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FH2-Dependent Localization of FHOD Formins in the Sarcomere

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FH2-Dependent Localization of FHOD Formins in the Sarcomere FH2-dependent Localization of FHOD Formins in the Sarcomere Item Type Dissertation Authors Hamilton, Elisabeth Rights Attribution-NonCommercial-NoDerivatives 4.0 International Download date 24/09/2021 09:36:01 Item License http://creativecommons.org/licenses/by-nc-nd/4.0/ Link to Item http://hdl.handle.net/20.500.12648/1892 FH2-dependent Localization of FHOD Formins in the Sarcomere By Elisabeth C. Hamilton A Dissertation in Cell and Developmental Biology Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Graduate Studies at the State University of New York, Upstate Medical University Approved_____________________ Date_____________________ TABLE OF CONTENTS Acknowledgements…………………………………………………………..… iv List of Figures……………………………………………..………….………… vi Abbreviations……………………………………………..………….…………. ix Thesis Abstract………………………………………………………….……… xi Chapter 1: General Introduction……………………………………………..… 1 Chapter 2: The multiplicity of human formins: Expression patterns in cells and tissues………………………………….……………………..……….. 31 Abstract………………………………………….………………………… 32 Introduction………………………………………….…………………… 33 Methods…………………………………………………………………… 37 Results…………………………………………………………………..… 63 Discussion…………………………………………….………………..… 78 References…………………………………………………………..…… 117 Chapter 3: Immunohistochemical Localization of Fhod1 and Fhod3 in the Sarcomere……………………………………….…..…………………………… 122 Introduction………………………………………….……………………. 123 Methods…………………………………………………………………… 125 Results…………………………………………………………………….. 128 Discussion………………………………………………………………... 139 References……………………………………………………………….. 141 Chapter 4: Exogenous Fhod1 and Fhod3 in the Sarcomere….……………. 143 Introduction………………………………………….……………………. 144 Methods……………………………………………………………….…... 145 Results…………………………………………………………………….. 151 Discussion………………………………………………………………... 164 References……………………………………………………………….. 168 Chapter 5: General Discussion………………………………………………... 171 References…………………………………………………………………189 ii Chapter 6: Localization of Exogenous FMNL1…………….………………… 192 Introduction………………………………………….……………………. 193 Methods…………………………………………………………………… 194 Results……………………………………………………………………. 197 Discussion………………………………………………………………… 201 References……………………………………………………………….. 203 iii ACKNOWLEDGEMENTS First and foremost I want to thank my thesis advisor and mentor, Dr. Scott Blystone for providing me the opportunity to learn and grow while pursuing a graduate degree in Cell & Developmental Biology in his lab. His guidance, insight, patience and sense of humor have been an invaluable resource on this journey to become an independently thinking scientist and individual. I would also like to thank the members of my thesis committee for offering up their time and expertise: Dr. Brian Haarer, Dr. David Pruyne, Dr. Jennifer Schwarz, Dr. Jean Sanger and Dr. Andrea Viczian. I am especially grateful to Dr. Jean Sanger and Dr. David Pruyne for providing guidance, knowledge and motivation throughout my graduate career. Dr. Joseph Sanger and the Department of Cell & Developmental Biology have provided resources and support throughout my time as a graduate student. I could not have completed this project without the help and previous work of all of the members of the Blystone laboratory past and present. Thank you to Ginny Graczyk for keeping the lab running smoothly, and most importantly for her untiring efforts to help clone cDNAs. I also want to thank my fellow graduate students in the lab, Dr. Akos Mersich, Dr. Matthew Miller and Eric Miller for their support and friendship inside and outside of the lab. A special thank you to Dr. Jessica Pecone for helping to get the project analyzing formin expression started that contributed to Chapter 2 of this thesis, and to Dr. George Contantineau for laying the groundwork in the lab for work with FHOD1 and skeletal muscle. The work in Chapter 2 could not have been completed without the generous help and sharing of equipment of Dr. Thomas Welch’s lab. I owe a special thank you to Lisa Blystone for her help with the RT-PCR experiments. I would also like to thank the people behind the scenes, my friends and family. The unwavering support and open doors of friends nearby have made the past seven years a joy. My parents and siblings, though far away, have been nothing but supportive of my pursuit of a graduate degree and my parents have been incredibly generous in their efforts to help me achieve my goals. Last, but iv not least, I would like to thank my husband, Bill for always believing in me and for taking care of our daughter, giving me the time and space to finish my project and write this thesis. v LIST OF FIGURES & TABLES Chapter 1: General Introduction Figure 1: Actin nucleation, elongation, hydrolysis and dissociation………… 3 Figure 2: Domain organizations of formin families…………………………… 5 Figure 3: Actin polymerization by formins…………………………………….. 7 Figure 4: Autoinhibition and activation of mDIA2……………………………... 8 Figure 5: Model of FHOD1 activation in endothelial cells…………………… 11 Figure 6: Electron micrograph of a sarcomere……………………………….. 15 Figure 7: Sarcomere structure in skeletal muscle……………………………. 16 Chapter 2: The multiplicity of human formins: Expression patterns in cells and tissues Table 1: Human formin family nomenclature and sequence references…… 35 Figure 1A: Formin alignments……………………..……………………………. 43 Figure 1B: PCR products………………………………………………………... 44 Figure 1C: Complete Formin Alignment with Primers……………………….. 45 Table 2: Expression level of human formins in cell and tissue types………. 65 Figure 2: Homeostatic formins expressed at detectable levels in all cells and tissue types……………………………………………………………….. 67 Figure 3: Least and most commonly expressed formins are closely related 68 Figure 4: Formin family expression is elevated in hematopoietic cells……. 70 Figure 5: Expression of most formins is similar between cells and tissues of mesenchymal, epithelial and neural origins……………………….. 72 Figure 6A: Total expression of all formins varies in human cell and tissue types……………………………………………………………….. 74 Figure 6B: Variation of individual formin expression in human cell and tissue types………………………………………………………………... 75 vi Chapter 3: Immunohistochemical Localization of Fhod1 and Fhod3 in the Sarcomere Figure 1: FHOD1 and FHOD3 expression in 22 cell types and tissues……. 128 Figure 2: Formin expression in cardiac and skeletal muscle……………….. 129 Figure 3A: FHOD1 and FHOD3 during C2C12 differentiation …………….. 130 Figure 3B: FHOD1 and FHOD3 intensity throughout C2C12 differentiation. 131 Figure 4: FHOD1 immunohistochemistry in C2C12 cells with myomesin…. 133 Figure 5: FHOD3 immunohistochemistry in C2C12 with alpha actinin…….. 135 Figure 6: FHOD3 immunohistochemistry in C2C12 cells……………………. 136 Figure 7: Endogenous FHOD1 and FHOD3 localization in the sarcomere... 137 Figure 7a: Components of the Sarcomere ……………………………………. 137 Figure 7b: FHOD1 and FHOD3 localization in the sarcomere model 1…... 137 Figure 7c: FHOD1 and FHOD3 localization in the sarcomere model 2…… 137 Chapter 4: Exgoenous Fhod1 and Fhod3 in muscle Figure 1: GFP-tagged plasmids expressed in HeLa cells…………………… 151 Figure 2: Localization of GFP-Fhod1 and GFP-Fhod1 actin-binding mutants……………………………………………………………………. 153 Figure 3: Width of Intensity Profile Peaks at mid-point with standard deviation ………………………………………………………………….. 155 Figure 4: Localization of GFP-Fhod3 and GFP-Fhod3 actin-binding mutants……………………………………………………………………. 157 Figure 5: Average Number of Nuclei per Myotube for Fhod1 and Fhod3 siRNA treatments and controls……………………………….. 159 Figure 6: Exogenous FHOD1 and FHOD3 localization in the sarcomere… 161 Figure 6a: Components of the Sarcomere ……………………………………. 161 Figure 6b: GFP-FHOD1 and GFP-FHOD3 in the Sarcomere Model 1……. 161 Figure 6c: GFP-FHOD1 and GFP-FHOD3 in the Sarcomere Model 2……. 161 vii Chapter 5: Discussion Figure 1 - Proposed localization of endogenous FHOD1 and FHOD3, and GFP- FHOD1, GFP-FHOD3 and GFP-FMNL1………………………. 187 Figure 1a – Components of the Sarcomere ………………………………….. 187 Figure 1b – Formin localization in the sarcomere model 1………………….. 187 Figure 1c – Formin localization in the sarcomere model 2............................ 187 Appendix 1: Localization of Exogenous FMNL1 Figure 1 – GFP-FMNL1 localization with sarcomeric alpha-actinin ………... 198 Figure 2 – GFP-FMNL1 in the Sarcomere……………………………………..200 Figure 2a – Components of the Sarcomere ………………………………….. 200 Figure 2b – Model of GFP-FMNL1 Localization in the Sarcomere…………. 200 viii LIST OF ABBREVIATIONS Arp2/3 Actin-Related Proteins 2/3 BLASTp Basic Local Alignment Search Tool for proteins BSA Bovine serum albumin CC Coiled coil domain cDNA Complementary DNA Cobl Cordon-bleu COBALT Constraint-based Multiple Alignment Tool CK2 Casein kinase 2 CRISPR Clustered Regularly Interspaced Short Palindromic Repeats DAAM 1, 2 Disheveled-associated activators of morphogenesis 1, 2 DAD Diaphanous-autoinhibitory-domain DAPI 4',6-diamidino-2-phenylindole DD Dimerization domain DEL Delphilin DID Diaphanous inhibitory domain (DID) also known as FH3 Dia1, 2, 3 Diaphanous formin 1, 2, 3 DMEM Dulbecco's Modified Eagle Medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid FSI Formin-Spire interaction domain FH1 Formin homology domain 1 FH2 Formin homology domain 2 FH3 Formin homology domain 3, also known as DID FHOD1, 3 Formin homology domain containing proteins 1, 3 FITC Fluorescein isothiocyanate FMN 1, 2 Original formins 1, 2 FRAP Fluorescence Recovery after Photobleaching FRL1, 2, 3 Formin-related
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