The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 5-2011 Analysis of Band 4.1B in Integrin-Mediated Cell Adhesion and Signaling Youngsin Jung Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Cell Biology Commons, and the Developmental Biology Commons Recommended Citation Jung, Youngsin, "Analysis of Band 4.1B in Integrin-Mediated Cell Adhesion and Signaling" (2011). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 108. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/108 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. Analysis of Band 4.1B in Integrin-Mediated Cell Adhesion and Signaling By Youngsin Jung, B.S. Approved: Joseph H. McCarty, Ph.D. Supervisory Professor Gary E. Gallick, Ph.D. Georg Halder, Ph.D. Victoria P. Knutson, Ph.D. Renhao Li, Ph.D. Approved: Dean, The University of Texas Graduate School of Biomedical sciences Analysis of Band 4.1B in Integrin-Mediated Cell Adhesion and Signaling A DISSERTATION Presented to the Faculty of The University of Texas Health Science Center at Houston and The University of Texas M.D. Anderson Cancer Center Graduate School of Biomedical Sciences In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY by Youngsin Jung, B.S. Houston, Texas May, 2011 DEDICATION I would like to dedicate this dissertation to my parents, Hyung Keun Jung and Hea Ran Chang, who have always been my greatest support, my sister, Yoon Seo Jung, who has been cheering me through the life’s ups and downs, my aunt, Young Ran Chang, who has always believed in me, and my grandparents, Seok Rok Chang and In Soon Kim, who have been my inspiration. iii ACKNOWLEDGEMENTS I express immense gratitude to my mentor, Dr. Joseph McCarty, for his support throughout my Ph.D. training. His guidance has helped me become a better scientist. I would also like to thank the University of Texas-Houston M.D./Ph.D. Program and the Smith Research Foundation for their funding during my training at MD Anderson Cancer Center. I am extremely thankful for the members of my advisory, examining, and supervisory committees, Drs. Gary Gallick, Georg Halder, Victoria Knutson, Renhao Li, Zhimin Lu, and Renata Pasqualini, for taking time from their busy schedules to support my scientific development. I also thank the past and present members of the McCarty lab, including Mohammad Hossain, Aaron Mobley, Jeremy Tchaicha, Jae-Kyung Shin, Steve Reyes, Drs. Adam Hsu and Roman Schinder, for their support. iv Analysis of Band 4.1B in Integrin-Mediated Cell Adhesion and Signaling Publication No. Youngsin Jung, B.S. Supervisory Professor: Joseph McCarty, Ph.D. Band 4.1B is a cytoskeletal adaptor protein that regulates various cellular behavior; however, the mechanisms by which Band 4.1B contributes to intracellular signaling are unclear. This project addresses in vivo and in vitro functions for Band 4.1B in integrin-mediated cell adhesion and signaling. Band 4.1B has been shown to bind to β8 integrin, although cooperative functions of these two proteins have not been determined. Here, functional links between β8 integrin and Band 4.1B were investigated using gene knockout strategies. Ablation of β8 integrin and Band 4.1B genes resulted in impaired cardiac morphogenesis, leading to embryonic lethality by E11.5. These embryos displayed malformation of the outflow tract that was likely linked to abnormal regulation of cardiac neural crest migration. These data indicate the importance of cooperative signaling between β8 integrin and Band 4.1B in cardiac development. The involvement of Band 4.1B in integrin-mediated cell adhesion and signaling was further demonstrated by studying its functional roles in vitro. Band 4.1B is highly expressed in the brain, but its signaling in astrocytes is not understood. Here, Band 4.1B was shown to promote cell spreading likely by interacting with β1 integrin via its band 4.1, ezrin, radixin, and moesin (FERM) v domain in cell adhesions. In astrocytes, both Band 4.1B and β1 integrin were expressed in cell-ECM contact sites during early cell spreading. Exogenous expression of Band 4.1B, especially its FERM domain, enhanced cell spreading on fibronectin, an ECM ligand for β1 integrin. However, the increased cell spreading was prohibited by blocking β1 integrin. These findings suggest that Band 4.1B is crucial for early adhesion assembly and/or signaling that are mediated by β1 integrin. Collectively, this study was the first to establish Band 4.1B as a modulator of integrin-mediated adhesion and signaling. vi Table of Contents Approval Signatures……………………………………………………………………..i Title Page……………………………………………………………..……………………ii Dedication……………………………………………………..…………………………..iii Acknowledgments……………………………………………………………………….iv Abstract………………………………………………………….…………………………v Table of Contents……………………………………………………………………….vii List of Figures…………………………………………………………………………….x List of Tables…………………………………………………………………………….xiii Chapter 1: Introduction………………………………………………………………….1 1.1. Cell migration…...…………………………………………………………..1 1.2. Protein 4.1 superfamily…………….………………………………………6 1.3. Band 4.1B………………………………………………………………….13 1.4. Integrin family………………..…………………………………………….17 1.5. Integrin activation and signaling……….………………………………..23 1.6. Integrin signaling and Band 4.1B……….……………………………….27 1.7. Integrins, Band 4.1B, and organogenesis….…………………………..29 1.8. Specific aims………………………………………………………………31 Chapter 2: Materials and Methods……………………………………………………33 2.1. Experimental mice...……………………………………………………...33 2.2. Astrocyte isolation………………………………………………………...34 2.3. Immunoblotting……………………………………………………………35 2.4. Biotinylation and immunoprecipitation………………………………….35 vii 2.5. Immunohistochemistry……………………………………………………36 2.6. Immunofluorescence……………………….…………………………….37 2.7. Whole-mount immunostaining….………………………………………..38 2.8. Adhesion assays……...….……………………………………………….39 2.9. Statistical analysis….……………………………………………………..40 Chapter 3: Specific Aim I………………………………………………………….……41 3.1. Introduction………………………………………...………………………41 3.2. Results……………………………………………………………………..42 3.2.1. Genetic knockouts of β8 integrin and Band 4.1B demonstrate lethal phenotypes by E11.5……………………………….……..42 3.2.2. Cardiovascular abnormalities contribute to the lethal phenotype of β8-/-;4.1B-/- embryos………………………………………….56 3.2.3. β8-/-;4.1B-/- embryos display abnormal morphogenesis of the outflow tract and myocardium of the heart……………………..68 3.2.4. Neural crest cell migration is impaired in β8-/-;4.1B-/- embryos……………………………………………………………76 3.3. Discussion…………………………………………………………………81 Chapter 4. Specific Aim II……………………………………………………………...87 4.1. Introduction………………………………………………………………...87 4.2. Results…………………………………………………………………..…88 4.2.1. Lack of Band 4.1B expression does not affect integrin expression in astrocytes………………………………………….88 viii 4.2.2. Band 4.1B shows time-dependent changes in its subcellular localization…………………………………………………………94 4.2.3. Band 4.1B and β1 integrin co-localize to cell-ECM contact sites………………………………………....…………………….100 4.2.4. Band 4.1B is not necessary for cell adhesion to the ECM……………………………………………………………….109 4.2.5. The FERM domain of Band 4.1B enhances cell spreading on fibronectin………………………………………………………...112 4.3. Discussion………………………………………………………………..118 Chapter 5. Summary and Future Directions………………………………………123 References…………………………………..………………………………………….132 Vita….……………………………………………………………………………...……..147 ix List of Figures Chapter 1. Introduction Figure 1. Cell migration……………………………………………………………….3 Figure 2. Protein 4.1 superfamily………….…………………………………………7 Figure 3. Each conserved domain of Band 4.1B interacts with unique binding partners……………………………………………………….……………16 Figure 4. Integrin family……………………………………………………………...19 Figure 5. Integrins are involved in various intracellular signaling pathways…...22 Figure 6. Talin regulates integrin inside-out activation by interacting with the β subunit cytoplasmic domain……………………………………………..24 Chapter 3. Specific Aim I Figure 7. The genotypes of embryos are determined by PCR-based gene amplication methods……………………………………………………..44 Figure 8. β8-/- and β8-/-;4.1B-/- mice develop intracerebral hemorrhage after E11.5……………………………………………………………………….48 Figure 9. Kaplan-Meier survival analysis…………………………………………..50 Figure 10. Adult β8-/- and β8-/-;4.1B-/- mice develop hydrocephalus…………...52 Figure 11. Double knockout embryos develop lethal cardiovascular phenotypes………………………………………………………………...55 Figure 12. CNS vascular pathologies are seen in β8-/- and β8-/-;4.1B-/- embryos…………………………………………...……………………….57 Figure 13. β8-/-;4.1B-/- mice lack an elaborate vascular network…..……………59 Figure 14. Yolk sac vascular pathologies in β8-/-;4.1B-/- embryos………………61 x Figure 15. The expression of Band 4.1B and αvβ8 integrin in yolk sacs…………………………………………………………………………63 Figure