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The Force Regulation on Binding Kinetics and Conformations of Integrin and Selectins Using a Bio-Membrane Force Probe

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The Force Regulation on Binding Kinetics and Conformations of Integrin and Selectins Using a Bio-Membrane Force Probe THE FORCE REGULATION ON BINDING KINETICS AND CONFORMATIONS OF INTEGRIN AND SELECTINS USING A BIO-MEMBRANE FORCE PROBE A Thesis Presented to The Academic Faculty By Wei Chen In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Bioengineering in the School of Mechanical Engineering Georgia Institute of Technology May 2009 COPYRIGHT ©WEI CHEN 2009 THE FORCE REGULATION ON BINDING KINETICS AND CONFORMATIONS OF INTEGRIN AND SELECTINS USING A BIO-MEMBRANE FORCE PROBE Approved by: Dr. Cheng Zhu, Advisor Dr. Levent F. Degertekin School of Biomedical Engineering School of Mechanical Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Evan A. Evans Dr. Nael McCarty Departments of Physics and Pathology, School of Biology University of British Columbia Georgia Institute of Technology Dr. Andres J. Garcia School of Mechanical Engineering Georgia Institute of Technology Date Approved: April 1st, 2009 I dedicate this thesis to My parents I am forever indebted to my parents, (Zichen Chen) and (Suhua Li). I would not have been able to finish my Ph.D. thesis without their priceless love, endless patience and unconditional supports. My wife I am so grateful to my wife, (Weiwei Yin), who was always there to support me unconditionally, comfort and encourage me to go through difficult times. ACKNOWLEDGEMENTS I would like to express my gratitude to many people who supported me in the passed 5 years. Without their helps, I could not believe I could finish my Ph.D. research smoothly. Firstly, I would like to thank my Ph.D. thesis advisor, Dr. Cheng Zhu. He is the most important person to my Ph.D. study and research. I would like to thank him for providing me a precious chance to enter this exciting bioengineering field five years ago, and for his continuous financial support, helpful guide, and encouragement through my whole Ph.D. period. More importantly, I would thank him for teaching me how to do science, for his enthusiasm and curiosity to science which lead me to explore this mysterious scientific world, for his strictness to the scientific research which taught me to patiently carry out and repeat tedious scientific experiments, carefully examine discoveries and thoughtfully formalize our interpretation and conclusions. Secondly, I would like to thank Dr. Evan Evans. As old saying says, “Standing on the Shoulder of Giants.” Dr.Evans is a talented giant, and I’m a dwarf standing on his shoulder. His creative invention, the BFP, is my major powerful tool for my Ph.D. research. His unconditional technical support helped me so much to quickly build up a new BFP in Dr.Zhu’s lab and smoothly finish my thesis works. His thoughts of the essence of scientific research benefit my Ph.D. study and my whole lifetime in future. His enthusiasm on science makes me realize that doing science is full of fun. In addition to Dr.Evan Evans, I would like to thank his lab members, Andrew Leung and Dr. Koji Kinoshita. They are giant’s two arms. They gave me tremendous helps on teaching me, building up the BFP and overcoming difficulties in BFP experiments. I would like to thank my other thesis committee members, Dr.Andres Garcia, Dr.Levent Degertegin and Dr. Nael McCarty for serving as my thesis committee and for taking time to make valuable suggestions on my thesis work. I would also like to thank I Dr. Garcia for his encouragement since my Ph.D. qualify examination and strong support on my whole Ph.D. thesis research, Dr. Degertegin for his creativity that impacted my research, and Dr. McCarty for introducing and leading me into the biological world. I would like to thank Dr. Rodger McEver for kindly proving me many important reagents for my Ph.D. research, for guiding me on how to carry out cutting-edge scientific research and for greatly supporting my on academic career development. I also would like to thank his members as well for supporting my research. I would like to thanks technical supporting staff in the Institute of Bioscience and Bioengineering and the Mechanical Engineering (ME) at Georgia Tech. I would like to thank Mr.John Gramham and his co-op students in ME’s machine shop for taking so much effort on manufacturing my designs for the BFP system. I would also like to thank Johnafel Crowe for supporting the equipments in the IBB and Dr. Jeffrey Donnell for revising my thesis. Special thanks to previous and current Zhu lab members. Firstly I would like to thank Dr. Kong Fang for helping me so much on solving technical problems when I was setting up the BFP, Dr. Jizhong Lou, Dr. Fang Zhang, Dr. Ning Jiang, Dr. Veronica, Wei Chen, Dr. Jun Huang and Dr. Tao Wu for their useful discussion on my research works, Larissa Doudy for supporting my BFP experiments in the last year of my Ph.D. research, and William Madison Parks for his help on revising my thesis. I als would like to thank my friends for their helps in the passed five years. Finally, I would like to thank my family for supporting me in the passed five years. Especially, I would like to thank my wife and my mother in-law when I am writing this Ph.D. thesis. Without your caring and love, it would be much harder to finish my Ph.D. works. II TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ..............................................................................................I LIST OF FIGURES ........................................................................................................VI LIST OF SYMBOLS AND ABBREVIATIONS......................................................... XII SUMMARY…… ............................................................................................................... 1 CHAPTER 1 OBJECTIVES......................................................................................... 5 1.1 Specific aim 1: to implement a bio-membrane force probe (BFP) and characterize its capacity to study specific interactions between receptors and ligands ................... 5 1.2 Specific aim 2: to develop a thermal fluctuation assay to monitor receptor-ligand interactions between two opposing surfaces................................................................ 6 1.3 Specific aim 3: to functionally study the force and force history dependent lifetimes of selectins-ligand interactions. .................................................................... 7 1.4 Specific aim 4: to characterize the force-dependent dissociation of LFA-1/ICAM-1 interaction on a living cell................................................................. 8 1.5 Specific aim 5: to probe force-regulated dynamic global conformational change of LFA-1 integrin on a living cell. ............................................................................... 9 CHAPTER 2 BACKGROUND................................................................................... 12 2.1 Cell adhesion and cell adhesion molecules (CAMs) ........................................... 12 2.1.1 Selectins and their ligands ......................................................................... 14 2.1.2 Integrins and their ligands.......................................................................... 16 2.1.3 Regulation of LFA-1 activity..................................................................... 20 2.1.4 Conformational changes of LFA-1 integrin............................................... 22 2.2 Receptor-ligand binding kinetics ......................................................................... 26 2.2.1 General receptor-ligand kinetics ................................................................ 26 2.2.2 Force-regulated off-rate ............................................................................. 27 2.2.3 Forward association rate (2D-on-rate) of surface-anchored molecules..... 29 2.3 Single molecule techniques and assays................................................................ 30 2.3.1 Single molecule techniques........................................................................ 30 2.3.2 Experimental assays to measure kinetics of receptor-ligand interactions.. 31 CHAPTER 3 MATERIALS AND METHODS ......................................................... 35 3.1 Cells, Proteins and Small Molecules ................................................................... 35 3.1.1 Cell isolation and culture ........................................................................... 35 3.1.2 Proteins, antibodies and small molecule antagonists................................. 36 3.2 Coupling biotins onto RBCs and proteins onto glass beads ................................ 37 3.2.1 Coupling biotins onto RBCs ...................................................................... 38 3.2.2 Coupling proteins onto glass beads via PEG polymer linkage.................. 38 3.2.3 Coupling proteins onto glass beads via biotin-streptavidin’s bond ........... 40 III 3.2.4 Coupling Fc-chimera proteins onto IgG coated glasses beads .................. 40 3.3 Measurement of molecular site density ............................................................... 41 3.3.1 Fluorescence staining................................................................................. 41 3.3.2 Data acquisition ......................................................................................... 41 3.3.3 Site density determination.......................................................................... 42 3.4 The BFP system ................................................................................................... 42 3.4 Measurement of binding frequency ....................................................................
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