Nucleocytoplasmic shuttling of ecdysteroid receptor [Elektronische Ressource] / vorgelegt von Katarzyna Maria Betańska

FAKULTÄT FÜR NATURWISSENSCHAFTEN
UNIVERSITÄT ULM








Nucleocytoplasmic shuttling of ecdysteroid receptor










DISSERTATION
Zur Erlangung des Doktorgrades (Dr. rer. nat.)
an der Fakultät für Naturwissenschaften
der Universität Ulm
vorgelegt von
Katarzyna Maria Beta ńska
aus
Wroc ław, Polen







Ulm, 2007



Amtierender Dekan:
Prof. Dr. Klaus-Dieter Spindler





Erstgutachter:
Prof. Dr. Klaus-Dieter Spindler, Abteilung Allgemeine Zoologie und Endokrinologie
Universität Ulm





Zweitgutachter:







Tag der Promotion:





Die Arbeiten im Rahmen der vorliegenden Dissertation wurden in der Abteilung
Allgemeine Zoologie und Endokrinologie der Universität Ulm durchgeführt und von
Herrn Prof. Dr. Klaus-Dieter Spindler betreut.


TABLE OF CONTENTS

TABLE OF CONTENTS............................................................................................. 3
ABBREVIATIONS...................................................................................................... 7
1. INTRODUCTION 11
1.1. Nuclear receptors ............................................................................................ 11
1.1.1. The ecdysteroid receptor............................................................................. 12
1.2. Nuclear protein transport................................................................................ 13
1.2.1. Nuclear import ............................................................................................. 14
1.2.2. Nuclear export 16
1.2.3. Nucleotides exchange on Ran controls intracellular protein transport ......... 18
1.2.4. Nuclear pore complex structure and function .............................................. 19
1.2.5. Functions of karyopherins beyond nuclear transport ................................... 20
1.3. Cell cycle.......................................................................................................... 21
1.3.1. The restriction point and checkpoints .......................................................... 22
1.3.2. Cell cycle regulation..................................................................................... 23
1.3.3. Cyclin D and transcriptional control ............................................................. 24
1.4. Aims of the project .......................................................................................... 26
2. RESULTS........................................................................................................ 27
2.1. Intracellular localization of ecdysteroid receptor and ultraspiracle ........... 27
2.1.1. Usp is present only in the nucleus ............................................................... 27
2.1.2. EcR is present in the nucleus and cytoplasm .............................................. 27
2.1.3. Usp promotes nuclear localization of EcR ................................................... 29
2.2. Interaction of EcR and Usp with transport proteins..................................... 29
2.2.1. Expression of importin α1 and exportin-1 in mammalian and insect cells ... 29
3 TABLE OF CONTENTS
2.2.2 Importin α1 binds to EcR, Usp and the heterodimer EcR / Usp expressed in
Cos7 cells.............................................................................................................. 32
2.2.3. EcR, but not Usp reacts with exportin-1. Usp impairs the interaction of EcR
with exportin-1. ...................................................................................................... 34
2.3. Energy requirement for nucleocytoplasmic transport of EcR and Usp...... 37
2.3.1. Co-immunoprecipitation of Ran with EcR and Usp...................................... 37
2.3.2. Effect of oligomycin on intracellular distribution of EcR and Usp ................. 39
2.3.3. Knock down experiments with Ran.............................................................. 42
2.3.4. Nuclear export of EcR is controlled by the export receptor exportin-1......... 43
2.3.5. Knock down experiments with exportin-1 .................................................... 44
2.4. Influence of the cell cycle on EcR expression and intracellular distribution
................................................................................................................................. 45
2.4.1. Cell cycle synchronization ........................................................................... 45
2.4.2. EcR expression ........................................................................................... 49
2.4.3. EcR distribution 56
2.5. The influence of insect moulting hormone (muristerone A) on the cell cycle
in CHO-K1 cells....................................................................................................... 56
3. DISCUSSION .................................................................................................. 60
3.1. Intracellular distribution of nuclear receptors .............................................. 60
3.2. Which mechanisms are responsible for the different intracellular
distribution of EcR and Usp? ................................................................................ 61
3.3. Nucleocytoplasmic shuttling and energy requirement ................................ 64
3.4. Components of intracellular transport machinery in mammalian cell lines
and S2 cells............................................................................................................. 66
3.5. Influence of the cell cycle on EcR expression and intracellular distribution
................................................................................................................................. 67
3.6. Influence of muristerone A on cell cycle....................................................... 69
3.7. Application of mammalian cell lines in studying insect receptors ............. 70
4 TABLE OF CONTENTS
4. MATERIALS AND METHODS ........................................................................ 71
4.1. Materials......................................................................................................... 71
4.1.1. Chemicals and reagents............................................................................ 71
4.1.2. Plasmids...................................................................................................... 73
4.1.2.1. pEYFP-C1 ............................................................................................. 73
4.1.2.2. pEYFP-C1 / EcR (YFP-EcR) and pEYFP-N1 / EcR (EcR-YFP)............. 73
4.1.2.3. PEYFP-C1 / Usp (YFP-Usp).................................................................. 74
4.1.2.4. PEYFP-N1 / Usp STOP (Usp) ............................................................... 74
4.1.2.5. “Filler DNA”............................................................................................ 74
4.1.3. Bacteria strain E.coli (Stratagene) 74
4.1.4. Cell lines ...................................................................................................... 75
4.1.5. Kits............................................................................................................... 76
4.1.6. Antibody....................................................................................................... 76
4.2. Methods............................................................................................................ 76
4.2.1. Cellular and biological methods................................................................... 76
4.2.1.1. Cell culture............................................................................................. 76
4.2.1.2. Counting cells using a microscope counting chamber (hemacytometer)77
4.2.1.3. Cell cycle synchronization ..................................................................... 77
4.2.1.4. Application of muristerone A.................................................................. 78
4.2.1.5. Experiments with oligomycin (ATP depletion) and leptomycin B ........... 78
4.2.1.6. RNA Interference................................................................................... 78
4.2.1.7. Transfection........................................................................................... 79
4.2.1.8. Harvesting mammalian cells 79
4.2.1.9. Flow cytometry....................................................................................... 80
4.2.2. Biochemistry ................................................................................................ 80
4.2.2.1. Bradford reaction (Bradford, 1976) ........................................................ 80
4.2.2.2. SDS polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970)81
4.2.2.3. Western blotting (Towbin et al., 1979; Burnette, 1981).......................... 83
4.2.2.4. Optimisation of Western blotting conditions for importin α2................... 85
4.2.2.5. Co-immunoprecipitation......................................................................... 85
4.2.3. Molecular biology................