Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2018 Biophysics of cadherin interactions and junction assembly Omer Shafraz Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biophysics Commons, and the Physics Commons Recommended Citation Shafraz, Omer, "Biophysics of cadherin interactions and junction assembly" (2018). Graduate Theses and Dissertations. 17312. https://lib.dr.iastate.edu/etd/17312 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Biophysics of cadherin interactions and junction assembly by Omer Lebbe M. Shafraz A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Physics Program of Study Committee: Sanjeevi Sivasankar, Major Professor James Evans John Lajoie Xuefeng Wang Robert Jernigan The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2018 Copyright © Omer Lebbe M. Shafraz, 2018. All rights reserved. ii DEDICATION To my late father Omer Lebbe and mother Zaibunnisa, To my wife Shifnaz and my little boy Ayaan. iii TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................. v ABSTRACT ................................................................................................................... vii CHAPTER 1. INTRODUCTION ....................................................................................... 1 1.1 General Introduction ............................................................................................ 1 1.2 Thesis Organization ............................................................................................. 3 1.3 Single Molecule Force Spectroscopy: Theory and Method ................................... 5 1.3.1 Principles of atomic force microscope .......................................................... 5 1.3.2 Cantilever sensitivity measurement .............................................................. 6 1.3.3 Spring constant of the cantilever .................................................................. 6 1.3.4 Force measurement ..................................................................................... 7 1.3.5 Dynamic force spectroscopy ........................................................................ 9 1.4 References ......................................................................................................... 10 CHAPTER 2. RESOLVING DESMOSOMAL CADHERIN INTERACTIONS AT SINGLE MOLECULE LEVEL ................................................................................... 15 2.1 Introduction ........................................................................................................ 15 2.2 Results ............................................................................................................... 18 2.2.1 Desmosomal cadherins have distinct binding properties ............................ 18 2.2.2 W2 is required for Dsc2 Ca2+ dependent homophilic binding not for heterophilic interaction with Dsg2 ................................................................... 20 2.2.3 Different requirements of desmosomal cadherins for the formation of punctate desmosomal structures ..................................................... 22 2.2.4 W2 is required for Dsc2 recruitment into desmosome puncta not for Dsg2 .............................................................................................................. 22 2.3 Discussion ..................................................................................................... 23 2.4 Materials and Methods ....................................................................................... 25 2.4.1 Protein Purification ..................................................................................... 25 2.4.2 Single Molecule Force Spectroscopy (SMFS) ............................................ 25 2.5 References ......................................................................................................... 26 CHAPTER 3. E-CADHERIN BINDS TO DESMOGLEIN TO FACILITATE DESMOSOME ASSEMBLY .......................................................................................... 32 3.1 Abstract .............................................................................................................. 32 3.2 Introduction ........................................................................................................ 33 3.3 Results ............................................................................................................... 34 3.3.1 Ecad interacts with Dsg2 to form a Ca2+-independent heterodimer ......................................................................................................... 34 3.3.2 Ecad/Dsg2 and Dsc2/Dsc2 dimers have lower lifetimes than Dsc2/Dsg2 dimers ............................................................................................... 38 3.3.3 Ecad is present in nascent desmosomes but not in mature desmosomes....................................................................................................... 40 3.3.4 Leu 175 mediates Ecad and Dsg2 interactions ........................................... 42 iv 3.3.5 Ecad L175 is essential for efficient intercellular Dsg2 recruitment and desmosome assembly .................................................................................. 44 3.4 Discussion .......................................................................................................... 48 3.5 Materials and Methods ....................................................................................... 52 3.5.1 Purification of cadherin ectodomains .......................................................... 52 3.5.2 Single molecule AFM force measurements ................................................ 53 3.5.3 SIM imaging and analysis of cadherin localization within desmosomes....................................................................................................... 54 3.5.4 Isolation, culture, transfection and confocal imaging of primary keratinocytes ....................................................................................................... 55 3.6 Acknowledgements ............................................................................................ 56 3.7 References ......................................................................................................... 57 CHAPTER 4. FAST FRAP TO STUDY ENDOCYTOSIS DRIVEN REMODELING OF ADHERENS JUNCTION ................................................................. 61 4.1 Introduction ........................................................................................................ 61 4.2 Results ............................................................................................................... 64 4.3 Discussion .......................................................................................................... 67 4.4 Materials and Method ......................................................................................... 68 4.4.1 Cell culture ................................................................................................. 68 4.4.2 FRAP experimental set up.......................................................................... 68 4.5 References ......................................................................................................... 69 CHAPTER 5. CONCLUSIONS AND FUTURE DIRECTIONS ........................................ 73 5.1 Conclusions........................................................................................................ 73 5.2 Future Directions ................................................................................................ 75 5.3 References ......................................................................................................... 76 v ACKNOWLEDGMENTS I would like to thank everyone who was part of this journey with me. Most importantly, I would like to express my heartfelt gratitude to my advisor Dr. Sanjeevi Sivasankar for his incredible mentorship, continuous encouragement and valuable advice throughout my PhD. Sanjeevi, thank you for having me as a member of your lab and supporting me to present my work at various conferences that motivated me greatly in pursuing a scientific career. I would also like to thank my program of study committee members Dr. James Evans, Dr. Robert Jernigan and Dr. Xuefeng Wang for their valuable time and feedback on my research. Special thanks to Dr. John Lajoie and my lab mates Patrick Schmidt and Hussam Ibrahim for helping me with collecting data for the FRAP experiments using FPGA, for data analysis and valuable discussions. I would also like to thank my collaborators, Dr. Molly Lowndes and Prof. James Nelson from Stanford University for making me a part of their JCS