Characterization of MP2 cell division and pins function on spindle asymmetry of drosophila central nervous system

CHARACTERIZATION OF MP2 CELL DIVISION AND PINS FUNCTION ON SPINDLE ASYMMETRY OF DROSOPHILA CENTRAL NERVOUS SYSTEM LIN SHUPING (M.Sc.) DEPARTMENT OF ANATOMY & INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2006 CHARACTERIZATION OF MP2 CELL DIVISION AND PINS FUNCTION ON SPINDLE ASYMMETRY OF DROSOPHILA CENTRAL NERVOUS SYSTEM LIN SHUPING (M.Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY & INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS I would first of all like to thank my supervisor, Associate Professor Yang Xiaohang for taking me under his wings and opening my mind to the fascinating world of Drosophila neurobiology I am also indebted to Professor William Chia for his scientific zeal and lateral thinking Second, I would like to thank the members of my post-graduate committee, A/P Cai Mingjie, A/P Thomas Leung, Ass Prof Sami Bahri, for their invaluable discussions and suggestions pertaining to these projects Third, I would like to thank the past and present members of BC/YXH lab, in particular, Dr Cai Yu, Ass.Prof Sami Bahri for encouraging discussions, suggestions and assistance In addition, I would like to acknowledge the contributions of the various administrative and technical staffs in IMCB, especially to DNA sequencing facility and Media-prep people Lastly, I owe my deepest thanks and appreciation to my husband for his love, understanding and support ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF FIGURES viii ABBREVATIONS x OVERALL SUMMARY xiv CAHPTER Introduction 1.1 Drosophila melanogaster as a model organism 1.2 Asymmetric cell division versus symmetric cell division 1.3 Asymmetric cell division in Caenorhabditis elegans 1.4 Asymmetric cell division in Drosophila melanogaster CNS 1.4.1 Asymmetric localization and segregation of cell fate determinants 17 19 1.4.2 Adaptor proteins Miranda and Partner of Numb (Pon) direct the proper localization of cell fate determinants Pros and Numb, respectively 23 1.4.3 Inscuteable, a pivotal regulator, coordinates asymmetric cell division in NBs 26 1.4.4 Bazooka, DaPKC and DmPar6 complex, a conserved machinery that directs asymmetric cell division 30 1.4.5 Heterotrimeric G-proteins and GDIs are involved in asymmetric cell division of Drosophila CNS 1.4.6 Apical-basal spindle orientation 33 37 iii 1.4.7 Unequal daughter cell sizes 44 1.5 Cell polarity and asymmetric cell division 47 1.6 Cytoskeleton elements are involved in asymmetric cell division 52 1.7 Cell cycle regulation during asymmetric cell division 53 1.8 Asymmetric cell division in vertebrate neurogenesis 54 1.9 Unsolved questions 59 CHAPTER Materials and Methods 2.1 Molecular work 62 63 2.1.1 Recombinant DNA methods 63 2.1.2 Strains and growth conditions 63 2.1.3 Cloning strategy 64 2.1.4 Transformation of E coli cells 64 2.1.4.1 Electroporation mediated transformation 64 2.1.4.2 Heat-shock induced transformation 65 2.1.5 Plasmid DNA preparation 2.1.5.1 Plasmid DNA minipreps (STET boiling method) 66 66 2.1.6 Enzymatic manipulation 67 2.1.7 PCR reaction 67 2.1.8 pKS-ds-T7 vector modification 67 2.2 Fly genetics 2.2.1 Basic fly keeping 68 68 iv 2.2.2 Embryo fixing 68 2.2.3 Embryo antibody staining 69 2.2.4 Double-stranded RNA interference 70 2.2.5 Mobilization of EP-element 71 2.2.6 Fly inverse PCR 71 2.2.7 Fly genomic DNA extraction 72 2.2.8 Genomic DNA Southern Blots 72 2.2.8.1 Radioactive labeling of DNA probes 72 2.2.8.2 Restriction enzyme digestion of genomic DNA 73 2.2.8.3 Gel electrophoresis and Southern blotting of genomic DNA 73 2.2.8.4 Southern hybridization 74 2.2.9 Single fly PCR 75 2.2.10 Generation of Germline clones 76 2.2.11 Generation of Germline clones for double mutants 76 2.2.12 Ectopic expression 77 2.2.12 Antibodies used 77 2.2.13 Confocal analysis and image processing 78 2.2.14 Fly stocks used 78 2.2.15 Primers used in this study 82 CHAPTER Insc-independent asymmetric divisions in the Drosophila embryonic Midline Precursor Cell 84 3.1 Background 85 v 3.2 Results 89 3.2.1 MP2 asymmetric cell division is Insc independent 89 3.2.2 MP2 asymmetric cell division is Pins independent 91 3.2.3 MP2 asymmetric cell division is Baz dependent 92 3.3 Discussion 95 CHAPTER Characterization of Pins function and G protein signaling on spindle asymmetry during Drosophila NB asymmetric cell division 100 4.1 Background 101 4.2 Results 103 4.2.1 Cortical Pins in Gβ13F mutants is directly responsible for the loss of spindle asymmetry 103 4.2.2 The ability of cortically localized Gαi or ectopic expressed Gαo to induce equal size divisions requires Pins 107 4.2.3 Overexpression of chimeric Pins-C-Pon In Gαi mutant embryos eliminate spindle asymmetry 111 4.2.4 Gαi is dispensable for Pins function in the presence of ectopic Gαo 115 4.2.5 Pins/G-protein signaling is involved in cell fate determinant localization 117 4.3 Discussion 120 CHAPTER5 Heterotrimeric G protein α subunit and Pins play a role in the spindle orientation during Drosophila NBs asymmetric cell division 5.1 Background 125 126 vi 5.2 Result 130 5.2.1 Overrexpression of heterotrimeric Gαi or Gαo subunit causes spindle uncoupling 130 5.2.2 Pins provides anchorage signal for spindle orientation together with Gα subunits 135 5.2.3 Overexpression of Gαo can target Pins to the cortical cortex and causes spindle uncoupling in the absence of Gαi 5.3 Discussion Reference list 138 141 144 Publications vii List of Figures Fig 1.1 Two models of asymmetric cell divisions Fig 1.2 Polarized distribution of proteins and displacement of mitotic spindle in C.elegans P0 division Fig 1.3 Delamination and asymmetric cell division of Neuroblasts 18 Fig 1.4 NBs asymmetric cell division 20 Fig 3.1 Numb protein is an asymmetrically localized determinant necessary and sufficient to cell-autonomously specify dMP2 neuronal identity 87 Fig 3.2 Confocal images of wt and mutant embryos 90 Fig 3.3 Localization of proteins asymmetrically localized in NBs in dividing MP2 93 Fig 4.1 Cortical Pins in Gβ13F mutant is responsible for the similar sized NB division phenotype 106 Fig 4.2 Pins is essential for cortically localized Gαi or Gαo to induce equal size divisions 110 Fig 4.3 Ectopic expression of chimeric Pins-C-Pon mimics Pins/Gαi functions in Gαi mutants 113 Fig 4.4 Overexpression of Pins-C-Pon in Gαi mutants can cause reversed NBs division 114 Fig 4.5 Gαi is dispensable for Pins function in the presence of overexpressed Gαo 116 Fig 4.6 G-protein signaling is involved in cell fate determinant localization 118 Fig 4.7 Diagrams depicting roles of heterotrimeric G protein subunits Gai, Gao47A, Gβ13F, Gγ and Pins in mitotic spindle geometry regulation 124 viii Fig 5.1 Cortical Pins/Gαi and cortical Pins/Gαo prevent spindle reorientation by metaphase 133 Fig 5.2 Time-lapse image of live epithelial cell divisions in wild-type and mutant embryos 134 Fig 5.3 Pins provides position cue for spindle orientation 136 Fig 5.4 Gαo can substitute Gαi in targeting Pins to the cell cortex and generating “spindle uncoupling” phenotype in the absence of Gαi Table 1-1 Proteins required for asymmetric cell divisions of the C.elegans and their homologs in Drosophila and mammals 140 ix References Kuchinke,U., Grawe,F and Knust,E (1998) Control of spindle orientation in 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A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY... is Pins independent 91 3.2.3 MP2 asymmetric cell division is Baz dependent 92 3.3 Discussion 95 CHAPTER Characterization of Pins function and G protein signaling on spindle asymmetry during Drosophila. .. asymmetric division come from the invertebrate model systems, Drosophila and C elegans The first division of C elegans one -cell embryos and the embryonic neuroblast (NB) divisions of Drosophila