Structural and functional organization of synaptic proteins in Drosophila melanogaster [Elektronische Ressource] / Rui Tian. Betreuer: Stephan Sigrist

Rui Tian

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2011
• Nombre de lectures: 26
• Langue: English
• Poids de l'ouvrage: 12 Mo

Extrait

Structural and Functional Organization of
Synaptic Proteins in Drosophila melanogaster

Thesis for a doctoral degree
Faculty of Biology, Julius-Maximilians-Universität
Würzburg,


Submitted by

Rui Tian

From Peking

Würzburg 2011





Submitted on:
Office stamp

Members of the Promotionskomitee:

Chairperson: Prof. Dr. Thomas Dandekar

Primary Supervisor: Prof. Dr. Stephan Sigrist

Supervisor (Second): Prof. Dr. Erich Buchner



Date of Public Defence:

Date of receipt of Certificates:


Erklärung



Erklärung gemäß § 4 Absatz 3 der Promotionsordnung der Fakultät für Biologie der
Bayerischen Julius-Maximilians-Universität Würzburg vom 15. März 1999.


Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig verfasst habe
und keine anderen als die von mir angegebenen Quellen und Hilfsmittel verwendet
habe. Alle aus der Literatur entnommenen Stellen sind als solche gekennzeichnet. Die
Dissertation wurde weder in gleicher noch in ähnlicher Form bereits in einem anderen
Prüfungsverfahren vorgelegt. Zuvor habe ich keine akademischen Grade erworben
oder zu erwerben versucht.



Rui Tian

Würzburg, den 15.03.2011  Acknowledgements

I would like to thank my instructor and supervisor Prof. Dr. Stephan Sigrist
for giving me the opportunity to conduct exciting cutting-edge scientific
studies in his research group. During my whole thesis period, I was
encouraged by our extensive and enthusiastic discussion, from which I
really have learned a lot. His talent and passion in science impressed me
really. I would also like to thank my supervisor Prof. Dr. Erich Buchner,
Prof. Dr. H. Hilger Ropers, Dr. Andreas W. Kuss and Prof. Dr. Volker
Haucke for sharing their experience in discussions and supporting me on
my project. Prof. Dr. Erich Buchner gave me deep impression of his
kindness and nice personality.

I am appreciating the nice cooperation with Jasmin Podufall, Till Andlauer,
Dr. Carolin Wichmann, Dr. Wernher Fouquet, Karen Liu, Omid
Khorramshahi, Harald Depner and Sara Mertel. I also thank Dr. Tobias
Schwarz, Frauke Christiansen-Engelhart, David Owald, Dr. Robert Kittel,
Dr. Carola Sigrist, Dr. Birgit Greiner and Christian Werner for the nice
working atmosphere, their contributions to this work, and constant support.
In addition, I thank Christine Quentin, Claudia Wirth and Franziska Zehe
for the indispensable and excellent technical assistance, and Eva Albero for
friendly and reliable coordination.

Thank you to my friends, particularly YuMei, DongJiao, HaiMing and Xue
for supporting me in difficult times, while always letting me feel positive and
happy. I would like to thank my parents MeiYe and HaoYi and my family,
which always encouraged me in my life. Sincerely, I thank my husband,
Zhen Liang, who supported me spiritwise always during these years.
Specially, I thank my dear son, Jia Hui for bringing me a wonderful life and
encouraging me in my life.
1Contents
CONTENTS ..................................................................................................................................... 2 
1. ZUSAMMENFASSUNG .............................................................................................................. 4 
1. SUMMARY ... 7 
2. INTRODUCTION ........................................................................................................................ 9 
2.1 SYNAPSE ............. 9 
2.2 PRESYNAPSE ..... 10 
2.2.1 Exocytosis and endocytosis ..................................................................................................... 10 
2+2.2.2 Ca  ions in synaptic transmission ........................................................................................... 11 
2.2.3 Structural and molecular organization of the AZ .................................................................... 11 
2.2.4 ELKS/CAST/ERC/Brp proteins .................................................................................................. 13 
2.3 POSTSYNAPTIC SPECIALIZATION ...................................................................................................... 15 
2.4 SYNAPTIC TRANSPORT ..................................................................................................................... 18 
2.5 APOPTOTIC DEATH OF NEURONS  20 
2.5.1 Cell death intracellular effectors ............................................................................................. 21 
2.6. FUNCTIONAL ANALYSIS OF SYNAPTIC AND NEURONAL PROTEINS IN DROSOPHILA MELANOGASTER . 23 
2.6.1 Drosophila melanogaster as a model organism for genetic analysis ................................... 23 
2.6.2 The Neuromuscular junction of Drosophila ............................................................................. 24 
2.6.3 Organization and development of Drosophila NMJ synapses ................................................. 26 
2.7 OBJECTIVES ..................................................................................................................................... 27 
2.7.1 DREP proteins .......................................................................................................................... 27 
2.7.2 GIT ............ 28 
2.7.3 DJM‐1 ....... 32 
3. MATERIALS AND METHODS ................................................................................................. 33 
3.1 MOLECULAR BIOLOGY ..................................................................................................................... 33 
3.1.1 Materials .. 33 
3.1.2 Buffer solutions ....................................................................................................................... 34 
3.1.3 Construction of fluorophore tagged proteins .......................................................................... 35 
3.1.4 In situ hybridization ................................................................................................................. 40 
3.1.5 Production of DREP‐2 antibody ............................................................................................... 44 
3.2 DROSOPHILA MELANOGASTER ........................................................................................................... 49 
3.2.1 Fly culturing ............................................................................................................................. 49 
3.2.2 Transgenesis ............................................................................................................................ 49 
3.2.3 The UAS/Gal4 system and drivers ........................................................................................... 49 
3.2.4 FLP‐FRT recombination deletion ............................................................................................. 51 
3.2.5 P‐element imprecise excision screen ....................................................................................... 52 
3.3 YEAST TWO-HYBRID ........................................................................................................................ 53 
3.4 CELL CULTURE AND COIP ............................................................................................................... 54 
3.4.1 Cell culture .............................................................................................................................. 55 
3.4.2 Cell lysis ................................................................................................................................... 55 
3.4.3 Immunoprecipitation............................................................................................................... 55 
3.4.4 Western blot  56 
3.5 NMJ IMMUNOHISTOCHEMISTRY ...................................................................................................... 56 
3.5.1 Material................................................................................................................................... 56 
3.5.2 Larval body wall preparation .......................................................................