Characterization of LINA, a new RING-Finger protein with axon growth promoting activity [Elektronische Ressource] / vorgelegt von Jieun Lee

Characterization of LINA a new RING-Finger
protein with axon growth promoting activity



Dissertation
zur Erlangung des Doktorgrades Dr. rer. nat.
der Fakultät für Naturwissenschaften der Universität Ulm



vorgelegt von


Jieun Lee
aus Samchok, Südkorea
2009 2


















Amtierender Dekan: Prof. Dr. Peter Bäuerle

Erster Gutachter: Prof. Dr. Harald Wolf
Zweiter Gutachter: Dr. Wolfgang Stein

Tag der mündlichen Prüfung: 15.06.2009
Contents 3
Contents
Contents....................................................................................................3
Figures ......................................................................................................6
Abstract.....................................................................................................7
Zusammenfassung...................................................................................8
Abbreviations............................................................................................9
1 Introduction ..................................................................................13
1.1 The visual system...........................................................................13
1.2 Difference of glial environment between CNS and PNS.................15
1.2.1 Myelin inhibitory proteins ........................................................................ 16
1.2.2 The glial scar .......................................................................................... 18
1.2.3 Schwann cells......................................................................................... 19
1.3 Changes of intrinsic capabilities for axonal regeneration ...............20
1.4 LINA, a new RING finger protein promoting neurite outgrowth.......21
2 Material..........................................................................................25
2.1 Technical equipments ....................................................................25
2.2 Consumable Materials....................................................................26
2.3 Plasmids.........................................................................................26
2.4 Enzymes.........................................................................................27
2.5 Antibodies.......................................................................................27
2.6 Reagent kits ...................................................................................28
2.7 Chemicals and reagents.................................................................28
2.8 Buffers............................................................................................30
2.9 Cell and Bacterial culture and medium...........................................31
2.9.1 PC12 cells and medium.......................................................................... 31
2.9.2 HEK293 cells and medium...................................................................... 32
2.9.3 RGCs and medium ................................................................................. 32
2.9.4 Hippocampus cells and medium ............................................................. 32
2.9.5 Escherichia coli (E. coli) DH 5 α ™ and medium ..................................... 32
2.10 Rat animal material ........................................................................32
2.11 Oligonucleotide...............................................................................33
2.11.1 Cloning primer for LINA and LINA variants ............................................. 33
3 Experimental Methods .................................................................36 Contents 4
3.1 Cell culture .....................................................................................36
3.1.1 Cell freezing and defreezing ................................................................... 36
3.1.2 PDL and laminin coating......................................................................... 36
3.1.3 Transfection of PC12 cells ...................................................................... 36
3.1.4 Transfection of HEK293 cells by calcium phosphate method.................. 37
3.1.5 Dissociated RGCs cell culture................................................................. 37
3.1.6 Hippocampal cell culture......................................................................... 37
3.1.7 Myelin isolation and preparation ............................................................. 38
3.2 Surgical methods............................................................................39
3.2.1 Optic nerve crush (ONC) ........................................................................ 39
3.2.2 Lens Injury (LI)........................................................................................ 39
3.3 Molecular biological methods .........................................................39
3.3.1 Cloning of LINA....................................................................................... 39
3.3.2 Cloning of LINA-GFP (Green fluorescence protein) and LINA-RFP
(Red fluorescence protein) fusion protein................................................ 41
3.3.3 Phenol/Chloroform DNA purification ....................................................... 41
3.3.4 Site-directed Mutagenesis ...................................................................... 42
3.3.5 In vitro Mutagenesis by Overlap Extension and Splicing PCR................. 42
3.4 Protein biochemical methods .........................................................43
3.4.1 Protein Isolation...................................................................................... 43
3.4.2 Bradford test........................................................................................... 44
3.4.3 Western Blot Analysis............................................................................. 44
3.4.4 Coomasie brilliant blue staining .............................................................. 46
3.4.5 Cross Linking.......................................................................................... 46
3.4.6 Co-immunoprecipitation.......................................................................... 46
3.4.7 Membrane fragmentation........................................................................ 47
3.4.8 GST-fusion protein.................................................................................. 47
3.4.9 Affinity Chromatography for purification of anti-LINA Antibody................ 49
3.5 Immunocytochemistry and Immunohistochemistry.........................49
3.5.1 Immunocytochemistry............................................................................. 49
3.5.2 Immunohistochemistry............................................................................ 50
3.5.3 Haematoxylin and eosin staining (HE staining) ....................................... 50
3.6 Whole Mount In situ Hybridization on post-implantation mouse
embryos..........................................................................................50
3.6.1 RNA probes............................................................................................ 51
3.6.2 Materials for hybridization ....................................................................... 51
3.6.3 Mouse whole mount in situ Hybridization ................................................ 52
4 Results ..........................................................................................55
4.1 Cloning and sequence analysis of LINA.........................................55
4.2 Functional characterization of LINA................................................58
4.2.1 LINA promotes NGF-induced neurite outgrowth of PC12 cells................ 58
C93A4.2.2 LINA abrogates the neurite outgrowth effect of LINA in PC12 cells ... 60
4.3 Biochemical characterization of LINA.............................................62 Contents 5
4.3.1 LINA is a membrane associated protein ................................................. 62
4.3.2 LINA has a glycosylation site at N20....................................................... 63
4.3.3 LINA forms a ternary complex................................................................. 64
4.3.4 LINA forms a ternary complex with its RING finger domain..................... 70
4.4 Subcellular localization of exogenous LINA....................................72
4.5 Investigation of endogenous LINA expression ...............................76
4.5.1 Verification of anti-LINA antibody............................................................ 76
4.5.2 Endogenous LINA expression in cultured primary RGCs and
hippocampal neurons.............................................................................. 79
4.5.3 Subcellular localization of endogenous LINA in Hippocampal neurons ... 81
4.5.4 Investigation of endogenous LINA expression in the rat brain................. 82
4.5.5 Upregulation of LINA expression in PC 12 cells after NGF treatment...... 86
4.5.6 Upregulation of LINA expression after optic nerve crush (ONC) and
lens injury (LI) in retina.......................................