HECT-type Ubiquitin Ligases in Nerve Cell Development Dissertation in partial fulfillment of the requirements for the award of the degree ”Doctor rerum naturalium” of the Georg August University Göttingen, Faculty of Biology within the doctoral program Neurosciences of the Georg August University School of Science submitted by Mateusz Cyryl Ambrożkiewicz Born in Puławy, Poland Göttingen, 2015 Thesis Committee Prof. Nils Brose, Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Göttingen Dr. Judith Stegmüller, Cellular and Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Göttingen Prof. Ahmed Mansouri, Research Group Molecular Cell Differentiation, Max Planck Institute for Biophysical Chemistry, Göttingen Members of the Examination Board Referee: Prof. Nils Brose, Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Göttingen 2nd Referee: Dr. Judith Stegmüller, Cellular and Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Göttingen 3rd referee: Prof. Ahmed Mansouri, Research Group Molecular Cell Differentiation, Max Planck Institute for Biophysical Chemistry, Göttingen Further members of the Examination Board Camin Dean, Ph. D., Trans-Synaptic Signaling, European Neuroscience Institute, Göttingen Prof. Thomas Dresbach, Department of Anatomy and Embryology, University Medical Center Göttingen Prof. Tobias Moser, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen Date of oral examination: 19th November 2015 II DECLARATION Herewith I declare, that I prepared the Doctoral Thesis "HECT-type Ubiquitin Ligases in the Nerve Cell Development" on my own, and with no other sources and aids than quoted. This work was partly supported by the Ph.D. program "Neurosciences" – International Max Planck Research School at the Georg August University Göttingen. Mateusz Ambrożkiewicz Göttingen, 21st September, 2015 III Niczego w życiu nie należy sie bać, należy to tylko zrozumieć. Nothing in life is to be feared, it is only to be understood. Maria Skłodowska-Curie (1867-1934) You better work. RuPaul Andre Charles (1960- ) Mojej siostrze i mamie. For my sister and my mum. IV I. TABLE OF CONTENTS I. TABLE OF CONTENTS ......................................................................................................... V II. LIST OF ABBREVIATIONS ................................................................................................. IX III. ABSTRACT .............................................................................................................................. X 1. INTRODUCTION................................................................................................................. 1 1.1. Development of cortical neurons in vivo ...................................................................... 1 1.1.1. Neuroepithelial cells ............................................................................................................................. 1 1.1.2. Radial glial cells ................................................................................................................................... 2 1.1.3. Basal progenitors .................................................................................................................................. 2 1.2. Neuronal migration in the developing brain ................................................................ 3 1.2.1. Initiation of radial migration and acquisition of cell polarity in vivo .................................................... 3 1.2.2. Maintenance of migration ..................................................................................................................... 4 1.2.3. Radial migration termination ................................................................................................................ 4 1.3. Neuronal development in vitro ..................................................................................... 6 1.3.1. Axon formation .................................................................................................................................... 6 1.4. Dendritic spines and structural basis of synaptic transmission ................................... 7 1.4.1. Structural plasticity of dendritic spines ................................................................................................ 7 1.4.2. Synaptic transmission and circuit formation......................................................................................... 8 1.5. In utero electroporation in studies of neuronal development ....................................... 8 1.6. Posttranslational control of gene expression: biology of ubiquitylation ................... 10 1.6.1. Nedd4 family ubiquitin ligases of HECT type.................................................................................... 11 1.6.1.1. Nedd4 ligases in neuritogenesis ............................................................................................... 13 1.6.1.2. The role of Nedd4 ligases in regulation of neurotransmitter receptors, ion channels and transporters 13 1.6.2. Molecular basis of Angelman syndrome and the role of Ube3a in developing and mature neurons .. 15 1.6.3. HECT ligases in intellectual disability syndromes ............................................................................. 16 1.6.3.1. Clinical symptoms of Kaufman oculocerebrofacial syndrome................................................. 17 1.6.3.2. UBE3B gene discovery ............................................................................................................ 18 1.6.3.3. Mutations in UBE3B identified in KOS ................................................................................... 18 1.7. Posttranscriptional regulation of gene expression: biology of microRNAs ............... 18 1.7.1. miRNA gene transcription and initial processing of miRNA precursors ............................................ 19 1.7.2. Nucleus-cytoplasm shuttle of pre-miRNAs ........................................................................................ 19 1.7.3. Cytosolic pre-miRNA processing ....................................................................................................... 20 1.7.4. Formation of RNA-induced silencing complex .................................................................................. 20 1.7.5. Strand selection and target mRNA recognition .................................................................................. 20 1.7.6. Molecular mechanisms of mRNA silencing ....................................................................................... 21 1.7.7. The role of Dicer in developing and mature neurons .......................................................................... 23 1.7.8. Biology of intragenic miRNAs ........................................................................................................... 24 1.7.9. Interplay of Wwp2 ubiquitin ligase and intragenic miR-140 in control of gene expression ............... 24 1.8. IN BRIEF .................................................................................................................... 25 2. MATERIALS AND METHODS ........................................................................................ 27 2.1. Animals ....................................................................................................................... 27 2.2. Bacterial and yeast strains ......................................................................................... 27 2.3. DNA ............................................................................................................................ 27 2.4. Oligonucleotides ......................................................................................................... 29 2.5. Antibodies ................................................................................................................... 31 2.6. Molecular biology methods ........................................................................................ 32 2.6.1. Electroporation of plasmid DNA into competent bacteria .................................................................. 32 2.6.2. Plasmid DNA purification .................................................................................................................. 33 2.6.3. Determination of DNA concentration................................................................................................. 33 2.6.4. DNA Sequencing ................................................................................................................................ 33 2.6.5. DNA digestion with endonucleases .................................................................................................... 33 2.6.6. DNA ligation ...................................................................................................................................... 33 2.6.7. DNA precipitation method ................................................................................................................. 34 2.6.8. Isolation of plasmid DNA with phenol/chloroform ............................................................................ 34 2.6.9. Isolation of genomic DNA from mouse tail biopsies.......................................................................... 35 2.6.10. Agarose gel electrophoresis..........................................................................................................