and experimental plans. I would like to thank my thesis committee: Drs. Melissa Gardner, Sivaraj Sivaramakrishnan, Hans Othmer, Dave Thomas, with help from Chad Myers. Your support of my project, and my development as a scientist was instrumental in completing this work. Finally thank you to my family and friends for their love and support. i Contents ACKNOWLEDGEMENTS ..................................................................................... i List of Tables ...................................................................................................... iv List of Figures ...................................................................................................... v 1. CHAPTER 1: Introduction ............................................................................. 1 Significance: ........................................................................................................ 1 Cellular interactions with the environment and filopodia function ......................... 1 Filopodia characterization and diversity ............................................................... 3 Key molecules in filopodia formation ................................................................... 4 Filopodia formation .............................................................................................. 7 Regulation of Myosin by Autoinhibition ................................................................ 8 Myosins and binding specialized actin structures ................................................ 9 Dictyostelium as a model for studying filopodia formation .................................. 11 Understanding MF myosin targeting for filopodia formation ............................... 12 2. CHAPTER 2: Optimized filopodia formation requires myosin tail domain cooperation ....................................................................................................... 14 Summary ........................................................................................................... 17 Keywords .......................................................................................................... 17 Significance Statement ...................................................................................... 18 Introduction ....................................................................................................... 19 Results and Discussion ..................................................................................... 20 The proximal tail has a dominant negative effect on filopodia and promotes weak dimerization .......................................................................................... 22 The distal tail of DdMyo7 is critical to promote filopodia length and number ... 23 Deletion of both N-PLA and MF2 domains abolishes activity ......................... 24 Shortening the lever arm and post lever arm disrupts filopodia formation ...... 26 Disruptions in the proximal tail cause DdMyo7 accumulation in filopodia tips . 28 ii Summary and Conclusion.................................................................................. 29 Materials and Methods ...................................................................................... 33 Figures .............................................................................................................. 37 3. CHAPTER 3: VASP mediated actin dynamics activate a filopodia myosin .. 55 Introduction ....................................................................................................... 57 Results .............................................................................................................. 60 DdMyo7 targets to actin in pseudopodia ........................................................ 60 DdMyo7 is localized to dynamic cortical actin ................................................ 61 Role of myosin motor function in targeting and filopodia formation. ................ 62 The role of VASP in DdMyo7 cortical recruitment........................................... 63 Myo7 - VASP cooperation is required for filopodia formation ......................... 64 VASP mediated actin polymerization recruits DdMyo7. .................................. 65 Discussion ......................................................................................................... 66 VASP activity promotes DdMyo7 motor domain to bind actin filaments. ......... 67 DdMyo7 actin binding, and release of autoinhibition ...................................... 69 Conserved and divergent models of filopodia myosin function ....................... 69 Figures .............................................................................................................. 71 Materials and Methods ...................................................................................... 87 4. CHAPTER 4 - DISCUSSION AND FUTURE AIMS ........................................ 93 .......................................................................................................................... 96 BIBLIOGRAPHY ................................................................................................ 96 Reprint permission........................................................................................... 115 iii List of Tables Table 1 – Quantification of DdMyo7 mutants in myo7- cells 47 Table 2 – Molecular Biology constructs 49 Table 3 Quantification with anti-actin drugs. 84 Table 4 Quantification of filopodia number and cortical targeting. 86 Table 5 Key Resources 91 iv List of Figures Figure 1 Characteristics of filopodia and invadopodia. ......................................... 2 Figure 2 Filopodia in diverse cell types ................................................................ 4 Figure 3 Actin properties and modifications ....................................................... 11 Figure 4 Filopodia initiation sequence ............................................................... 13 Figure 5 Recruitment to the cortex and release of head-tail autoinhibition promotes filopodia formation. ..................................................................... 38 Figure 6 The proximal tail regulates DdMyo7 activity. ........................................ 39 Figure 7 The proximal tail domain has a dominant negative effect on filopodia and weakly dimerizes in solution. ............................................................... 40 Figure 8 Filopodia formation promoted by a forced dimer of the motor .............. 41 Figure 9 Functional cooperation of the proximal tail and MF2 regions. .............. 42 Figure 10 Shortening the lever arm and post lever arm disrupts filopodia formation. ................................................................................................... 43 Figure 11 Deletion of the proximal tail region causes DdMyo7 accumulation in filopodia tips. .............................................................................................. 45 Figure 12 Model of DdMyo7 Inhibition and Activation States of DdMyo7. .......... 46 Figure 13 Western blots Dictyostelium cell lines ................................................ 53 Figure 14 Localization of various deletion mutants. ........................................... 54 Figure 15 DdMyo7 is localizes with cortical actin. .............................................. 72 Figure 16 Actin dynamics
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