Addressing the role of the cytoskeletal molecules Diaphanous and Profilin in dendritic morphogenesis in Drosophila melanogaster [Elektronische Ressource] / vorgelegt von Madhuri Shivalkar

Madhuri Shivalkar

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Addressing the role of the cytoskeletal
molecules Diaphanous and Profilin in
dendritic morphogenesis in
Drosophila melanogaster







Dissertation
der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München










Vorgelegt von
Madhuri Shivalkar
München 2007



































Dissertation eingereicht am 11.07.2007
Tag der mündlichen Prüfung: 12.09.2007


Erstgutachter: PD Dr. Rüdiger Klein
Zweitgutachter: PD Dr. Angelika Böttger Erklärung


Ich versichere, da β ich meine Dissertation selbstständig, ohne unerlaubte
Hilfe angefertigt, und mich dabei keiner anderen als der von mir ausdrücklich
bezeichneten Hilfen und Quellen bedient habe.

Die Dissertation wurde in der jetzigen oder ähnlichen Form bei keiner anderen
Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken
gedient.



______________________ ______________________
(Ort, Datum) (Madhuri Shivalkar)
























1. Gutachter: Dr Rüdiger Klein
2. Gutachter: Dr Angelika Böttger











To my family The work presented in this thesis was performed from July 2003 to June 2007
in the laboratory of Dr. Gaia Tavosanis at Max-Planck Institute of
Neurobiology Munich, Germany. Index
INDEX………………….....................................................................i
Figures and tables.……………..……………………………....…………………..vi
Abbreviations…................................................................................................ix
SUMMARY....................................................................................xii
Chapter I: INTRODUCTION........................................................... 1
1.1 Dendrites and Dendritic morphology……………………………...…………..1
1.2 Molecular players of dendritic morphology…………………………...………1
1.3 Cytoskeletal molecules and dendritic morphogenesis………………………5
1.4 Model system: Peripheral Nervous System of Drosophila melanogaster...7
1.5 Assay System: RNA interference…...…………………………………...…..11
1.6 RNAi assay: positive products- Diaphanous and Profilin………………….14
1.6.1 Diaphanous...…………………………………………………..…………….14
1.6.2 Profilin…………...……………………………………………………………17
1.6.3 The role of Profilin in Formin function………………………………...…...19
1.6.4 Neuronal role of Diaphanous and Profilin…………………………………20
1.7 Analysis of dendritic branching in vivo and distribution of actin and
microtubule in dendrites…………………………………………………………..22


iChapter II: MATERIALS AND METHODS...……………………….25
2.1 RNA intereference assay……………………………………………………..25
2.1.1 Genomic DNA extraction……………………………………………………25
2.1.2 dsRNA preparation………………….………………………………………25
2.1.3 Injecting dsRNA in embryos……………………………………….……….28
2.2 Fly genetics………………………………………………………………….…29
2.2.1 Drosophila Stocks……………………………...……………………………31
2.2.2 Generation of recombinants………….…………………………………….32
2.2.3 Transgenic flies……………………………………………………………...32
2.3 MARCM (mosaic analysis with repressible cell marker)……….………….33
2.4 Time Lapse Imaging…………………………………………………….…….33
2.5 Image acquisition and processing…………………………………………...34
2.6 Quantitative analysis……………………………………………………….….34
2.7 Immunohistochemistry………………………………………………….….….35
2.8 Western blot analysis………………………………………….………………36

Chapter III: RESULTS……............................................................37
RNA INTERFERENCE SCREEN………...………………………………………37
3.1 Standardizing RNAi assay system………………………………………….37
3.1.2 Selection of molecules………………………………………………………42
ii3.1.3 RNAi assay…………………………………………………………………..47
3.1.4 Interesting candidates with no significant dendritic phenotype…………49
3.1.5 Interesting candidates with interesting phenotype……………………….50
GENETIC ANALYSIS…………………………………………………...………...53
4.1 Diaphanous………………..…………………………………….……………..53
4.1.1 Gain of function analysis……………………………………………………54
4.1.1.1 Overexpression of dia in class I neurons……………………………….55
4.1.1.2 Overexpression of diaphanous in class IV neurons………………...…58
4.1.2 Loss of function………………………………………………………………60
K071354.1.2.1 Dendritic phenotype of null mutant dia ……………………………60
K07135 4.1.2.1.1 dia – dendritic over branching phenotype in class I neurons…60
K07135 4.1.2.1.2 dia – no dendritic phenotype in class IV neurons……………...64
54.1.2.2 Dendritic phenotype of null mutant dia …………………………………65
5 4.1.2.2.1 dia – dendritic over branching phenotype in class I neurons……...65
5 4.1.2.3 Dendritic phenotype in trans-allelic combination of null mutants- dia
K07135and dia ………………………………………………………………………...67
4.1.4 Western blot analysis: no Diaphanous protein in null mutants…………68
4.1.5 Expression pattern using antibody staining………………………………69
4.1.6 MARCM- generating homozygous mutant clones in heterozygous
animals………………………………………………………………………………70
4.1.7 Analysis with deficiency…………………………………………………….70
iii2-214.1.8 Dystrophin: insertion of Gal4 causes a dendritic phenotype!............72
4.1.9 Dendritic over branching phenotype of dia null mutants is lost in
heterozygous marker condition……………………….…………………………..76
4.2 Profilin (chickadee)…………………………………………………………….78
4.2.1 Gain of function analysis……………………………………………………79
4.2.1.1 Overexpression of chic in class I and class IV neurons………………79
4.2.2 Loss of function analysis……………………………………………………81
221 11 374.2.2.1 Dendritic phenotype of null chic and hypomorphic chic and chic
alleles……………………………………………………...………………………...81
4.2.3 Expression pattern using antibody staining……………………………....83
2214.2.4 MARCM- generating homozygous mutant clones in heterozygous chic
animals………………………………………………………….…………………..83
4.3 chickadee and diaphanous: analyzing the interaction…………….……….85
4.4 Time lapse analysis……………………………………………………………88
4.4.1 Standardizing time lapse assay………………………………...………….88
4.4.2 Imaging dendrites in vivo over time………………………………………..89
4.5 How do actin and microtubule contribute to dendrite formation?..............91

Chapter IV: DISCUSSION.…........................................................93
5.1 RNA interference screen……………………………………………………...93
5.2 Diaphanous: Role in dendritic morphogenesis……………………………..96
iv5.3 Dystrophin……………………………………………………………….……101
5.4 Diaphanous: Ambiguous results from loss of function analysis………...102
5.5 Profilin…………………………………………………………………………103
5.4 Higher order branches of vpda neuron are dynamic at late larval
stages………………………………………………………………………………105
5.5 Localization of actin and tubulin varies in class I vpda neuron…….……106

CONCLUDING REMARKS..……………………………………….108

REFERENCES............................................................................109

ACKNOWLEDGEMENTS ..........................................................119

CURRICULUM VITAE.................................................................120
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