Molecular characterization of the Drosophila mitotic inhibitor Frühstart [Elektronische Ressource] / presented by: Paweł Gawliński

Kaminski

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Dissertation

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences




























presented by:
Pawe ł Gawli ński
Diploma: Master of Biotechnology
born in Olsztyn, Poland
Oral examination:




Molecular characterization of the Drosophila mitotic
inhibitor Frühstart





























Referees: Prof. Dr. Herbert Steinbeisser
PD Dr. Jörg Großhans











































Moim Rodzicom oraz
Prof. dr hab. Annie J. Podhajskiej dedykuj ę
Contents
Contents
Contetns………………………………............…………………………………………… 4
Acknowledgements ...…………………………………............…………………..……… 8
Summary …………………………………………………….............……………………. 9
Zusammenfassung ………………………………….………………............……………. 10
Abbreviations .………………………………………………………………...........…...... 11
Introduction……………………………………………………………………….............. 14
1. Drosophila melanogaster as a model organism……………………………….............. 14
2. The Drosophila egg as a model system…………………………………….............…. 14
3. Cell cycle and its modifications during Drosophila development…………….............. 15
4. Synchronization of cell proliferation with morphogenetic movements in early Drosophila
embryo as an example of switch in developmental programs….................................… 17
5. The role of Drosophila zygotic genes frühstart and tribbles in mid-blastula transition..18
6. Regulation of cell cycle……………………………………............……… 21
Aim of the studies……………………………………………………………............……. 29
Materials…………………………………………………………………………............... 30
1. Reagents……………………………………………………………………….............. 30
2. Radioactivity…………………………………………………………………............... 30
3. Antibiotics……………………………………………………………………............... 30
4. Enzymes……………………………………………………………………….............. 30
5. Peptides………………………………………………………………………............... 30
6. Antibodies and immunochemicals…………………………………………….............. 30
7. Buffers………………………………………………………………………............…. 31
8. Media………………………………………………………………………….............. 32
9. Bacterial strains………………………………………………………………............... 34
10. Yeast strains………………………………………………………………..............… 34
11. Animal cell strains used in the cell cultures………………………………..............… 34
12. Fly stocks…………………………………………………………………….............. 34
13. PCR primer sequences……………………………………………………….............. 35
14. Constructs………………………………………………………………….............… 37
15. Chromatography…………………………………………………………...............… 39
16. Kits for the molecular biology……………………………………………….............. 39
17. Equipment…………………………………………………………………............…. 39

4 Contents
Methods……………………………………………………………………………............. 41
1. Standard methods in molecular biology………………………………………............. 41
2. Isolation of DNA from yeast cells…………………………………………….............. 41
3. Polymerase Chain Reaction (PCR)……………………………………………............. 41
4. Error prone Polymerase Chain Reaction…………………………………….............… 41
5. DNA sequencing…………………………………………………………….............… 42
6. 2-Nitrophenyl- β-galactopyranoside (ONPG) test……………………………............... 42
7. Protein expression and purification…………………………………………................ 43
8. Antibody staining of embryos and tissues…..……………………………............…… 44
9. Estimation of the Frühstart concentration in the embryo……………………................ 44
10. Yeast-Two-Hybrid frühstart ovarian library screen………………………............…. 45
11. Yeast-Tw* mutants library screen………………………............... 46
12. In situ hybridization of nup50 gene………………………………………..............… 47
13. Polyclonal antibodies against DmNup50 protein………………………….............… 48
14. Polyclonal antibodies against C-terminus of DmCdk1 protein……………............… 49
15. Immunoprecipitation of Frs from embryonic lysate……………………….............… 50
16. In vitro binding test with TNT Coupled Reticulocyte Lysate System expressed
proteins……………………………………………………………………............….… 51
17. Surface plasmon resonance (SPR)………………………………............…………… 52
18. Kinase assay……………………………………………………………............…….. 54
19. In-gel tryptic digestion and LC-MS/MS analysis………………………….............… 54
20. FACS analysis……………………………………………………………….............. 55
21. Ventral furrow frs rescue phenotype…………………………………………............ 55
22. In vitro GFP-Frs nuclear export assay in permeabilized HeLa cells………................ 56
23. Transgenic flies…………………………………………………………….............… 57
24. Preservation and analysis of adult Drosophila wings……………………...............… 57
25. Cell cultures………………………………………………………………..............… 58
Results……………………………………………………………………………............... 59
1. Frühstart interacts with Nucleoporins, export factor Crm1 and CyclinE……............... 59
1.1 Yeast-two-hybrid ovarian library screen for Frühstart protein interactors............… 59
1.2 nup50 mRNA and Nup50 protein are present at mid-blastula transition…............... 61
1.3 Frühstart interacts with the N-terminal part of Nup50 in Y2H system….................. 65
351.4 Frühstart interacts with [S ] labelled Nup50 protein in an in vitro binding assay.... 67
1.5 Nup214 affects Frühstart cytoplasmic localization in vivo………………................ 68
5 Contents
1.6 Crm1 affects Frühstart cytoplasmic localization in vivo…………………………….. 70
2. Frühstart directly interacts with the hydrophobic patch of the cyclins but does not stably
interact with Cdk1 in vitro ……………………………………….....................………… 72
2.1 Frühstart specifically interacts with CycE in the Y2H system……………....…...….. 72
352.2 Frühstart interacts with [S ] labelled CyclinA, B, B3 and E, but does not interact with
Cdk1 in an in vitro binding assay……………………………..................................….. 73
2.3 Frühstart interacts with human CyclinA1 and CyclinB1 in the in vitro binding
assay………………………………………………………………………….........….... 74
2.4 Frühstart interacts 2.5 times more strongly with CycA compared to CycE.......…...... 75
2.5 Frühstart reaches a physiological concentration of 100nM in the mid-cellularising
embryo…………………………………………………………...............................….. 79
2.6 Frühstart has two different activities……………………………………..….........…. 80
2.7 Mutation of the Frs KxL motif severely affects Frs-CyclinA complex formation in
vitro…...…………………………………….……………….................................…… 83
2.8 Frühstart interacts with hydrophobic patch of CycA in vitro……..……...........…….. 84
2.9 Frühstart forms a complex with CycA and Cdk1 in vivo………………..............…... 87
2.10 Frs affects Cdk1 and Cdk2 kinase activity………………………………..........…... 88
2.11 The hydrophobic patch is not required for HistoneH1, Rb and LaminDmO substrates
interaction with Cdk1…………………………………………….............................…. 94
2.12 Frs is a Cdk substrate in vitro……………………………………………..........…... 95
2.13 Binding of Frs to the Cyclins is essential for its function in vivo………………….. 101
3. Frühstart is G2/M specific inhibitor in vivo………………………………….....………. 104
3.1 Ectopically expressed Frs during the last (16th) zygotic division blocks mitosis but does
not affect S-phase……..…………………………………………........................…….. 104
3.2 Wing imaginal disc epithelial cells that ectopically expressed frs are bigger and have a
several fold higher DNA content compared to the wild-type…………………….….... 104
3.3 Larval salivary gland cells ectopically expressed frs show normal growth of the tissue
compare to the wild-type………………………………………...........................……. 105
4. Frühstart genetically interacts with CycA, CycB, CycB3 and CycE………….........….. 106
4.1 Frühstart genetically interacts with CycB3 in the Drosophila eyes…….…......……. 106
4.2 Frühstart genetically interacts with cycA, cycB and cycE in the Drosophila wing
imaginal discs…………………………………………………………................……. 107
Discussion……………………………………………………