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Obscurin Mediates Ankyrin Complex Formation in the Heart

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Obscurin Mediates Ankyrin Complex Formation in the Heart 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 8-2019 OBSCURIN MEDIATES ANKYRIN COMPLEX FORMATION IN THE HEART Janani Subramaniam Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Biochemistry Commons, Integrative Biology Commons, and the Molecular Biology Commons Recommended Citation Subramaniam, Janani, "OBSCURIN MEDIATES ANKYRIN COMPLEX FORMATION IN THE HEART" (2019). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 961. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/961 This Thesis (MS) 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]. OBSCURIN MEDIATES ANKYRIN COMPLEX FORMATION IN THE HEART by Janani Subramaniam, B.S. APPROVED: ______________________________ Shane R. Cunha, Ph.D. Advisory Professor ______________________________ Darren F. Boehning, Ph.D. ______________________________ Carmen W. Dessauer, Ph.D. ______________________________ Jeffery A. Frost, Ph.D. ______________________________ M. Neal Waxham, Ph.D. APPROVED: ____________________________ Dean, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences 1 OBSCURIN MEDIATES ANKYRIN COMPLEX FORMATION IN THE HEART A THESIS Presented to the Faculty of The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE by Janani Subramaniam, B.S. Houston, Texas August, 2019 Dedication To my family: Mom, Dad, Utsi, and Manga, Your unconditional love and support allow me to ―burrow burrow‖ my way through life. I am so deeply grateful for you! iii Acknowledgements The phrase ‗It takes a village,‘ could not be more applicable to my graduate career thus far. I am thankful for all the people I have met and the all opportunities I‘d had to grow. I would not be here without the love, support and guidance of my family. Despite the distance you are there for me no matter what, and I am so grateful for that. Thank you for the encouragement, for celebrating my highs with me, and for being there for me during my lows. I love you more than I can express! To all my friends from Houston, thank you for making this city feel like home for the last few years. You‘ve been there for me through it all and I‘ve had so much fun in the time we‘ve spent together. Meredith, you were my first friend in Houston and grew to be one of my best. Thank you for always believing in me and inviting me into your family with open arms! To Katie, thanks for helping me learn how to use Illustrator and answering my design questions- I enjoyed that almost as much as our wine nights. To all my grad school friends, thank you for listening to my presentations, helping me with experiments, encouraging me when I needed it, and commiserating with me when experiments/things didn‘t work out! I‘d have a lot more grey in my hair if it weren‘t for you. To everyone in the BCB program and IBP department, thank you for helping me learn techniques, providing me with reagents and cells, and all the feedback on my projects and experiments. Special shout-out to the admin for always ensuring that things run seamlessly. Thank you, Drs. Jeff Frost, Neal Waxham, Carmen Dessauer, and Darren Boehning, for providing a supportive yet challenging atmosphere at committee meetings. Thank you for iv your guidance and insight along the course of my project. Dr. Carmen Dessauer, thank you for all the encouragement along the way. It always meant a lot to me as you are someone I hope to emulate one day. Dr. Darren Boehning, thank you so much teaching me techniques, letting me use your lab like I was a part of it, and answering my numerous questions. And last but not least, thank you, Shane! You have been both a mentor and a friend these last few years and I know I wouldn‘t be here today without you. You have been patient with me when I needed it and pushed me when I needed that. Thank you for encouraging me to think critically, be an ethical and creative scientist, and deliver my ideas with clarity- all while having a good time! v OBSCURIN MEDIATES ANKYRIN COMPLEX FORMATION IN THE HEART Janani Subramaniam, B.S. Advisory Professor: Shane R. Cunha, Ph.D. Distinctly organized domains of receptors, ion channels, transporters, signaling molecules, cell adhesion molecules, and contractile proteins are crucial to cardiac function. Interactions between adaptor proteins such as ankyrins and cytoskeletal proteins such as obscurin play a pivotal role in organizing these functional domains in cardiomyocytes. Therefore, dysfunction of both ankyrin as well as obscurin lead to a host of cardiovascular diseases such as arrhythmias and cardiomyopathies. Alternative splicing of ankyrin yields numerous isoforms that interact with obscurin at various sub-cellular domains. And while some of these obscurin-ankyrin complexes have been studied, many others have not been characterized. Further, previous studies have focused on these ankyrin-obscurin complexes individually; however, how ankyrin-mediated macromolecular complexes are integrated together within the cardiomyocyte is not clear. Thus, the work in this thesis describes mechanisms by which obscurin organizes two separate ankyrin-mediated macromolecular complexes simultaneously. Two muscle-specific ankyrin isoforms, sAnk1.5 and AnkG107/130, which do not interact with each other, were used to test if obscurin could serve to bridge these ankyrins. Through a series of in vitro binding assays, co-precipitation assays, and FLIM-FRET analysis we demonstrate that obscurin binds both isoforms simultaneously via interactions with two ankyrin binding sites in its C-terminus. We also show that β1-spectrin, a protein that does not vi directly interact with obscurin or sAnk1.5, can be recruited to a macromolecular complex via an interaction with AnkG107/130. Lastly, both ankyrin isoforms localize to the M-line of cardiomyocytes suggesting that obscurin could serve to target two separate ankyrin protein complexes to the same domain (M-line). In sum, this work provides a novel, compelling mechanism for ankyrin complex integration and coordination in cardiomyocytes. vii Table of Contents Approval Page.............................................................................................................................i Title Page...................................................................................................................................ii Dedication.................................................................................................................................iii Acknowledgements...................................................................................................................iv Abstract.....................................................................................................................................vi Table of Contents....................................................................................................................viii List of Figures...........................................................................................................................xi Abbreviations……………………….......................................................................................xii Chapter 1: Introduction..............................................................................................................1 1.1: Spotlight on cardiovascular disease........................................................................2 1.2: Features of cardiomyocytes....................................................................................2 1.3: Ankyrins..................................................................................................................4 1.4: Ankyrin functional domains...................................................................................5 1.5: Ankyrin diversity: genes and alternatively spliced isoforms..................................8 1.6: Ankyrins in the heart.............................................................................................11 1.7: Obscurin................................................................................................................17 1.8: Significance and scope of this thesis....................................................................21 viii Chapter 2: Materials and Methods...........................................................................................23 2.1: Plasmids and antibodies........................................................................................24 2.2: RNA isolation, reverse transcription, and PCR amplification of AnkG107/130 mRNA transcripts.................................................................................................26 2.3: In-vitro
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