The function of DTRAF1 in drosophila neuroblast asymmetric cell division

THE FUNCTION OF DTRAF1 IN DROSOPHILA NEUROBLAST ASYMMETRIC CELL DIVISION WANG HUASHAN NATIONAL UNIVERSITY OF SINGAPORE 2006 THE FUNCTION OF DTRAF1 IN DROSOPHILA NEUROBLAST ASYMMETRIC CELL DIVISION WANG HUASHAN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF ANATOMY NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS Firstly, I would like to thank my parents, sister, wife and all the relatives I love and love me for their support, love and encouragement throughout all these years. Secondly, I would like to thank A/P Xiaohang Yang and Prof. William Chia, for their taking me as a student in the lab, their continuous guidance and supervision throughout these years. Thirdly, I would like to thank the members of my post-graduate committee, A/P Mingjie Cai, A/P Li Benjamin and Dr. Sami Bahri, for their invaluable discussions and suggestions pertaining to the project. Fourthly, I would like to thank the past and present members of BC/YXH lab and LSC lab, especially Dr. Yu Cai and Dr. Chanhe Chen, for encouraging discussions, suggestions, assistance and all the happy time spent together. In addition, I would like to acknowledge the contributions of the various administrative and technical staffs in IMCB, especially Mohd Sharudin bin for his help on the generation of antibodies. All to my grandmother, you are always in my heart. 4th April, 2006 Wang Huashan i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii ABBREVATIONS vi SUMMARY xiii Chapter1. Introduction 1. Asymmetric cell division in C. elegans 1.1 The establishment of the anterior-posterior (AP) body axis 1.2 The formation of cortical domains 1.3 Cortical domains are required for asymmetric events in the early embryo 1.3.1 The control of spindle positioning during the first mitotic division of the C. elegans zygote 1.3.2 The polarized distribution of cell-fate determinants along the AP axis 2. Asymmetric cell division in Drosophila melanogaster 10 13 2.1 Asymmetric division of neuroblasts in the Drosophila central nervous system 2.1.1 Establishment of apical–basal NB polarity 2.1.2 Asymmetric localization of cell fate determinants and ii 13 15 the control of spindle orientation in NBs 17 2.1.3 Cell size regulation during NB divisions 22 2.1.4 Asymmetric localization and function of cell-fate determinants 25 2.1.5 Telophase rescue and Insc-independent mechanism 28 2.2 Asymmetric division of sense organ precursor cells in the Drosophila peripheral nervous system 2.3 Establishing polarity in Drosophila germline cyst during oogenesis 3. The cell polarity and asymmetric cell division in vertebrate 30 33 37 3.1 Asymmetric cell divisions during neurogenesis in developing vertebrate central nervous system 3.2 Establishing cell polarity in mammalian epithelial cells 4. TNF pathway 37 45 48 4.1. TNF 49 4.2. TNF receptor 50 4.3. Tumor necrosis factor receptor associated factor 53 4.3.1. TRAF family members 53 4.3.2. Domains and structures of TRAF proteins 54 4.3.3. Recruitment of TRAFs to signaling receptors 55 4.3.4. TRAF-activated signal transduction pathways 57 4.3.4.1. TRAF-mediated activation of NF-κB 57 4.3.4.2. TRAF-mediated activation of JNK 58 iii Chapter2. Materials and Methods 60 2.1 Molecular work 61 2.1.1 Recombinant DNA methods 61 2.1.2 Strains and growth conditions 61 2.1.3 Cloning strategy 62 2.1.4 Transformation of E. coli cells 63 2.1.5 Plasmid DNA preparation 64 2.1.6 Site-directed mutagenesis 68 2.1.7 PCR reaction 69 2.1.8 Protein analysis 70 2.1.9 Generation of polyclonal antibody 70 2.1.10 In vitro protein binding assay 72 2.2 Fly genetics iv 72 2.2.1 Embryo fixing 73 2.2.2 Whole embryo RNA in-situ hybridization 75 2.2.3 Embryo antibody staining 77 2.2.4 Double-stranded RNA interference 78 2.2.5 Mobilization of EP-element 79 2.2.6 Inverse PCR 80 2.2.7 Fly genomic DNA extraction 81 2.2.8 Single fly DNA extraction 82 2.2.9 Southern blot for the detection of deletion in fly genome 82 2.2.10 Germ line transformation 84 2.2.11 Ectopic expression 85 2.2.12 Antibodies 85 2.2.13 Confocal analysis and image processing 85 Chapter3. Result and discussion 87 3.1 Background 88 3.2 Results 92 3.2.1 DTRAF1 is apically localized in mitotic NBs 92 3.2.2 Cell fate determinants Mira/Pros and Pon/Numb are normal in DTRAF1 mutant NBs 96 3.2.3 DTRAF1 is required for Mira/Pros normal crescent formation at metaphase in insc NBs 97 3.2.4 Mira telophase rescue is compromised in the absence of DTRAF1 99 3.2.5 Apical localization of DTRAF1 is required for Mira/Pros telophase rescue 102 3.2.6 Egr, Drosophila homolog of TNF, is involved in Mira telophase rescue 3.2.7 DTRAF1 interacts with Baz in vitro 3.3 Discussion 104 107 107 Reference 114 Appendix 131 v ABBREVIATIONS a.a. amino acid APC adenomatous polyposis coli protein Baz bazooka bp base pair BSA bovine serum albumin CIP calf intestinal phosphatase Crb crumbs CRD cysteine-rich domain C-terminus carboxy-terminus DaPKC Drosophila atypical protein kinase C DASK Drosophila apoptosis signal-regulating kinase DD death domain DIAP Drosophila inhibitor-of-apoptosis protein DISC death-inducing signaling complex DNA deoxyribonucleic acid DLG discs Large DmPar6 Drosophila melanogaster Par6 E-APC epithelial-cell-enriched APC ECM extracelluar matrix E.coli Esherichia coli EDTA ethylenediamine tetraacetic acid Esg escargot F-actin filamentous actin FADD fas associated death domain GDI guanine-nucleotide dissociation inhibitor GFP green fluorescent protein GJ gap junction GMC ganglion mother cell vi GST glutathione S-transferase Gαi α subunit of heterotrimeric G protein Gβ13F β subunit of heterotrimeric G protein on chromosome 13F Hid head involution defective IgG immunoglobin G Insc inscuteable IP immunoprecipitation IPTG isopropyl-1-thio-β-D-galactopyranoside Kb kilobases Lgl lethal giant larvae LT lymphotoxin Mira miranda MTOC microtubule-organizing center MZ marginal zone MAGUK membrane-associated guanylate kinase N-terminus amino-terminus PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction PDZ domain PSD-95, discs large, ZO-1 domain Pins partner of Inscuteable Pon partner of Numb Pros prospero Rpr reaper Scrib scribbled Sna snail SPCC sperm pronucleus/centrosomal complex Std stardust TIM TRAF-interacting motif TIR Toll/IL-1R homology region TLR Toll-like receptor vii TNF tumor necrosis factor TNFR tumor necrosis factor receptor TRAF tumor necrosis factor associated factors TRADD TNFR-associated death domain WT wild-type Wor worniu ZA zonula adherens Summary In the past decade, Drosophila melanogaster has been proven to be an excellent model for studying the mechanisms of asymmetric cell division, which generates cell diversity during animal development. 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Antibiotics Stock solutions Working Antibiotic concentration Concentration Storage (dilution) Ampicillin (sodium 50 mg/ml in water –20°C salt) 100 µg/ml (1/500) 34 mg/ml in Chloramphenicol –20°C 170 µg/ml (1/200) ethanol Kanamycin 10 mg/ml in water –20°C 50 µg/ml (1/200) Streptomycin 10 mg/ml in water –20°C 50 µg/ml (1/200) Tetracycline HCl mg/ml in ethanol –20°C 50 µg/ml (1/100) Table2. Laemmli SDS-PAGE Gel Unit: ml Lower Gel(20ml) 5% 8% 10% 12% 15% 20% 30% Acry-Bis 3.33 5.33 6.67 10 13.3 65% Sucrose 0.77 1.18 1.56 2.5 3.13 4.66 10X Lower Buffer 20 H2 O 13.8 11.48 9.75 7.5 4.88 10% APS 1/100 TEMED 1/100 131 Appendix Unit: ml Stacking Gel (10 ml) 4% 30%Acrylamide-bis 1.34 X Stacking buffer 2.5 H2 O 6.1 10% APS 1/100 TEMED 1/500 Table3. EST clones used in RNA in-situ hybridization Cloning Sites Sequence Primers RNA Vector EST Digestion enzyme 5’-end 3’-end 5’ 3’ Polymerase GH pOT2 EcoRI XhoI T7 PM001 SP6 EcoRI LP pOT2 EcoRI XhoI T7 PM001 SP6 EcoRI SD pOT2 EcoRI XhoI T7 PM001 SP6 EcoRI AT pOTB7 EcoRI XhoI PM001 T7 T7 EcoRI RE pFLC-I XhoI BamHI T7 T3 T3 NotI(SacI, EcoRI) RH pFLC-I XhoI BamHI T7 T3 T3 NotI(SacI, EcoRI) EcoRI XhoI T3 T7 T7 NotI(EcoRI) EcoRI XhoI T7 PM001 SP6 EcoRI pBlue-SK(01001LD 21096) LD pOT2(21101~) pFLC-I~3.0kb 132 pOT2-1665bp pOTB7-1815bp Appendix Table4. Fly stocks used Name Genotype Source DTRAF1L2 Deletion of the DTRAF1 coding region Make in this study nem22-DTRAF1L2 Double mutant of Insc and DTRAF1 Make in this study eiger66 Deletion of the Eiger coding region Make in this study nem22-eiger66 Double mutant of Insc and Eiger Make in this study eiger66--DTRAF1L2 Double mutant of Eiger and DTRAF1 Make in this study nem22 EMS allele for inscuteable laboratory collection BazXi106 Null allele for bazooka gene laboratory collection Transgenic fly carrying UAS-Gαi construct UAS-Gαi laboratory collection for UAS/Gal system overexpression Delete N-terminus about 300 aa of Pins, pins laboratory collection p89 antigen-minus allele Delete most coding region of pins locus, pins laboratory collection p62 antigen-minus allele gal4 driver inserted into the downstream of laboratory collection sca-gal4 scabrous promoter region Transgenic fly carrying UAS-insc (full UAS-insc length) construct for UAS/Gal system laboratory collection overexpression Table5. Primers used in this study 133 Appendix Name Sequence Pwht1 5’-ACG CTA ATC ACT CCG AAC AGG TCA CA-3’ Plac1 5’-CAC CCA AGG CTC TGC TCC CAC AAT-3’ Plac4 5’-ACT GTG CGT TAG GTC CTG TTC ATT GTT-3’ Pry1 5’-CCT TAG CAT GTC CGT GGG GTT TGA AT-3’ Pry2 5’-CTT GCC GAC GGG ACC ACC TTA TGT TAT T-3’ Pry4 5’-CAA TCA TAT CGC TGT CTC ACT CA-3’ Plw3-1 5’-TGT CGG CGT CAT CAA CTC C-3’ Sp1 5’-ACA CAA CCT TTC CTC TCA ACA A-3’ Spep-1 5’-GAC ACT CAG AAT ACT ATT C-3’ T3 5’-GTA ATC CGA CTC ACT ATA GGG C-3’ T7 5’-AAT TAA CCC TCA CTA AAG GG-3’ FLP-5’ 5’- GAG AAG TTC CTA TTC CGA AGT TCC -3’ FLP-3’ 5’- ACT TTC TAG AGA ATAG GAA CTT CGG-3’ FLP-5’ -1 5’-CGC ACT AGT T TC TCG GTA CTA TGC -3’ FLP-3’-1 5’-GCA GCA TAT TAC AGC CGT ATG GGT C-3’ malE 5’-GGTCGTCAGACTGTCGATGAAGCC-3´ M13 5’-CGCCAGGGTTTTCCCAGTCACGAC-3’ Eiger-NdeI-5’ 5’-ccatATGACTGCCGAGACCCTCAAGCCG -3’ Eiger-3’ 5’-ccTTACACCTTGAAGATGCCAAAGTAGCTTCGG -3 Eiger-180-3’ 5’-ccTTACTGCCAGATCGTTAGTGCGAG-3’ Eiger-180-NdeI-5’ 5’-ccatATGACAACGCGTGTATCGCATCTGGAC-3’ Wengen-NdeI-5’ 5’-ccatATGATGCCGCCAAGACTGCCAGGCGG -3’ Wengen-3’ 5’- ccTCAGCCCTTCAGGCCGGAACAGCCGC-3’ Wengen-600-3’ 5’-ccTCAAGTCTGCCAGTCAAGGACCCAGG-3’ 134 Appendix Wengen-600-NdeI-5’ 5’-ccatATGGGCGTTCTTTACGTGGCCGTGC-3’ DTRAF1-NcoI-5’ 5’-cgcgccATGGTTCGAAGTTTGGCCCAGTGG-3’ DTRAF1-999-3’ 5’-ccTTACAGAGTGCCTGTGTAGTTGATGG-3’ DTRAF1-670-EcoRI-5’ 5’-gatcGAATTCTCGGCTGACACACTGCCC-3’ DTRAF1-1000-NdeI-5’ 5’-cgcgcatatgTTGTGGAAGATCACCGACTGGTCG-3’ DTRAF1-XhoI-3’ 5’-ggccctcgagTTAGACGGCCACTATCTTGCTG-3’ 135 [...]... embryonic cells 12 Chapter1 Introduction 2 Asymmetric cell division in Drosophila melanogaster Drosophila melanogaster provides another excellent model for understanding the mechanisms behind asymmetric cell division during animal development The complexity of neuronal cell types in the central nervous system (CNS) of Drosophila is generated by the asymmetric cell division of stem-celllike precursors, neuroblasts... proteins are present in the NB cortex and could thus be more directly involved in the targeting or tethering of cell- fate determinants to the basal cortex Interestingly, Lgl binds to the non-muscle myosin II Zipper and restricts the protein to the apical cortex Myosin II is activated by Rho kinase and regulates the basal localization of the determinants by excluding them from the apical cortex During... central region of Insc, which is sufficient for the asymmetric localization of Insc, is also required for its interaction with Baz and Pins At the same time, the carboxyl terminus of Insc, containing the predicted a-helices, binds to the carboxyl terminus of Staufen (Tio et al., 1999) Insc is necessary for the asymmetric segregation of basal cell fate determinants and the spindle reorientation In insc mutant... localization of cell fate determinants and the control of spindle orientation in NBs Within dividing NBs, two conserved apical complexes act together with the actin cytoskeleton to divide the cell cortex into apical and basal domains One consists of the atypical protein kinase C (DaPKC) and two PDZ (PSD95/Discs large/ZO1 domain)-containing proteins, DmPar6 and Bazooka (Baz), the other is composed of the GoLoCo... motif-containing protein, ‘Partner of Inscuteable’ (Pins) and its associated G-protein subunit (Gαi) An adaptor protein, Inscuteable (Insc), forms the ‘apical complex’ by linking these two apical complexes by the direct interaction of Insc with Pins and Baz (Allison et al., 2004) All these six proteins are colocalized in the apical cortex of NBs In each group, the apical localization of each member is interdependent... restricted to the anterior cortex of the one -cell zygote after the SPCC-induced polarization of the AP axis The RING-finger protein PAR-2 and the serine/threonine kinase PAR-1 become restricted to the posterior cortex (Levitan et al., 1994; Guo & Kemphues, 1995; Boyd et al., 1996) The boundaries of these two cortical domains abut roughly midway along the AP axis In the absence of any one of the anterior... the faithful segregation of determinants Extrinsic mechanism involves cell cell communication In metazoans, interactions between daughter cells or between a daughter cell and other nearby cells could specify daughter cell fate Recent studies have indicated that a combination of intrinsic and extrinsic mechanisms specify distinct daughter cell fates during asymmetric cell divisions I will focus my introduction... myosin II prevents determinants from localizing apically At anaphase and telophase, myosin II moves to the cleavage furrow and appears to “push” rather than carry the determinants into the GMC Therefore, the movement of 21 Chapter1 Introduction myosin II to the contractile ring not only initiates cytokinesis but also completes the partitioning of the cell- fate determinants from the NB to its daughter cells... centrosome away from the cortex, leading to basal displacement of the spindle 24 Chapter1 Introduction 2.1.4 Asymmetric localization and function of cell- fate determinants The invariability of the lineage of neurons and glia that each NB produces could be a function of either invariant extrinsic cues each NB and its progeny receive or a stereotyped segregation pattern of intrinsic cell fate determinants Recent... Recent studies indicate that intrinsic factors play important roles in cell fate determination during NB division During the asymmetric division of NBs, the cell- fate determinants and their adaptor proteins are segregated into future GMCs and control the GMC development Two cell- fate determinants, Prospero (Pros) and Numb, have been characterized in the NB Pros is a homeodomain-containing transcription . to the anterior cortex of the one -cell zygote after the SPCC-induced polarization of the AP axis. The RING-finger protein PAR-2 and the serine/threonine kinase PAR-1 become restricted to the. pole. In the absence of centrosomes, the Figure 1. Cell polarity in the C. elegans zygote. The cell cortex of the zygote is divided into distinct anterior (red) and posterior (green) domains. The. during asymmetric cell divisions. I will focus my introduction on the asymmetric cell divisions that occur during the early divisions of Caenorhabditis elegans embryos and the development of