Miranda localization during asymmetric cell division of neuroblasts in Drosophila melanogaster [Elektronische Ressource] / Veronika Erben

Erben

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• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 49
• Poids de l'ouvrage: 2 Mo

Extrait

Lehrstuhl für Zellbiologie der Technischen Universität München
Wissenschaftszentrum Weihenstephan


MIRANDA LOCALIZATION DURING ASYMMETRIC CELL
DIVISION OF NEUROBLASTS IN DROSOPHILA MELANOGASTER

Veronika Erben


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernäh-
rung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des
akademischen Grades eines

Doktors der Naturwissenschaften (Dr. rer. nat.)

genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. Dr. h. c. (Univ. Kaposvári/Ungarn) Johann Bauer

Prüfer der Dissertation: 1. Univ.-Prof. Dr. Bertold Hock, em.
2. Univ.-Prof. Angelika Schnieke, Ph.D. (Univ. of Edinburgh/UK)






Die Dissertation wurde am 08.03.2007 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 30.04.2007 angenommen.
.
INDEX
LIST OF ABBREVIATIONS V
1 ZUSAMMENFASSUNG VIII
2 ABSTRACT X
3 INTRODUCTION 1
3.1 The Development of Drosophila melanogaster 1
3.1.1 Stages of Embryogenesis 1
3.1.2 Early Development (Oogenesis) 1
3.1.3 Maternal Contribution 4
3.2 The Nervous System of Drosophila melanogaster 5
3.2.1 Structure of the Nervous System 5
3.2.2 Development of the Ventral Nerve Cord 5
3.2.2.1 Overview 5
3.2.2.2 Patterning of the Ventral Nerve Cord 6
3.2.2.3 Neuroblast Formation 7
3.2.2.4 Neuroblast Delamination and Division 8
3.2.2.5 Neuroblast Specification 9
3.2.2.6 Ganglion Mother Cell Specification 9
3.3 Asymmetric Cell Division 10
3.3.1 Mechanism of Asymmetric Stem Cell Division 10
3.3.2 Asymmetric Cell Division in Drosophila Neuroblasts 11
3.3.2.1 Regulation of Cell Division by an Apical Protein Complex 11
3.3.2.2 Proteins show Dynamic Localization during Neuroblast Mitosis 13
3.3.2.3 Characterization of the Miranda Protein 14
3.3.2.4 The Miranda Complex 16
3.3.2.5 Miranda Localization Depends on Myosin II and Myosin VI 16
3.3.2.6 The PON/Numb Complex 17
3.3.2.7 Regulation of the Mitotic Spindle during Neuroblast Mitosis 17
4 AIM OF THE WORK 19
5 MATERIAL AND METHODS 20
5.1 Chemicals and Other Materials 20
5.1.1 Chemicals 20
5.1.2 Ready-to-Use Systems 20
5.1.3 Vectors 20
5.1.4 Bacterial Strains 20
5.1.5 Molecular Weight Markers 21
5.1.6 Enzymes 21
5.1.7 Antibodies 21
5.1.7.1 Primary Antibodies 21
5.1.7.2 Secondary Antibodies 22
5.2 Cultivation of Drosophila Melanogaster 22
5.2.1 Keeping Drosophila
5.2.2 Fly Food 23
5.2.2.1 Standard Fly Food 23
5.2.2.2 Apple Agar 24
5.2.2.3 Yeast-Sugar Solution 24
5.3 Genetics 25
5.3.1 Drosophila Stocks 25
5.3.1.1 UAS-Lines 25
II INDEX
5.3.1.2 GAL4-Lines 26
5.3.1.3 Other Stocks 26
5.3.1.4 Balancer Stocks 27
5.3.2 Genetic Methods 27
5.3.2.1 The Basics of a Cross 27
5.3.2.2 Nomenclature 27
5.3.2.3 Genetic Tools 28
5.3.2.4 Microinjection of Drosophila Embryos 29
5.3.2.4.1 Germline Transformation 29
5.3.2.4.2 Production of Injection Mix 30
5.3.2.4.3 Injection Needles 30
5.3.2.4.4 Microinjection 30
5.3.2.4.5 The “Set-Up” 31
5.3.2.4.6 Isolation and Balancing of Transgenic Flies 31
5.3.2.5 RNA Interference 31
5.3.2.5.1 Mechanism of RNAi 31
5.3.2.5.2 RNAi of Myosin VI in Drosophila Embryos 32
5.3.2.6 Injection of Rho-Kinase Inhibitor (RKI) 32
5.4 Immunohistochemistry 32
5.4.1 Embryo Staining with Antibodies 32
ZZ1765.4.2 miranda Germline Clones 33
5.4.3 Whole Mount In-situ Hybridization 34
5.5 Live Imaging and Fluorescence Recovery After Photobleaching 34
5.5.1 Technical Data 34
5.5.2 Live Imaging 34
5.5.3 Fluorescence Recovery After Photobleaching 35
5.6 Molecular Biology 35
5.6.1 Polymerase Chain Reaction (PCR) 35
5.6.1.1 Conditions 35
5.6.1.2 Primer 36
5.6.2 Sequencing 37
5.6.3 Competent Bacteria 37
5.6.4 Transformation
5.6.5 Preparation of Plasmid DNA 38
5.6.6 Ligation 38
5.6.7 Restriction Digest 38
5.6.8 In vitro Transcription 38
5.7 Biochemistry 39
5.7.1 Production of Drosophila Embryo Extract 39
5.7.2 Immunoprecipitations 39
5.7.2.1 Principle 39
5.7.2.2 Immunoprecipitation of Miranda 40
5.7.3 SDS-PAGE and Western Blotting 40
5.7.3.1 Sample Preparation 40
5.7.3.2 Gel Electrophoresis 40
5.7.3.2.1 Blotting, Blocking and Staining 41
5.7.3.2.2 Developing 41
6 RESULTS 42
6.1 Regulation of Miranda Localization by Myosin Dependent Mechanism 42
6.1.1 Underlying Genetic Mechanism: The UAS-GAL4 system 43
6.1.2 Miranda Localizes via the Cytoplasm to the Basal Cortex 44
6.1.3 Mechanism of Dynamic Miranda Localization 46
III INDEX
6.1.3.1 Miranda is not Associated with the Endoplasmatic Reticulum 46
6.1.3.2 miranda mRNA and Protein Show Distinct Localization 48
6.1.3.3 Miranda is not Locally Degraded at the Apical Side of the Cell 49
6.1.3.4 Distinct Modes of Miranda Localization in the Cytoplasm and on the Cortex 49
6.1.3.5 Myosin II and Myosin VI Act at Distinctive Steps During ACD 53
6.1.3.6 PON Localization Depends on Myosin II but not on Myosin VI 55
6.1.3.7 The N-terminal 300 Amino Acids of Miranda are Required for Interaction with the
Cortex 56
6.2 Miranda is Required for Embryonic Development 58
6.2.1 Underlying Genetic Mechanism: The FLP/FRT System 58
ZZ1766.2.2 Cross Breeding to Generate miranda Germline Clones 60
ZZ1766.2.3 Phenotype of miranda Germline Clones 61
6.3 Regulation of Miranda Localization by Post-Translational Modifications 62
6.3.1 Miranda Encodes Four Isoforms 63
6.3.2 Miranda is Phosphorylated In vivo 64
7 DISCUSSION 66
7.1 Miranda Moves via the Cytoplasm to the Basal Side of the Cell 66
7.2 PON and Miranda Localization are Differently Regulated 67
7.3 miranda mRNA and the ER are Asymmetrically Inherited in Drosophila Neuroblasts 68
7.4 Apical Degradation does not Contribute to Dynamic Miranda Localization 69
7.5 Miranda Localization is Regulated by Diffusion, Myosin II, and Myosin VI 69
7.6 Model of Miranda Localization 71
7.7 Miranda is Essential During Early Embryogenesis 71
7.8 Miranda Localization may be Regulated by Phosphorylation 72
7.9 Relevance of ACD for Mammalian Stem Cell Biology and Cancer 73
8 REFERENCES 75
9 ACKNOWLEDGEMENTS 89
IV ABBREVIATIONS
LIST OF ABBREVIATIONS
α anti
aa amino acid
ac achaete
ACD asymmetric cell division
ac/scachaete–scute complex
A/P anterior-posterior
APC adenomatous polyposis coli
aPKC atypical protein kinase C
ase asense
Baz Bazooka
BMP bone morphogenic protein
BSA Bovine serum albumin
Cas Castor
CIAP calf intestine alkaline phosohatase
CLIP cytoplasmic linker protein
CNS central nervous system
CyO curly wing
ddonkey
DEB Drosophila embryo buffer
DEX Drosophila embryo extract
DIGdigoxygenin
Dlg Discs large
DppDecapentaplegic
dsRNA double stranded RNA
DTS dominant temperature sensitive
DTT dithiothreitol
D/Vdorsal-ventral
e ebony
EB1end-binding protein 1
eGFPenhanced GFP
EGFR epidermal growth factor receptor
ElpEllipsoid
ER endoplasmatic reticulum
E(spl) Enhancer of split
FLP flippase
FRT flippase recognition target
FRAP fluorescence recovery after photobleaching
g goat
V ABBREVIATIONS
GAL genes induced by galactose
Gh/Grh Grainyhead
GFP green fluorescent protein
GMC ganglion mother cell
GRP glucose-regulated protein
Hb Hunchback
HRPhorseradish peroxidase
hs-flpheat shock-flippase
IHC immunohistochemistry
Ind intermediate neuroblast defective
InscInscuteable
IP immunprecipitation
KDELlysine (K)-aspartic acid(D)-glutamic acid (E)-leucine (L)
KrKrüppel
LB Luria broth
Lgl Lethal giant larvae
L´sc Lethal of scute
m mouse
msh muscle segment homeobox
NEB nuclear envelope breakdown
nls nuclear localization signal
NGS normal goat serum
NFnuclear factor
O/N over night
pa pro analysis
Par partitioning defective
PAGE polyacrylamid gel electrophoresis
PBS phosphate buffered saline
PBT phosphate buffered saline with triton
pc pole cells
PCR polymerase chain reaction
pdmPOU domain proteins
PTB phosphotyrosine-binding
PDZ Post synaptic density protein/Discs large/Zonula occludens protein
Pins Partner of Inscuteable
PNS peripheral nervous system
PON Partner of Numb
PreIS preimmune serum
PTB phosphotyrosine binding domain
PVDF Polyvinylidene fluoride
r rat
VI
ABBREVIATIONS
rb rabbit
RFP red fluorescent protein
RISCRNA-induced silencing complex
RKIRho-kinase inhibitor
RLC regulatory light chain
RNAi RNA interference
ROIregion of interest
RRX Rhodamine Red-X
RT room temperature
Sb Stubble
sc scute
sca scabrous
SDS sodium