The effect of tubulin acetylation and detyrosination on the in vitro motility of kinesin-1 by Neha Kaul A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Mechanical Engineering) in The University of Michigan 2012 Doctoral Committee: Professor Edgar Meyhofer, Chair Professor Ellen M. Arruda Associate Professor Katsuo Kurabayashi Associate Professor Kristen J. Verhey ACKNOWLEDGEMENTS Binding assays were performed by Virupakshi Soppina, research scientist in the Verhey Lab. COS cell lysates were prepared by him and Steve Norris from the same lab. I have been fortunate in closely collaborating with Viru and I would like to thank both him and Steve for their continued support. I am also grateful to Lynne Blasius and other members of the Verhey lab for valuable guidance with biochemistry and cell biology techniques. My development as a graduate student would not have been the same without the influence of Kristen Verhey, collaborator and mentor. Her approach to experiments opened up a whole new world to my initially engineering-oriented one-track mind. Committee members Ellen Arruda and Katsuo Kurabayashi have been highly supportive of my work throughout. I am extremely thankful to them for their time, which they have never hesitated to share with me, be it in discussing my research directly, or in advising me about graduate school and beyond. I would like to thank my labmates, Chang Jiang and Jenna Campbell for making the lab a great place to be at all times, including 5am for tubulin preps. Being in the mechanical engineering department has brought with it the fortune of working with some very kind people in the staff and I am thankful to them for being themselves. I am especially thankful to my adviser, Edgar Meyhofer for his continued support and patience as my mentor. Finally, I would like to thank my family and friends from the bottom of my heart for their love and support without which none of this would have been possible. ii CONTENTS ACKNOWLEDGEMENTS ............................................................................................ ii LIST OF TABLES .......................................................................................................... v LIST OF FIGURES ........................................................................................................ vi GLOSSARY ................................................................................................................ viii CHAPTER 1 Introduction ......................................................................................................... 1 What is Intra-cellular Transport and Why is it Important? ........................ 1 What Do We Know About Cargo-carrying Motor Proteins? .................... 3 Kinesin-1 ................................................................................................. 5 What Do We Know About Microtubules, the Cytoskeletal Highways? .... 7 What Do We Know About PTMs of Tubulin? ....................................... 10 Acetylation ................................................................................ 11 Detyrosination ........................................................................... 12 Polyglutamylation and Polyglycylation ...................................... 12 Do Microtubule PTMs Affect Kinesin-based Transport? .................................... 14 The Role of Acetylation in Kinesin-1 Motility ........................................ 17 The Role of Detyrosination in Kinesin-1 Motility ................................... 18 The Role of the Poly-modifications in Kinesin-1 Motility ....................... 19 Summary of the Problem .................................................................................... 20 Thesis Statement ................................................................................................ 21 CHAPTER 2 The Effect of Tubulin Acetylation on the in vitro Motility of Kinesin-1 ......................... 22 Preliminary Studies with Doublet Microtubules ................................................. 24 Materials and Methods ........................................................................... 25 Results.................................................................................................... 27 Gliding Assays ............................................................................ 28 iii Single-molecule TIRF Motility Assays ....................................... 29 Control Experiment ..................................................................... 30 Discussion for Preliminary Studies .............................................. 32 Acetylated and Deacetylated Microtubules ......................................................... 33 Materials and Methods ....................................................................................... 34 Results.................................................................................................... 38 Binding Assays .......................................................................... 39 Single-molecule TIRF Motility Assays ...................................... 40 Control Experiments .................................................................. 42 Discussion ................................................................................. 48 CHAPTER 3 The Combined Effect of Tubulin Acetylation and Detyrosination on the in vitro Motility of Kinesin-1 ................................................................................................................... 53 Materials and Methods ....................................................................................... 54 Results ............................................................................................................... 57 Single-molecule TIRF Motility Assays ................................................... 58 Control Experiments .............................................................................. 60 Discussion ............................................................................................. 64 CHAPTER 4 Device and/or Software Development ............................................................................ 69 Novel Dual-color Imaging Device ...................................................................... 69 Sample-heater Device ........................................................................................ 76 Image Processing Single-particle Tracking Program .......................................... 80 CHAPTER 5 Conclusions and Future Work ............................................................................ 83 BIBLIOGRAPHY ......................................................................................................... 86 iv LIST OF TABLES TABLE 1. Kinesin super-families ......................................................................................... 5 2. Cellular functions of PTMs ................................................................................ 17 v LIST OF FIGURES 1. Cartoon of a cell ................................................................................................... 2 2. Transport machinery ............................................................................................ 3 3. Cartoon for kinesin, dynein and myosin ............................................................... 5 4. Kinesin and its crystal structure ............................................................................ 7 5. Microtubules and the crystal structure of tubulin .................................................. 9 6. PTM locations on α- and β- tubulin .................................................................... 11 7. PTM distribution in cells .................................................................................... 14 8. Tetrahymena and their cilia ................................................................................ 24 9. SDS-PAGE for doublet purification ................................................................... 28 10. Velocity of WT and K40R doublets in gliding assays with NKHK560 ............... 29 11. Velocity and run length of RnKHC560 on WT and K40R doublets .................... 30 12. Motility parameters for RnKHC560 on WT and K40R doublets in the same assay .......................................................................................................................... 32 13. Western blot for acetylation and deacetylation of tubulin ................................... 39 14. Motility parameters for RnKHC560 on acetylated and deacetylated microtubules in the same assay ................................................................................................ 42 15. Motility parameters for RnKHC560 on unlabeled acetylated and deacetylated microtubules ..................................................................................................... 44 16. Motility parameters for RnKHC560 on GMPCPP-stabilized acetylated and deacetylated microtubules ................................................................................. 45 17. Motility parameters for RnKHC560 on acetylated and deacetylated microtubules in a physiological buffer ..................................................................................... 47 18. Velocity of acetylated and deacetylated microtubules in gliding assays with NKHK560.......................................................................................................... 48 19. SDS-PAGE and Western