INVESTIGATION OF THE FUNCTION OF MYOTUBULARIN THROUGH EXAMINATION OF PROTEIN-PROTEIN INTERACTIONS AND EXCLUSION OF MTMR1 AS A FREQUENT CAUSE OF X-LINKED MYOTUBULAR MYOPATHY DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By LaRae Meschelle Copley, B.S. ***** The Ohio State University 2004 Dissertation Committee: Approved by Gail Herman, MD/PhD, Advisor Arthur Burghes, PhD ______________________________________ Charis Eng, MD/PhD Advisor Thomas Sferra, MD Molecular, Cellular & Developmental Biology ABSTRACT X-linked myotubular myopathy (MTM1, MIM# 310400) is a rare neuromuscular disorder presenting at birth with hypotonia, respiratory distress, and characteristic facies. Many patients do not survive past infancy. However, long-term survivors of MTM1 are known and have presented with additional medical problems later in life such as advanced bone age, mild spherocytosis, and peliosis hepatis. In 1996, an MTM1 gene encoding a predicted protein called myotubularin was isolated. Based on the structure of the gene, it was postulated that myotubularin would be a dual specificity phosphatase. Recently, researchers have discovered that myotubularin can act as a lipid phosphatase by dephosphorylating phosphatidylinositol-3-phosphate [PI(3)P], an intracellular membrane trafficking signal. Additionally, myotubularin is predicted to contain GRAM, PDZ and coiled coil domains which may be relevant for protein-protein interactions. To further elucidate the function of myotubularin in vivo, we employed a yeast two- hybrid system to screen for proteins with which it interacts. We generated two binding domain fusion constructs, both containing the phosphatase domain of myotubularin. One ii of the constructs harbors a C375S mutation at the active site of the enzyme and is known to abolish catalytic activity. Candidate interacting proteins were identified using a human fetal brain library (Clontech, Palo Alto, California). Our results yielded several clones, including the catalytic subunit of protein phosphatase 2A (PP2Ac), SCG10, and four novel ESTs. PP2A, a serine/threonine phosphatase, is involved in a variety of functions within the cell. SCG10 is a stathmin like protein implicated in microtubule depolymerization. Binding of the MTM1 protein with these candidates was determined to be specific in the yeast system, but confirmation of specific interactions in mammalian cells using coimmunoprecipitation and GST pulldown strategies was unsuccessful. A second goal of this work was to develop antibodies to myotubularin. Regions of antigenicity were predicted in the myotubularin primary structure and corresponding peptides were generated and injected into rabbits. Upon screening and purifying sera, an antibody that specifically detects human myotubularin but not mouse myotubularin was generated as determined by immunoblotting. To date, mutations in more than three hundred MTM1 families worldwide have been characterized, including many from our laboratory. However, no mutation within this gene has been found in approximately 20% of “MTM1” patients, including several males with an X-linked pattern of inheritance. MTMR1, a gene highly homologous to MTM1, is located 50KB telomeric to MTM1 on the X chromosome. We have screened 16 exons of MTMR1 in genomic DNA from 14 males with biopsy proven myotubular myopathy in whom we found no mutation in MTM1. This includes two males with X-linked iii pedigrees and two with affected male siblings. No mutations were found in these patients. These results suggest that mutations in MTMR1 are not a frequent cause of X-linked myotubular myopathy in males for whom no mutation is found in MTM1. Finally, we evaluated changes in cell shape in the context of myotubularin overexpression. Recently, researchers discovered that myotubularin localizes to the plasma membrane and Rac1 induced membrane ruffles. We developed HeLa cell lines that stably express Flag-tagged myotubularin in a doxycycline inducible fashion using the tet-on system (Clontech). These cell lines, with variable expression levels of Flag-MTM1 protein, were used to quantitate cell spreading and membrane ruffling. We found a statistically significant decrease in spreading over 6 hours in cells with a high level of myotubularin overexpression compared to untreated cells. This effect was lessened as the level of Flag-MTM1 protein levels decreased. Cell lines with little to no inducible expression of Flag-MTM1 protein exhibited spreading levels similar to untreated cells. This system was also used to quantitate membrane ruffling. A decrease in membrane ruffling was observed in cells expressing high levels of the myotubularin, but statistical significance could not be established with two independent observers. iv For Christopher v ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Gail Herman, for the opportunity to work in her lab. Without her, none of this would have been possible. I have learned much from her. I would also like to acknowledge my committee for their continuous assistance and encouragement. I especially thank Dr. Charis Eng for acting as my "Medical Scientist Mentor" and for many discussions regarding my work, career, and life as a graduate student. I also thank Dr. Arthur Burghes for being my "Basic Scientist Mentor" and providing me with multiple suggestions for my project. I am also grateful for the straightforwardness and enthusiasm for this project displayed by Dr. Thomas Sferra. I extend my special thanks to MCDB’s Program Director, Dr. David Bisaro, for always taking the time to discuss questions and concerns along the way. I must also thank my lab mates, past and present, for making each day in the lab more interesting. Without their support and encouragement, this entire process would have been much less enjoyable. I am honored to have worked alongside and learned enumerable lessons in science and politics from my friend, Dr. David Cunningham. I am lucky to be leaving the lab having made lifelong friends with Marsha Lucas, Tessa Carrel and Marlene Parker. Their abilities to listen, advise, and reassure amaze me. I thank Dr. Hugo Caldas and Tara Grove for their support and helpful technical discussions. I am vi grateful for the friendship and humor of Leon Humphries and Allison Parent. I am also thankful to Fenglei Jiang for the encouraging discussions and willingness to be "Independent Observer #2". Heartfelt thanks goes to David Zhao for much of my initial training in molecular biology. I cannot express how thankful I am to have had the support, love, and friendship of my husband, Christopher Kelly. He has managed to grin and bear the late nights and frustrating days, without ever losing sight of a goal that started out as mine, but ended as ours. I am also very grateful to my parents, Larry and Davida Copley, who at every stage of my education assured me that I could make it. I also appreciate the assistance in predicting peptides from Dr. Pravin Kaumaya, the tet vectors from Dr. Reed Clark, and the GST vectors and protocols from Dr. Gregory Taylor and Dr. Jack Dixon. I am thankful for the support received by way of the Distinguished University, Molecular Life Sciences, and Bennett Fellowships from The Ohio State University, and the General Mason Award from Children's Research Institute. vii VITA September 7, 1974. Born –Columbus, Ohio, USA June, 1997……….. Bachelor of Science, Pharmacy The Ohio State University magna cum laude, with Honors and Distinction 1997-1999. ……… Medical Scientist Fellow The Ohio State University. 1999-2000. …… Distinguished University Fellow The Ohio State University 1999-2003. .Molecular Life Sciences Fellow 1999-2003. Bennett Fellow 2000-2003…………………………………… Mason Fellow Children’s Research Institute 2003-2004……………………………………...Distinguished University Fellow The Ohio State University PUBLICATIONS Research Publications 1. Copley, LM., Zhao, WD., Kopacz K., Herman GE., Kioschis, P., Poustka, A., Taudien, S., Platzer, M. (2002) Exclusion of mutations in the MTMR1 gene as a frequent cause of X-linked myotubular myopathy. Am J Med Genet., 107(3):256-8. viii FIELDS OF STUDY Major Field: Molecular, Cellular & Developmental Biology ix TABLE OF CONTENTS P a g e Abstract. .ii Dedication. v Acknowledgments . .. vi Vita . viii List of Tables. xiii List of Figures . .xiv List of Abbreviations. .. .xvi Chapters: 1. Introduction 1.1 Clinical Features of MTM1. 1 1.2 Histopathology of MTM. 5 1.3 Differential Diagnosis of MTM1. .6 1.4 Muscle Differentiation and Development. 7 1.5 Manifestations in Carrier Females . 8 1.6 Autosomal Forms of Myotubular Myopathies .. .9 1.7 Isolation of MTM1, a Gene Mutated in X-Linked Myotubular Myopathy . 10 1.8 Protein Structure of Myotubularin .. .11 1.9 Mutations in the Human MTM1 Gene. .12 1.10 Genotype/Phenotype Correlations . .. 14 1.11 Substrate Identification for Myotubularin . 15 1.12 Myotubularin Expression During Myoblast Differentiation . 18 1.13 Identification of Other MTM Family Proteins . .19 1.14 Subcellular Localization of MTM1 . 25 1.15 Mouse Models for MTM1 . .. 27 x 2. Screening for Protein-Protein Interactions Involving Myotubularin 2.1 Introduction . .29 2.2 Materials and Methods . .. 35 2.2.1 General Reagents . 35 2.2.2 Yeast 2-Hybrid. 35 2.2.2 A. Yeast Strains and General Yeast Protocols i. Media . .35 ii. Strains . .37 2.2.2 B. Cloning i. Generation of Bait Vectors for Use in Yeast 2-Hybrid Screenings. .. .37 ii. Generation of pGBKT7-MTM1341-544 (C375S) by Site Directed Mutagenesis. .. .40 2.2.2 C. Strain Development i. Generation of Bait Strains PJ692A- pGBKT7- MTM1341-544 and PJ692A- pGBKT7-MTM1341-544(C375S). .41 ii. Verification of Yeast Bait Strains. .. .42 2.2.2 D. Yeast 2-Hybrid Screening i. Library Mating. .44 ii. Isolation of Positive Clones. .. .45 2.2.3 Coimmunoprecipitation Strategy. .. .48 2.2.3 A. Cloning i. Cloning of pcDNA4His/Max TOPO-SCG10 and pcDNA4His/Max TOPO-PP2Ac. .48 ii. Generation of pCMV-tag2B-hMTM1 and pTRE2pur-hMTM1. .50 2.2.3 B. Generation of Stable Clones that Express Flag-MTM1 in a Tet-Inducible Fashion .