Neuronal differentiation and epithelial integrity [Elektronische Ressource] : the role of Drosophila short stop / Wolfgang Bottenberg

Bottwahn

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2007
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 18 Mo

Extrait

“Neuronal differentiation and epithelial integrity:
The role of Drosophila Short Stop”




Dissertation
zur Erlangung des Grades
Doktor der Naturwissenschaften




Am Fachbereich Biologie
Der Johannes Gutenberg-Universität Mainz


Wolfgang Bottenberg
Geboren am 06.12.1973
in Wiesbaden, Deutschland


Mainz und Manchester, Juni 2006 Chapter Index I
I Chapter Index

1. Introduction 1

1.1. The importance and principle features of neuronal growth 1
1.2. The cytoskeleton as sub-cellular prerequisite for cellular growth 1
1.3. Spectraplakins: Cytoskeletal linker proteins 2
1.4. The Importance of Spectraplakins 4
1.5. The architecture of Spectraplakins 5
1.6. Shot function in the nervous system 7
1.7. Drosophila melanogaster as model system to study neuronal growth and the function of 8
Spectraplakins
1.8. Aim of this study, experimental approach and brief summary 10

2 Materials and methods: 14

2.1. Fly genetics and cell biology 14

2.1.1. Fly stock maintenance: 14
2.1.2. Fly stocks 14
2.1.3. Virgin collection and genetic crosses 16
2.1.4. Embryo collection and dechorionisation 16
2.1.5. Ectopic gene expression in embryos and larvae using the Gal4/UAS system 16
2.1.6. Selection of shot mutant embryos 16
2.1.7. Dissection of stage 17 Drosophila embryonic brains 17
2.1.8. Flat preparation of stage 17 mutant Drosophila embryos 17
2.1.9. Flat preparation of L2-L3 Drosophila larvae 17
2.1.10. Whole mount embryo preparation for in-situ hybridisation or antibody staining 18
2.1.11. Antibody staining 18
2.1.12. X-Gal staining of unfixed Drosophila embryos to select homozygous mutant animals 18
2.1.13. DNA extraction from unfixed Drosophila embryos for “single embryo PCR” 18
2.1.14. Alkaline Phosphatase staining 19
2.1.15. In-situ hybridisation of whole mount embryos 19
2.1.15.2. RNAse inactivation using DEPC (diethylpyrocarbonate) 19
2.1.15.3. In-situ hybridisation 19
2.1.15.4.Hybridisation 19
2.1.15.5. Washing 20
2.1.15.6. Staining 20
2.1.16. Alkaline Phosphatase staining of whole mount embryos 20
2.1.17. Phalloidin staining of embryonic and larval Drosophila flat preps 20 Chapter Index II
2.1.18. Phalloidin injection into stage 17 embryos 21
2.1.19. Documentation of stained specimens

2.2. Generation of transgenic flies 22

2.2.1. Preparation of injection plasmids 22
2.2.1.1. Injection mixture 22
2.2.2. Preparation of embryos: 22
2.2.3. Injection of plasmid DNA 22
2.2.4. Raising of injected embryos 22
2.2.5. Determination of the insertion chromosomes of transgenic flies 23

2.3. Molecular Biology 23

2.3.1. General applied methods 23
2.3.1.1. Sterilisation of solutions and utensils 23
2.3.1.2. Photometric measurements 23
2.3.1.3. Optic density (OD) of bacterial cultures 23
2.3.2. Microbiological methods 23
2.3.2.1. Cultivation of cells 23
2.3.2.2. Competent cells 24
2.3.2.3. Preparation of competent cells 24
2.3.2.4. Transformation of chemical competent cells 24
2.3.3 Molecular methods 24
2.3.3.1. Generation of Primers 24
2.3.3.2. Polymerase chain reaction (PCR) 25
2.3.3.3. Hot start PCR 25
2.3.3.4. Single colony PCR 25
2.3.3.5. Agarose gel electrophoresis 26
2.3.3.6. Gel purification of DNA fragments 26
2.3.3.7. Isolation of plasmid DNA 26
2.3.3.8. DNA isolation from flies 26
2.3.3.9. Restriction digestion and dephosphorylation of DNA 27
2.3.3.10. Ethanol precipitation of DNA 27
2.3.3.11. DNA ligation 27
2.3.3.12. A-Tailing of DNA for TA ligation and cloning 27
2.3.3.13. DNA Sequencing 28
2.3.3.14. RNA gel electrophoresis 28
Chapter Index III
V1042.4. Single embryo PCR (sePCR) to confirm and identify chromosomal lesion points of shot 29
V168and shot

V1042.5. Inverse PCR (iPCR) to map the precise molecular breakpoints of shot 29

2.5.1. Isolation of embryonic DNA 29
2.5.2. Restriction digestion of isolated embryonic DNA 29
2.5.3. Inverse PCR 29

2.6. Generation of digoxygenin (DIG) labelled shot RNA antisense probes 30

2.6.1. Primers and probes 30
2.6.2. Probe synthesis and labelling: After the orientation of the template inserts was 30
2.6.2.1. Labelling of probes 30
2.6.2.2. Precipitation of the RNA probe 31

2.7. Generation of digoxygenin labelled shot domain DNA antisense probes and Dot blot 31

2.7.1. Probe generation 31
2.7.2. Dot blot to asses the labelling success of digoxygenin labelled DNA probes 31

2.8. Generation of Shot constructs for Cell culture expression studies 31

2.8.1. Generation of UAS-coupled expression constructs for S2 cell transfection 31
2.8.1.1. Generation of p{UAST}-Gas2 31
2.8.1.2. Generation of p{UAST}-Gas2-6cmyc 32
2.8.1.3. Generation of p{UAS-EB1aff}-2HA 32
2.8.1.4. Generation of p{UAST}-mRFP 32
2.8.1.5. Generation of p{UAST}-mRFP-GSR 32
2.8.1.6. Generation of p{UAST}-mRFP-GSR+ 33
2.8.2 Generation of Shot construcst for expression studies in fibroblasts 33
2.8.2.1. Generation of pcDNA3-Gas2/GSR/EB1-GFP 33
2.8.2.2. Generation of pcDNA3-Gas2/GSR-GFP 33
2.8.2.3. Generation of pcDNA3-Gas2/EB1-GFP 34

2.9. Cell culture: 34

2.9.1. Drosophila S2 cell culture 34
2.9.1.1. S2 cell maintenance 34
2.9.1.2. Transient cell transfection 34 Chapter Index IV
2.9.1.3. Transfection plasmids 35
2.9.1.4. Immobilisation of S2 cells on coverslips 35
2.9.2. NIH3T3 murine Fibroblast cell culture 35
2.9.2.1. Fibroblast maintenance 35
2.9.2.2. Transfection protocol 35
2.9.2.3. Vertebrate expression vector 36
2.9.3. Immunohistocemistry and Life imaging of cell cultures 36
2.9.3.1. Fixation and immunofluorescence of cells 36
2.9.3.3. Documentation of stained cells 36
2.9.3.4. Life imaging of NIH3T3 Fibroblasts 36

3. Results 37

3.1. Motorneuronal dendrites of Drosophila represent postsynaptic compartments 37

3.1.1. Choice of strategy 37
3.1.2. Generation of p{UAST}-RDL-HA transgenic flies 37
3.1.3. Analysis of motorneurons using p{UAST}-RDL-HA transgenic flies 38

3.2. Shot is required and strongly enriched in developing dendrites 41

3.3. Methodological consideration for the use and generation of constructs for structure-function 45
analysis of shot

3.3.1. Principal strategy and available tools 45
3.3.2. Generation of novel Shot constructs 47
3.3.3. Cloning strategies – general considerations 48

3.4. Structure-function analyses of Shot localisation and function in motorneuronal dendrites 56

3.4.1. Shot-L(C)-GFP localises normally and rescues dendrites 56
3.4.2. The plakin domain is dispensable for Shot localisation to dendrites 59
3.4.3. Constructs with deletions in the rod domain 60
3.4.3.1. Shot-L(A)∆rod1-GFP shows abnormal and normal features at dendrites 60
3.4.3.2.. Partial deletions of the rod domain show stronger aberrations 64
3.4.4. The EF hand is required for proper localisation and function of Shot in dendrites 66
3.4.5. The Gas2 domain is essential for the localisation and function of Shot in dendrites 69
3.4.6. Shot-L(C)∆Gas2-GFP does not affect dendrites 70
3.4.7. The EB1 domain is not crucial for dendritic enrichment of Shot 71
3.4.8. Localisation studies with isolated Shot domains 72 Chapter Index V
3.4.8.1. N-terminal domains seem dispensable for dendritic localisation 73
3.4.8.2. C-terminal domains are not sufficient for dendritic localisation 73
3.4.9. Lessons learned from structure-function analyses of Shot 75

3.5. The F-Actin and microtubule binding capabilities of Shot are essential for the proper 77
development of NMJs and required to organise FasII localisation

3.5.1. Motivation and strategy 77
3.5.2. The N-terminus of Shot is required in neurons 78

3.6. A cross tissue comparison highlights specific nervous system requirements only for the EF- 79
hand domain

3.6.1. Motivation, strategy and preparational work 79
3.6.2. Structure-function analysis of Shot in tendon cells reveals domain requirements 81
3.6.3. Expression of Shot-L(C) ∆Gas2-GFP in tendon cells induces a strong dominant effect 83
3.6.4. Neither the isolated Gas2 nor the isolated EB1aff domain localises specifically in tendon 84
cells

3.7. Comparative analysis of shot phenotypes in different shot mutant alleles 85

3.7.1. Strategic considerations for these analyses 85
kakP23.7.2. Analyses of shot confirm results obtained with UAS-Shot-L(C)-GFP 85
V168 V1043.7.3. Analyses of shot and shot suggest a specific requirement of C-terminal Shot 86
sequences in tendon cells

3.8. Molecular mapping of the breakpoint of the shot alleles V104 and V168 89

V1043.8.1. The Gas2 domain in the shot allele is not affected 90
3.8.1.1. Strategy and preparational work 90
3.8.1.2. Mapping the break points of inversion V104 93
V1043.8.1.3. In silico analysis of the shot gene product 94
V1683.8.2. Mapping the molecular breakpoint of shot 95

3.9. Dissection of the Shot C-terminus: importance