DOCSLIB.ORG

Investigation of the Function of Myotubularin Through

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Investigation of the Function of Myotubularin Through 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 .
Load more

Recommended publications
  • The Role of PI3P Phosphatases in the Regulation of Autophagy
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 584 (2010) 1313–1318 journal homepage: www.FEBSLetters.org Review The role of PI3P phosphatases in the regulation of autophagy Isabelle Vergne a,*, Vojo Deretic a,b a Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA b Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA article info abstract Article history: Autophagy initiation is strictly dependent on phosphatidylinositol 3-phosphate (PI3P) synthesis. Received 31 December 2009 PI3P production is under tight control of PI3Kinase, hVps34, in complex with Beclin-1. Mammalian Revised 15 February 2010 cells express several PI3P phosphatases that belong to the myotubularin family. Even though some Accepted 16 February 2010 of them have been linked to serious human diseases, their cellular function is largely unknown. Two Available online 24 February 2010 recent studies indicate that PI3P metabolism involved in autophagy initiation is further regulated by Edited by Noboru Mizushima the PI3P phosphatases Jumpy and MTMR3. Additional pools of PI3P, upstream of mTOR and on the endocytic pathway, may modulate autophagy indirectly, suggesting that other PI3P phosphatases might be involved in this process. This review sums up our knowledge on PI3P phosphatases and Keywords: Autophagy discusses the recent progress on their role in autophagy. Myotubularin Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. PI3P Phosphatase Jumpy MTMR14 1. Introduction PI3P phosphatases were one of the likely candidates as it is known that the phosphoinositide, PI3,4,5P3 and its signaling can be down- PI3P synthesis has long been recognized as one of the key regulated by PI3,4,5P3 phosphatase, PTEN.
    [Show full text]
  • Myotubularin-Related Protein (MTMR) 9 Determines the Enzymatic Activity, Substrate Specificity, and Role in Autophagy of MTMR8
    Myotubularin-related protein (MTMR) 9 determines the enzymatic activity, substrate specificity, and role in autophagy of MTMR8 Jun Zoua,1, Chunfen Zhangb,1,2, Jasna Marjanovicc, Marina V. Kisselevab, Philip W. Majerusb,d,2, and Monita P. Wilsonb,2 aDepartment of Pathology and Immunology, bDivision of Hematology, Department of Internal Medicine, and dDepartment of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110; and cDivision of Basic and Pharmaceutical Sciences, St. Louis College of Pharmacy, St. Louis, MO 63110 Contributed by Philip W. Majerus, May 1, 2012 (sent for review February 24, 2012) The myotubularins are a large family of inositol polyphosphate myotubularin proteins (16–21). One mechanism that regulates 3-phosphatases that, despite having common substrates, subsume the myotubularins is the formation of heterodimers between unique functions in cells that are disparate. The myotubularin catalytically active and inactive proteins. The interaction between family consists of 16 different proteins, 9 members of which different myotubularin proteins has a significant effect on en- possess catalytic activity, dephosphorylating phosphatidylinositol zymatic activity. For example, the association of myotubularin 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphos- (MTM1) with MTMR12 results in a threefold increase in the 3- phate [PtdIns(3,5)P2] at the D-3 position. Seven members are in- phosphatase activity of MTM1, alters the subcellular localiza- active because they lack the conserved cysteine residue in the tion of MTM1 from the plasma membrane to the cytosol, and CX5R motif required for activity. We studied a subfamily of homol- attenuates the filopodia formation seen with MTM1 overex- ogous myotubularins, including myotubularin-related protein 6 pression (21, 22).
    [Show full text]
  • Myotubularin-Related Phosphatase 5 Is a Critical Determinant of Autophagy in Neurons
    bioRxiv preprint doi: https://doi.org/10.1101/2021.07.20.453106; this version posted July 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Myotubularin-related phosphatase 5 is a critical determinant of autophagy in neurons Jason P. Chua*1,8, Karan Bedi2,3,4, Michelle T. Paulsen2,4, Mats Ljungman2,4, Elizabeth M. H. Tank1, Erin S. Kim1, Jennifer M. Colón-Mercado7, Michael E. Ward7, Lois S. Weisman5,6, and Sami J. Barmada*1,8 1Department of Neurology, University of Michigan, Ann Arbor, MI, USA 2Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA 3Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA 4Rogel Cancer Center and Center for RNA Biomedicine, University of Michigan, Ann Arbor, MI, USA 5Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA 6Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA 7National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA 8Lead contact *Correspondence: [email protected] (J.P.C.), [email protected] (S.J.B.) 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.20.453106; this version posted July 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT Autophagy is a conserved, multi-step process of capturing proteolytic cargo in autophagosomes for lysosome degradation. The capacity to remove toxic proteins that accumulate in neurodegenerative disorders attests to the disease-modifying potential of the autophagy pathway.
    [Show full text]
  • Phosphatidylinositol-5-Phosphate Activation and Conserved Substrate Specificity of the Myotubularin Phosphatidylinositol 3-Phosphatases
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Current Biology, Vol. 13, 504–509, March 18, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0960-9822(03)00132-5 Phosphatidylinositol-5-Phosphate Activation and Conserved Substrate Specificity of the Myotubularin Phosphatidylinositol 3-Phosphatases 1 2 Julia Schaletzky, Stephen K. Dove, displayed activity toward PtdIns3P and PtdIns(3,5)P2, Benjamin Short,1 Oscar Lorenzo,3 but not the other naturally occurring phosphoinositides 3 1 Michael J. Clague, and Francis A. Barr (Figure 1A). The activity toward PtdIns(3,5)P2 adds a 1Department of Cell Biology novel substrate specificity for MTM1 [6] and is consis- Max-Planck-Institute of Biochemistry tent with reports that MTMR2 and MTMR3 use PtdIns3P Am Klopferspitz 18a and PtdIns(3,5)P2 as substrates [10, 11]. Purified MTMR6 Martinsried, 82152 showed the same substrate specificity as MTM1 (Figure Germany 1C), while the active site mutant protein MTMR6C336S 2 School of Biosciences lacked any detectable activity toward either PtdIns3P University of Birmingham or PtdIns(3,5)P2. To confirm that MTM1 hydrolyses The Medical School PtdIns(3,5)P2, we performed an alternate analysis of activ- Birmingham B15 2TT ity described previously for MTMR3 [10]. This assay is 3 Physiological Laboratory carried out in living yeast cells and is useful for establishing University of Liverpool 3-phosphatase activity against PtdIns(3,5)P2, since it Crown Street allows detection of the reaction product PtdIns5P, which Liverpool L69 3BX is not normally detectable in yeast [10, 12, 13].
    [Show full text]
  • Genes Retina/RPE Choroid Sclera
    Supplementary Materials: Genes Retina/RPE Choroid Sclera Fold Change p-value Fold Change p-value Fold Change p-value PPFIA2 NS NS 2.35 1.3X10-3 1.5 1.6X10-3 PTPRF 1.24 2.65X10-5 6.42 7X10-4 1.11 1X10-4 1.19 2.65X10-5 NS NS 1.11 3.3X10-3 PTPRR 1.44 2.65X10-5 3.04 4.7X10-3 NS NS Supplementary Table S1. Genes Differentially Expressed Related to Candidate Genes from Association. Genes selected for follow up validation by real time quantitative PCR. Multiple values for each gene indicate multiple probes within the same gene. NS indicates the fold change was not statistically significant. Gene/SNP Assay ID rs4764971 C__30866249_10 rs7134216 C__30023434_10 rs17306116 C__33218892_10 rs3803036 C__25749934_20 rs824311 C___8342112_10 PPFIA2 Hs00170308_m1 PTPRF Hs00160858_m1 PTPRR Hs00373136_m1 18S Hs03003631_g1 GAPDH Hs02758991_g1 Supplementary Table S2. Taqman® Genotyping and Gene Expression Assay Identification Numbers. SNP Chimp Orangutan Rhesus Marmoset Mouse Rat Cow Pig Guinea Pig Dog Elephant Opossum Chicken rs3803036 X X X X X X X X X X X X X rs1520562 X X X X X X rs1358228 X X X X X X X X X X X rs17306116 X X X X X X rs790436 X X X X X X X rs1558726 X X X X X X X X rs741525 X X X X X X X X rs7134216 X X X X X X rs4764971 X X X X X X X Supplementary Table S3. Conservation of Top SNPs from Association. X indicates SNP is conserved.
    [Show full text]
  • Inherited Neuropathies
    407 Inherited Neuropathies Vera Fridman, MD1 M. M. Reilly, MD, FRCP, FRCPI2 1 Department of Neurology, Neuromuscular Diagnostic Center, Address for correspondence Vera Fridman, MD, Neuromuscular Massachusetts General Hospital, Boston, Massachusetts Diagnostic Center, Massachusetts General Hospital, Boston, 2 MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology Massachusetts, 165 Cambridge St. Boston, MA 02114 and The National Hospital for Neurology and Neurosurgery, Queen (e-mail: [email protected]). Square, London, United Kingdom Semin Neurol 2015;35:407–423. Abstract Hereditary neuropathies (HNs) are among the most common inherited neurologic Keywords disorders and are diverse both clinically and genetically. Recent genetic advances have ► hereditary contributed to a rapid expansion of identifiable causes of HN and have broadened the neuropathy phenotypic spectrum associated with many of the causative mutations. The underlying ► Charcot-Marie-Tooth molecular pathways of disease have also been better delineated, leading to the promise disease for potential treatments. This chapter reviews the clinical and biological aspects of the ► hereditary sensory common causes of HN and addresses the challenges of approaching the diagnostic and motor workup of these conditions in a rapidly evolving genetic landscape. neuropathy ► hereditary sensory and autonomic neuropathy Hereditary neuropathies (HN) are among the most common Select forms of HN also involve cranial nerves and respiratory inherited neurologic diseases, with a prevalence of 1 in 2,500 function. Nevertheless, in the majority of patients with HN individuals.1,2 They encompass a clinically heterogeneous set there is no shortening of life expectancy. of disorders and vary greatly in severity, spanning a spectrum Historically, hereditary neuropathies have been classified from mildly symptomatic forms to those resulting in severe based on the primary site of nerve pathology (myelin vs.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Datasheet Blank Template
    SAN TA C RUZ BI OTEC HNOL OG Y, INC . MTMR3 (N-20): sc-47187 BACKGROUND RECOMMENDED SECONDARY REAGENTS Myotubularin and the myotubularin-related proteins (MTMR1-9) belong to a To ensure optimal results, the following support (secondary) reagents are highly conserved family of eukaryotic phosphatases. They are protein tyrosine recommended: 1) Western Blotting: use donkey anti-goat IgG-HRP: sc-2020 phosphatases that utilize inositol phospholipids, rather than phosphoproteins, (dilution range: 1:2000-1:100,000) or Cruz Marker™ compatible donkey as substrates. MTMR family members hydrolyze both Phosphatidylinositol anti- goat IgG-HRP: sc-2033 (dilution range: 1:2000-1:5000), Cruz Marker™ 3-phosphate (PtdIns3P) and PtdIns P2. MTMR2 interacts with MTMR5, an Molecular Weight Standards: sc-2035, TBS Blotto A Blocking Reagent: inactive family member that increases the enzymatic activity of MTMR2 and sc-2333 and Western Blotting Luminol Reagent: sc-2048. 2) Immunoprecip- dictates its subcellular localization. Mutations in MTMR2 cause autosomal itation: use Protein A/G PLUS-Agarose: sc-2003 (0.5 ml agarose/2.0 ml). recessive Charcot-Marie-Tooth type 4B1 (CMT4B1), which is characterized 3) Immunofluorescence: use donkey anti-goat IgG-FITC: sc-2024 (dilution by reduced nerve conduction velocities, focally folded myelin sheaths and range: 1:100-1:400) or donkey anti-goat IgG-TR: sc-2783 (dilution range: demyelination. MTMR3 and MTMR4 can either interact with each other or self 1:100-1:400) with UltraCruz™ Mounting Medium: sc-24941. associate. MTMR6 regulates the activity of the calcium-activated potassium channel 3.1. MTMR9 regulates the activity of MTMR7 and MTMR8.
    [Show full text]
  • Downloaded from [266]
    Patterns of DNA methylation on the human X chromosome and use in analyzing X-chromosome inactivation by Allison Marie Cotton B.Sc., The University of Guelph, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2012 © Allison Marie Cotton, 2012 Abstract The process of X-chromosome inactivation achieves dosage compensation between mammalian males and females. In females one X chromosome is transcriptionally silenced through a variety of epigenetic modifications including DNA methylation. Most X-linked genes are subject to X-chromosome inactivation and only expressed from the active X chromosome. On the inactive X chromosome, the CpG island promoters of genes subject to X-chromosome inactivation are methylated in their promoter regions, while genes which escape from X- chromosome inactivation have unmethylated CpG island promoters on both the active and inactive X chromosomes. The first objective of this thesis was to determine if the DNA methylation of CpG island promoters could be used to accurately predict X chromosome inactivation status. The second objective was to use DNA methylation to predict X-chromosome inactivation status in a variety of tissues. A comparison of blood, muscle, kidney and neural tissues revealed tissue-specific X-chromosome inactivation, in which 12% of genes escaped from X-chromosome inactivation in some, but not all, tissues. X-linked DNA methylation analysis of placental tissues predicted four times higher escape from X-chromosome inactivation than in any other tissue. Despite the hypomethylation of repetitive elements on both the X chromosome and the autosomes, no changes were detected in the frequency or intensity of placental Cot-1 holes.
    [Show full text]
  • Genetic Heterogeneity of Motor Neuropathies
    Genetic heterogeneity of motor neuropathies Boglarka Bansagi, MD ABSTRACT Helen Griffin, PhD Objective: To study the prevalence, molecular cause, and clinical presentation of hereditary motor Roger G. Whittaker, MD, neuropathies in a large cohort of patients from the North of England. PhD Methods: Detailed neurologic and electrophysiologic assessments and next-generation panel Thalia Antoniadi, PhD testing or whole exome sequencing were performed in 105 patients with clinical symptoms of Teresinha Evangelista, distal hereditary motor neuropathy (dHMN, 64 patients), axonal motor neuropathy (motor MD Charcot-Marie-Tooth disease [CMT2], 16 patients), or complex neurologic disease predominantly James Miller, MD, PhD affecting the motor nerves (hereditary motor neuropathy plus, 25 patients). Mark Greenslade, PhD Natalie Forester, PhD Results: The prevalence of dHMN is 2.14 affected individuals per 100,000 inhabitants (95% – Jennifer Duff, PhD confidence interval 1.62 2.66) in the North of England. Causative mutations were identified in Anna Bradshaw 26 out of 73 index patients (35.6%). The diagnostic rate in the dHMN subgroup was 32.5%, Stephanie Kleinle, PhD which is higher than previously reported (20%). We detected a significant defect of neuromus- Veronika Boczonadi, PhD cular transmission in 7 cases and identified potentially causative mutations in 4 patients with Hannah Steele, MD multifocal demyelinating motor neuropathy. Venkateswaran Ramesh, Conclusions: Many of the genes were shared between dHMN and motor CMT2, indicating identical MD disease mechanisms; therefore, we suggest changing the classification and including dHMN also as Edit Franko, MD, PhD a subcategory of Charcot-Marie-Tooth disease. Abnormal neuromuscular transmission in some Angela Pyle, PhD genetic forms provides a treatable target to develop therapies.
    [Show full text]
  • Redefining the Specificity of Phosphoinositide-Binding by Human
    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.20.163253; this version posted June 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins Nilmani Singh1†, Adriana Reyes-Ordoñez1†, Michael A. Compagnone1, Jesus F. Moreno Castillo1, Benjamin J. Leslie2, Taekjip Ha2,3,4,5, Jie Chen1* 1Department of Cell & Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801; 2Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205; 3Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218; 4Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205; 5Howard Hughes Medical Institute, Baltimore, MD 21205, USA †These authors contributed equally to this work. *Correspondence: [email protected]. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.20.163253; this version posted June 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. ABSTRACT Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employed a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates.
    [Show full text]
  • Association of Gene Ontology Categories with Decay Rate for Hepg2 Experiments These Tables Show Details for All Gene Ontology Categories
    Supplementary Table 1: Association of Gene Ontology Categories with Decay Rate for HepG2 Experiments These tables show details for all Gene Ontology categories. Inferences for manual classification scheme shown at the bottom. Those categories used in Figure 1A are highlighted in bold. Standard Deviations are shown in parentheses. P-values less than 1E-20 are indicated with a "0". Rate r (hour^-1) Half-life < 2hr. Decay % GO Number Category Name Probe Sets Group Non-Group Distribution p-value In-Group Non-Group Representation p-value GO:0006350 transcription 1523 0.221 (0.009) 0.127 (0.002) FASTER 0 13.1 (0.4) 4.5 (0.1) OVER 0 GO:0006351 transcription, DNA-dependent 1498 0.220 (0.009) 0.127 (0.002) FASTER 0 13.0 (0.4) 4.5 (0.1) OVER 0 GO:0006355 regulation of transcription, DNA-dependent 1163 0.230 (0.011) 0.128 (0.002) FASTER 5.00E-21 14.2 (0.5) 4.6 (0.1) OVER 0 GO:0006366 transcription from Pol II promoter 845 0.225 (0.012) 0.130 (0.002) FASTER 1.88E-14 13.0 (0.5) 4.8 (0.1) OVER 0 GO:0006139 nucleobase, nucleoside, nucleotide and nucleic acid metabolism3004 0.173 (0.006) 0.127 (0.002) FASTER 1.28E-12 8.4 (0.2) 4.5 (0.1) OVER 0 GO:0006357 regulation of transcription from Pol II promoter 487 0.231 (0.016) 0.132 (0.002) FASTER 6.05E-10 13.5 (0.6) 4.9 (0.1) OVER 0 GO:0008283 cell proliferation 625 0.189 (0.014) 0.132 (0.002) FASTER 1.95E-05 10.1 (0.6) 5.0 (0.1) OVER 1.50E-20 GO:0006513 monoubiquitination 36 0.305 (0.049) 0.134 (0.002) FASTER 2.69E-04 25.4 (4.4) 5.1 (0.1) OVER 2.04E-06 GO:0007050 cell cycle arrest 57 0.311 (0.054) 0.133 (0.002)
    [Show full text]