Analysing the role of short stop during the formation of synaptic terminals in Drosophila melanogaster [Elektronische Ressource] / Michael Mende

Technau

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2004
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 31 Mo

Extrait

“Analysing the Role of Short Stop during the
Formation of Synaptic Terminals in
Drosophila melanogaster”
Dissertation
zur Erlangung des Grades
Doktor der Naturwissenschaften
Am Fachbereich Biologie
Der Johannes Gutenberg-Universität Mainz
Michael Mende
Geboren am 29.01.1974
in Abidjan, Elfenbeinküste
Mainz, März 2004Tag der mündlichen Prüfung: 07 Juni 2004INDEX I
CHAPTER INDEX

1. INTRODUCTION_________________________________________________________ 1
1.1. Significance of synapses for the function of the nervous system______________________ 1
1.2. Problem leading up to this work ___________________________________________________ 3
1.3. Drosophila melanogaster as a model system for studying the cellular and molecular
mechanisms of synaptic development_______________________________________________ 4
1.4. Short stop phenotypes and its relevance for synapse formation_______________________ 9
1.5. Aim of this study ________________________________________________________________ 12

2. MATERIALS AND METHODS ____________________________________________ 13
2.1. Fly genetics and Cellbiology _____________________________________________________ 13
2.1.1. Fly stock maintenance________________________________________________________ 13
2.1.2. Fly stocks ___________________________________________________________________ 13
2.1.3. Virgin collection and genetic crosses __________________________________________ 15
2.1.4. Ectopic gene expression in embryos and larvae ________________________________ 15
2.1.4.1. Localisation of GFP tagged DCdc42 isoforms in shot mutant background___ 16
2.1.4.2. Generation of mys/shot double mutant_______________________________ 16
GFP2.1.4.3. Recombination of Df(2L)VA23 with UAS-DPxn ____________________ 17
2.1.5. Embryo collection ___________________________________________________________ 18
2.1.6. Whole mount preparation of 0-to 17hr embryos ________________________________ 18
2.1.7. Hand dissection of living embryos and larvae __________________________________ 19
2.1.7.1. Hand dissection of stage 16 embryos _______________________________ 19
2.1.7.2. Hand dissection of stage 17 embryos 19
2.1.7.3. Hand dissection of third instar larvae________________________________ 20
2.1.7.4. Dissection of larval CNSs ________________________________________ 20
2.1.8. Antibody staining ____________________________________________________________ 21
2.1.8.1. Fluorescence staining ____________________________________________ 23
2.1.8.2. Biotin Staining _________________________________________________ 23
2.1.8.3. Alkaline Phosphatase staining _____________________________________ 23
2.1.9. Mounting of preparations _____________________________________________________ 23
2.1.10. Analysis of embryos and documentation 24
2.2. Generation of an antibody specific for Shot________________________________________ 24
2.2.1. Western Analysis ____________________________________________________________ 25
2.2.1.1. Protein extraction from third instar larvae ____________________________ 25 INDEX II
2.2.1.2. SDS polyacrylamide gel electrophoresis (PAGE) ______________________ 25
2.2.1.3. Western blotting ________________________________________________ 26
2.2.1.4. Immunodetection on the blotted membrane___________________________ 27
2.3. Molecular Biology _______________________________________________________________ 27
2.3.1. Generally applied methods ___________________________________________________ 27
2.3.1.1. Sterilisation of solutions and utensils________________________________ 27
2.3.1.2. Photometric measurements________________________________________ 27
2.3.1.4. Optic density (OD) of bacterial and yeast cultures _____________________ 28
2.3.2. Bacteriological methods ______________________________________________________ 28
2.3.2.1. Cultivation of bacteria ___________________________________________ 28
2.3.2.2. Making of competent cells 28
2.3.2.3. Transformation of competent cells__________________________________ 29
2.3.3. Mating-based Yeast Two-Hybrid Screening ___________________________________ 29
2.3.3.1. Construct fusion genes 30
2.3.3.2. Generation of primers____________________________________________ 31
2.3.3.3. Polymerase Chain Reaction (PCR) _________________________________ 31
2.3.3.4. Agarose gel electrophoresis _______________________________________ 32
2.3.3.5. Cloning of the PCR amplified shot gene fragments_____________________ 32
2.3.3.6. Isolation of plasmid DNA from bacteria _____________________________ 33
2.3.3.7. Restriction enzyme digestion of plasmid DNA ________________________ 33
2.3.3.8. Gel purification of restriction enzyme digestions ______________________ 34
2.3.3.9. Ligation of digestion products into plasmid vector pAS2-1 ______________ 34
2.3.3.10. Amplification of the bait-BD vectors_______________________________ 34
2.3.3.11. Sequencing ___________________________________________________ 34
2.3.3.13. Cultivation of yeast cells ________________________________________ 35
2.3.3.14. Colony-lift Filter Assay _________________________________________ 35
2.3.3.15. Plasmid isolation from yeast _____________________________________ 36
2.3.3.16. Large scale plasmid DNA isolation from bacteria _____________________ 36
2.3.3.17. Identification of putative interaction partners of the distinct Shot domains _ 37

3. RESULTS ______________________________________________________________ 38
3.1. Comparative morphological study of different shot mutant alleles___________________ 39
3.2. shot mutants show defects in the organisation of the cytoskeleton in outgrowing
motor neurones___________________________________________________________________ 56 INDEX III
3.3. N- but not C-terminal domains of Shot localise at presynaptic sites of NMJs_________ 60
3.4. Immunohistochemical study using anti-sera to different domains of the Shot protein _ 70
3.4.1. Analysis of shot mutant alleles using antibodies specific to different regions
of Shot _________________________________________________________________________ 76
3.5. Yeast two-hybrid analysis: screening for interaction partners of the N-terminal
domains of Shot __________________________________________________________________ 78
3.5.1. Studies of DPxn in situ _______________________________________________________ 80
3.5.2. DPxn mutant analysis ________________________________________________________ 88
3.6. Genetic strategy to uncover potential factors of the pathway of Shot function ________ 97
3.6.1. Functions of Shot and activated Rho GTPases seem to converge on
common factors________________________________________________________________ 109

4. DISCUSSION __________________________________________________________ 112
4.1. The N-terminus of Shot is essential for the formation of synaptic terminals and its
modular domains mediate different types of interactions ___________________________ 113
4.2. DPxn interacts with the Shot Plakin domain and is potentially required for the
formation of synaptic terminals __________________________________________________ 118
4.3. Genetic interaction between Shot, Rho-like GTPases and DPxn?___________________ 122
4.4. Shot function during synaptogenesis: Conclusions and future prospects ____________ 125

5. SUMMARY ___________________________________________________________ 127

6. APPENDIX I 128
6.1. Chemicals ______________________________________________________________________ 128
6.2. Kit-systems_____________________________________________________________________ 128
6.3. Enzymes and buffers ____________________________________________________________ 128
6.3.1. Restriction Enzymes ________________________________________________________ 128
6.3.2. Other Enzymes _____________________________________________________________ 129
6.4. Equipment 129
6.5. Buffers, solutions and media_____________________________________________________ 130
6.6. Fixative Solutions ______________________________________________________________ 132
6.7. Other materials _________________________________________________________________ 133
6.7.1. Sharpened tungsten wires____________________________________________________ 133
6.7.2. Sylgard_____________________________________________________________________ 133 INDEX IV
6.7.3. Dissection glass needles _____________________________________________________ 133
6.7.4. Membranes for Western Analysis ____________________________________________ 133
6.7.5. Microdissection tools________________________________________________________ 134
6.8. Bacterial strains ________________________________________________________________ 134
6.9.Yeast strains ____________________________________________________________________ 134
6.10.Vectors ________________________________________________________________________ 134
6.11.Oligonucleotides _______________________________________________________________ 136
6.12. DNA/protein markers and quantifying standards_________________________________ 138

7.APPENDIX II___________________________________________________________ 139
7.1. Biochemical confirmation of the interaction between the Shot Plakin domain and
DPxn as revealed by yeast two-