Identification and characterization of Syndapin I, Vacuolar protein sorting 35 and Neurobeachin as new interaction partners of the glycine receptor [Elektronische Ressource] / von Isabel del Pino Pariente

Isabel Meredith

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2010
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 53 Mo

Extrait

Identification and characterization of
Syndapin I, Vacuolar protein sorting 35 and
Neurobeachin as new interaction partners
of the glycine receptor


Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften

vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe -Universität
in Frankfurt am Main



von Isabel del Pino Pariente
aus Valencia (Spanien)



Frankfurt 2010
(D30) Die vorliegende Arbeit wurde in der Abteilung Neurochemie am Max-Planck-Institut für
Hirnforschung in Frankfurt am Main unter Anleitung von Prof. Dr. Heinrich Betz durchgeführt
und vom Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe -
Universität in Frankfurt am Main als Dissertation angenommen.










Dekan: Prof. Dr. Dieter Steinhilber

Gutachter: Prof. Dr. Ernst Bamberg
Prof. Dr. Heinrich Betz

Datum der Disputation:


Table of contents 
Abbreviations....................................................................................................................... I 
1  SUMMARY.................... 1 
2  INTRODUCTION ........................................................................................................... 3 
2.1  Inhibitory neurotransmitters in the central nervous system...........4 
2.2  Ligand-gated ion channels .................................................................................................4 
2.3  Glycine receptors (GlyRs)...5 
2.3.1  Molecular structure and diversity of GlyRs.....................................................................................5 
2.3.2  GlyR assembly ................................................................6 
2.3.3  Localization of glycine receptors in the central nervous system....................7 
2.3.4  Clustering of glycine receptors at postsynaptic sites.....................................8 
2.3.5  GlyR neuronal trafficking and diffusion...........................................................9 
2.4  Objectives of the present study.......................12 
2.4.1  Syndapin .......................................................................................................................................13 
2.4.2  Vacuolar protein sorting 35 (Vps35)..............................17 
2.4.3  Neurobeachin20 
3  MATERIALS AND METHODS .................................................................................... 22 
3.1  MATERIALS ........................................................22 
3.1.1  Chemicals and plastic materials...................................22 
3.1.2  Enzymes........................................................................22 
3.1.3  Kits ................................................................................23 
3.1.4  DNA standard ...............................................................23 
3.1.5  Protein standard...........................................................................................23 
3.1.6  Membranes and films ...................................................23 
3.1.7  Oligonucleotides24 
3.1.8  Organisms.....................................................................................................................................25 
3.1.9  Cell lines........26 
3.1.10  Antibodies...26 
3.1.11  Solutions and media ...................................................................................................................28 
3.1.12  Vectors........................................30 
3.1.13  Plasmid constructs.....................31 
3.2  MOLECULAR BIOLOGY METHODS .................................................................................32 
3.2.1  Alcohol precipitation of nucleic acids...........................32 
3.2.2  Isolation and purification of plasmid DNA from E. coli XL1-Blue..................32 
3.2.3  Determination of DNA concentration by spectrophotometry .......................................................33 

3.2.4  DNA sequencing ...........................................................................................................................33 
3.2.5  Polymerase chain reaction (PCR).................................33 
3.2.6  Cloning procedures.......................34 
3.2.7  Preparation of glycerol stocks ......................................................................38 
3.2.8  Preparation of chemo-competent bacterial cells..........................................38 
3.2.9  Genotyping of SdpI -/- mice.........38 
3.3  PROTEIN BIOCHEMISTRY METHODS.............................................40 
3.3.1  Colorimetric determination of protein concentration....................................................................40 
3.3.2  Discontinuous Polyacrylamide Gel Electrophoresis (PAGE).........................40 
3.3.3  Coomassie staining of protein gels...............................................................40 
3.3.4  Silver staining of protein gels........................................................................41 
3.3.5  Western blot analysis....................................................41 
3.3.6  Expression of recombinant proteins in E. coli BL21.....42 
3.3.7  Affinity purification of GST- and His- fusion proteins...................................................................43 
6
3.3.8  GST pull-down assay for the analysis of protein-protein interactions..........45 
3.3.9  Co-immunoprecipitation ...............................................................................................................45 
3.3.10  Production of polyclonal antibodies...........................46 
3.3.11  Purification of polyclonal antibodies by antigen affinity chromatography ..................................46 
3.4  METHODS FOR VIRAL INFECTION ..................................................................................47 
3.4.1  Calcium phosphate transfection for the production of recombinant adeno-associated virus (rAAV)
47 
3.4.2  Preparation of HEK 293T detergent extract for rAAV purification ................................................48 
3.4.3  rAAV purification through an iodixanol gradient ...........................................48 
3.4.4  rAAV infection in primary cultures.................................................................48 
3.5  CELL BIOLOGY METHODS...............................49 
3.5.1  Coating of coverslips and well plates with poly-L-ornithine.........................................................49 
3.5.2  Culture and maintenance of HEK 293T and COS-7 cells.............................49 
3.5.3  Freezing and thawing of cell lines.................................................................49 
3.5.4  Preparation of rat hippocampal neuron cultures ..........................................50 
3.5.5  Preparation of spinal cord neuron cultures...................................................50 
3.5.6  Lipofection of HEK 293T and COS-7 cells51 
3.5.7  Preparation of detergent extract from HEK 293T cells.................................................................51 
3.5.8  Fractionation of spinal cord neuron homogenate.........51 
3.5.9  Immunocytochemistry ..................................................................................................................52 
3.5.10  Immunohistochemistry................52 
3.5.11  Confocal microscopy, image acquisition and analysis...............................................................53 
4  RESULTS..................................................................................... 55 
4.1  Analysis of the interaction between the Sdp protein family and the GlyRβ subunit ...55 
4.1.1  Characterization of the interaction between Sdp protein family members and the GlyRβ subunit55 
4.1.2  Interaction between Sdp proteins and GlyRβ in a mammalian cell expression system ...............62 
4.1.3  Interaction between Sdp proteins and gephyrin...........................................................................68 

4.1.4  SdpI competes with gephyrin for binding to GlyRβ .....................................................................71 
78
4.1.5  Localization of SdpI in spinal cord neurons..................73 
4.1.6  Analysis of SdpI function at inhibitory synapses..........75 
4.2  Analysis of the interaction between vacuolar protein sorting 35 (Vps35) and the GlyRβ
subunit..........................................................................................................................................86 
4.2.1  In vitro analysis of the