PTPA is required for MIRA complex localization during drosophila neuroblasts asymmetric divisions

PHOSPHOTYROSOYL PHOSPHATASE ACTIVATOR IS REQUIRED FOR MIRANDA LOCALIZATION DURING DROSOPHILA NEUROBLASTS ASYMMETRIC DIVISIONS HUANG ZHENXING (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTER OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY & DEPARTMENT OF ANATOMY NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS I would like to express my heartfelt thanks to my supervisor Prof. Yang Xiaohang for his guidance, patience and encouragement during the entire course of my PhD study. I also like to extend my sincere thanks and gratitude to A/P Liang Fengyi who went beyond his call of duty to look after me in the final stage of my PhD study. My sincere gratitude goes to Dr. Cai Yu for his invaluable help and insightful advices along the way and Chai Phing Chian for his helpful discussion and generously sharing reagents. I also like to thank Dr. Greg Somers and Prof. William Chia for giving me EMS collections to work on. A big thank you goes to Dr. Wang Huashan, Dr. Lin Shupin, Ying Ying, Li Hui, and Cai Ling for their help and friendship. Most importantly, I would like to thank my family for always being supportive throughout my PhD study. I thank my beloved wife for her love, her support and her encouragement especially during the toughest times. I thank my parents for always being understanding and encouraging. Without their support, I would not be able to complete my study. All my love to my late grandfather, thank you for watching over me from above! i    TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii ABBREVIATIONS vi LIST OF FIGURES x LIST OF TABLES xi SUMMARY xii CHAPTER 1: INTRODUCTION 1.1 Drosophila melanogaster as a model organism 1.2 Asymmetry in cell division 1.3 Neuroblasts in Drosophila neurogenesis 1.4 Asymmetric cell division in Drosophila neuroblasts 1.4.1 Cell polarity setting up 1.4.2 Mitotic spindle orientation 12 1.4.3 Asymmetric segregation of cell fate determinants 13 1.4.4 Other proteins required for asymmetric localization of cell fate determinants 16 1.5 Drosophila neuroblasts asymmetric division is regulated by phosphorylation and dephosphorylation 1.5.1 Protein kinases involved in neuroblast asymmetric division 21 21 1.5.2 Protein phosphatases involved in neuroblast asymmetric division 23 1.6 Phosphotyrosyl phosphatase activator (PTPA) & its functions 24 1.7 Objectives 28 CHAPTER 2: MATERIALS AND METHODS 29 2.1 Molecular biology 29 2.1.1 Recombinant DNA methods 29 ii    2.1.2 Bacterial host strains and growth conditions 29 2.1.3 Cloning strategies 30 2.1.4 Transformation of E. coli cells 30 2.1.4.1 Heat shock transformation of E. coli 30 2.1.4.2 Electroporation transformation of E. coli 31 2.1.5 Plasmid DNA preparations 31 2.1.6 Isolation of total genomic DNA 32 2.1.7 Genomic DNA resequencing 33 2.1.8 Site-directed mutagenesis 35 2.2 Biochemistry 36 2.2.1 Frequently used buffers and solutions 36 2.2.2 PAGE and Western transfer of protein samples 37 2.2.3 Immunological detection of proteins and antibodies used 37 2.2.4 Generation of anti-PTPA polyclonal antibody 38 2.2.5 Fusion protein expression 39 2.3 Immunohistochemistry and microscopy 40 2.3.1 Frequently used reagents and buffers 40 2.3.2 Antibodies 40 2.3.3 Fixing and staining of Drosophila larval brains 41 2.3.4 Neuroblast quantification and brain orientation 41 2.3.5 Quantification of Mira localization 42 2.3.6 Scoring of ectopic nuclear Mira 42 2.4 Fly Genetics 42 2.4.1 Fly stocks and growth conditions used in this study 42 2.4.2 Generation of positively labeled neuroblast MARCM clones 43 iii    CHAPTER 3: PTPA IS REQUIRED FOR MIRA COMPLEX LOCALIZATION DURING DROSOPHILA NEUROBLAST ASYMMETRIC DIVISION 45 3.1 Introduction 45 3.2 Results 48 3.2.1 Mapping of the causative mutation in the EMS mutant 48 3.2.2 Disruption of ptpa specifically affects basal cortical localization of Mira complex 50 3.2.3 Subcellular localization of endogenous PTPA 54 3.2.4 PTPA transgene can fully rescue the mislocalization of Mira in ptpa mutant 56 3.2.5 ptpa mutant neuroblasts exhibit ectopic nuclear Mira and Pros during interphase 58 3.2.6 Ectopic nuclear localization of Mira in ptpa mutant neuroblast is Pros dependent 59 3.2.7 PTPA prevents premature Pros-dependent cycle exit in Type I larval brain neuroblasts 60 3.2.8 PTPA acts downstream from or in parallel to Grh in preventing nuclear Pros 63 3.2.9 ptpa mutant neuroblasts generate fewer progenies 65 3.2.10 ptpa is epistatic to aPKC in localizing Mira complex to the basal cortex 66 3.2.11 Mts is upregulated in ptpa mutant neuroblasts. 70 3.2.12 A specific function of Mts that mediates Mira localization requires PTPA 73 3.2.13 PTPA and Flfl not depend on each other for their nuclear localization in neuroblasts 76 3.2.14. PTPA regulates Flfl level but is not required in Flfl-Mira complex formation 77 iv    3.3 Discussion 80 3.3.1 PTPA mediates Mira basal cortical localization as a phosphatase regulator 80 3.3.2 Phosphorylation of Mira and its cortical association 81 3.3.3 PTPA could be downstream from the temporal system in the control of neuroblast aging 82 3.3.4 PP2A independent function of PTPA 83 CHAPTER 4: CONCLUSION 84 REFERENCES 88 v    ABBREVIATIONS aa Amino acid aPKC Atypical protein kinase C APS Ammonium persulphate Ase Asense ATP Adenosine 5’ Triphosphate Aur-A Aurora-A Baz Bazooka bp Base pairs Brat Brain tumor BSA Bovine serum albumin C. elegans Caenorhabditis elegans CaCl2 Calcium chloride cDNA Complementary DNA ChIP Chromatin immunoprecipitation CIP Calf intestinal phosphatase CNN Centrosomin CNS Central nervous system Cy3 Cyanine conjugated Cyc Cyclin DEPC Diethyl Pyrocarbonate DGRC Drosophila Genomics Resource Center Dlg Disc large DNA Deoxyribonucleic acid dNTP Deoxynucleotide triphosphate vi    Dpn Deadpan DSHB Developmental Studies Hybridoma Bank dsRNA Double-stranded ribonucleic acid DTT 1, 4-Dithio-DL-threitol E. coli Escherichia coli ECL Enhanced Chemiluminescence EDTA Ethylenediaminetetraacetic acid EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’- tetraacetic acid ELAV Embryonic Lethal Abnormal Vision EMS Ethlymethane sulfonate Flfl Falafel FRT FLP recombinase recombination target g Grams grh Grainy head GDI Guanine-nucleotide-dissociation inhibitor GFP Green fluorescent protein GMC Ganglion mother cell GST Glutathione-S-Transferase HCl Hydrochloric acid hr Hour IEF Isoelectric focusing IgG Immunoglobulin Insc Inscuteable IPG immobilized pH gradient IPTG Isopropyl-β-thiogalactopyranoside Kb Kilobase vii    KCl Potassium chloride Khc73 Kinesin heavy chain 73 Kv Kilovolts L3 Third-instar larval LB Luria bertani LiCl Lithium chloride Lgl Lethal giant lavea Loco Locomotion defects M Molar MgCl2 Magnesium chloride mins Minutes Mira Miranda ml Millilitre mM Millimolar Mts Microtubule star Mud Mushroom body defective NB Neuroblast N-terminal Amino (NH2) terminal OD Optical density PAGE Polyacrylamide gel electrophoresis PAN Posterior Asense-Negative Par Partitioning defective PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PH3 Phospho-histone H3 Pins Partner of Inscuteable PON Partner of Numb viii    PP2A Protein phosphatase 2A PP4 Protein phosphatase Pros Prospero PTPA Phosphotyrosyl phosphatase activator RNA Ribonucleic acid RNAi RNA interference RT Room temperature RT-PCR Real-time polymerase chain reaction S Serine SDS Sodium Dodecyl Sulphate secs Seconds SOP Sensory organ precursor stau Staufen TE Tris EDTA TEMED N, N, N’, N’ tetramethylethylene diamine Tris Tris (hydroxymethyl) aminomethane Tws Twins UAS Upstream Activator Sequence V Volts Wdb Widerborst wt Wild type β-Gal β-Galactosidase μg Microgram μl Microlitre μM Micromolar 2-D PAGE Two-dimensional polyacrylamide gel electrophoresis ix    Results: PTPA is required for Mira complex localization during Drosophila neuroblast asymmetric divisions Mts protein which requires PTPA, as neither up-regulation of Mts synthesis by the cell nor overexpression of Mts transgene can rescue the Mira mislocalization phenotype in ptpa mutant neuroblast. Moreover, Flfl, which itself is required in Mira basal localization, is also upregulated in ptpa mutant. Although the binding between Flfl and Mira is not disrupted, the downstream steps in localizing Mira must be blocked in the absence of PTPA, so that the up-regulation of Flfl could be the response to the feedback signal in the pathway. How PTPA regulates Flfl or PP4 activity demands further investigation. 3.3.2 Phosphorylation of Mira and its cortical association Atwood and his colleagues have elegantly showed that aPKC phosphorylation of Mira on the five serine residues on its C-terminal is necessary and sufficient to displace Mira from apical cortex (Atwood et al., 2009). However, it has only solved half of the puzzle. In order to asymmetrically localize Mira in neuroblasts, cytoplasmic Mira has to regain its membrane association ability at the basal cortex. Recent discovery of phosphatases involved in asymmetric division of neuroblast provided a very attractive model: dephosphorylation of Mira in cytoplasm allows it to associate to the cell cortex at the basal domain. Results from this study also showed that indeed phosphatases are involved and they have to be carefully regulated by their regulators such as PTPA to localize Mira. However, there is still no direct evidence to show that aPKC-phosphorylated Mira 81 Results: PTPA is required for Mira complex localization during Drosophila neuroblast asymmetric divisions is a substrate of any phosphatase. This study showed that Mira remains in cytoplasm in ptpa mutant neuroblast in metaphase even when aPKC activity is attenuated. This result does not agree well with the phosphorylation and dephosphorylation model, as Mira cannot be phosphorylated by aPKC at first place when aPKC is knocked down, but still mislocalized into cytoplasm in ptpa mutant neuroblasts. Other modifications on Mira or its cortical targeting machinery responsible for appropriate Mira localization must be impaired in ptpa mutant as well. Further investigation using non-phosphorylatable Mira (Mira5A) and ptpa mutant line would definitely shed light on whether dephosphorylation of aPKC-phosphorylated Mira is sufficient to target Mira to cell cortex. 3.3.3 PTPA could be downstream from the temporal system in the control of neuroblast aging Several temporal transcriptional factors and their targets have been recently identified to schedule the termination of post-embryonic neuroblast division in Drosophila. The transcription factor Grh was identified to be a neuroblast target of temporal transcriptional factors to prevent premature nuclear localization of Pros in larval brain neuroblasts (Maurange et al., 2008). The neuroblast phenotypes observed in the ptpa mutant larval brain, such as cytoplasmic Mira/Pros in metaphase and nuclear Pros/Mira in interphase, resemble reported phenotypes of grh370 mutant larval brain neuroblasts and aged neuroblasts in the pupal brain (Maurange et al., 2008). Furthermore, Grh is not prematurely depleted from neuroblasts in the ptpa mutant. Therefore, PTPA 82 Results: PTPA is required for Mira complex localization during Drosophila neuroblast asymmetric divisions could be another target of the temporal transcription factors downstream from or in parallel to Grh to prevent premature neuroblast aging. 3.3.4 PP2A independent function of PTPA The results from my study showed that loss of a specific PP2A activity is, at least partially, responsible for the Mira mislocalization phenotype in ptpa mutant larval brain neuroblasts. Since I have not found a fully active form of Mts that can rescue the neuroblast phenotype in the ptpa mutant, I cannot rule out the possibility that the PP2A independent function of PTPA also contributes in Mira localization. Furthermore, larval brain staining with specific antibodies revealed that PTPA is not co-localized with Mts before NEBD, so it is possible for PTPA to have a PP2A-independent function in the nucleus. Currently, data on the PP2A independent function of PTPA are limited. It has been reported only once in a recent work on the effect of PTPA overexpression in human cancer cell lines (Azam et al., 2007). Structural-directed mutagenesis has identified the amino acid residues in PTPA protein that are crucial for its interaction with PP2A (Chao et al., 2006). Introducing PTPA variants that cannot interact with PP2A into the ptpa mutant identified in this study would provide an excellent opportunity to investigate the PP2A-independent function of PTPA in asymmetric cell division and other biological processes in vivo. 83 Conclusion CHAPTER CONCLUSION Drosophila neuroblasts have been used as model system for studying asymmetric division of stem cells for more than two decades. In earlier years, most investigations on asymmetric division were carried out in embryonic neuroblasts, and many important players involved have been identified. Larval brain neuroblasts have been increasingly used in recent years for basically two reasons. One is that larval brain neuroblasts are more suitable for studying the relationship between defects in asymmetric division and tumorigenesis. The other is that the phenotypes of many mutations can be masked by maternal deposit during embryonic stage and only can be detectable at larval stage. Moreover, recent developments of clonal assays such as MARCM (Lee and Luo, 1999) make the larval brain a valuable system to study the cell autonomous effect of certain mutation. Asymmetric localization of cell fate determinants is one of the most important processes during asymmetric division of Drosophila neuroblasts. Previous works have shown that cell fate determinants are asymmetrically localized on basal cortex in two different complexes. One is adaptor protein Mira and its associated cell fate determinants Pros and Brat (Ikeshima-Kataoka et al., 1997; Lee et al., 2006c); the other is the cell fate determinant Numb and its adaptor protein Pon (Lu et al., 1998). The localization of both basal complexes are controlled by apical proteins such as aPKC (Atwood and Prehoda, 2009; 84 Conclusion Betschinger et al., 2003; Smith et al., 2007; Wirtz-Peitz et al., 2008). Despite recent advances in the asymmetric cell division field, the molecular mechanism of the basal localization of cell fate determinants is still elusive. In this work, I identified PTPA as a novel player involved in asymmetric division of Drosophila neuroblast, which not only solved a piece of the puzzle but also revealed new perspectives to our understanding of the mechanism of basal protein asymmetric localization. In this thesis I show that PTPA specifically mediates the localization of Mira and its associated cell fate determinants but not the other asymmetrically localized cell fate determinant, Numb. In ptpa mutant neuroblasts, Mira is mislocalized into cytoplasm in metaphase. By reducing aPKC activity in both wild type and mutant neuroblasts, I also show that this mislocalization of Mira is not due to the cortical displacement by aPKC phosphorylation reported previously (Atwood et. al.,2009). Thus PTPA acts in the specific Mira cortical localizing machinery that is downstream from or in parallel to aPKC phosphorylation pathway. Another Mira mislocalization phenotype of the ptpa mutant described in this thesis is ectopic nuclear Mira and Pros in interphase neuroblasts. Cell type specific analysis revealed that only Type I neuroblasts exhibit nuclear Mira and Pros; whereas Type II neuroblasts, which not express Pros, have normal interphase Mira localization. Thus, the mislocalization of Mira to the nucleus is likely dependent on ectopic nuclear Pros. Similar results were also reported recently in flfl and jar mutant neuroblasts (Sousa-Nunes et al., 2009). 85 Conclusion The nuclear Pros is correlated with reduced proliferation and cell cycle exit as reported in aged neuroblasts and other mutant neuroblasts (Maurange et al., 2008). Consistently, clonal analysis revealed that the ptpa mutant neuroblast generated fewer progenies and ptpa mutant larval brain also showed premature neuroblast loss. However, the temporal transcription factors that schedule the cell cycle exit of neuroblast seem intact in the ptpa mutant as their downstream target Grh is not prematurely depleted from neuroblasts. Therefore, PTPA acts downstream from or in parallel to the temporal transcription factors and their neuroblast target Grh in preventing premature neuroblast aging. Another striking phenotype in ptpa mutant larval brain neuroblasts is drastic up-regulation of Mts. Although the status of the inhibitory phosphorylation on Y307 is not altered in the ptpa mutant, the Mira mislocalization phenotype is not due to excessive PP2A activity. Genetic analysis showed that reducing Mts in the ptpa mutant exacerbated the Mira mislocalization phenotype. Thus, I conclude that the mislocalization of Mira and Pros is, at least partially, due to loss of a specific activity of PP2A in the ptpa mutant; and PTPA is required for PP2A to acquire such activity to mediate Mira localization. The larval brain phenotype of the ptpa mutant resembles that of the previously reported flfl mutant (Sousa-Nunes et al., 2009), and PTPA and Flfl have same subcellular localizations. Unexpectedly, Flfl protein level in the ptpa mutant brain is significantly higher than that in wild type brain. Subsequent immune-staining and biochemistry analysis showed that the subcellular localization of Flfl and complex formation between Flfl and Mira were not effected 86 Conclusion in the ptpa mutant. I propose that PTPA may functionally interact with Flfl and the up-regulation of Flfl in the ptpa mutant may be the response to the feedback signal from the downstream of the pathway. However, further investigation is required to establish the mechanistic link between PTPA and Flfl. Another immediate future work of this study would be re-introducing mutant PTPA transgenes with altered functional sites, as many functional sites and their corresponding activities have been revealed in recent years, especially by structural-directed mutagenesis. For instance, mutating the sites required for PP2A binding, would uncover the PP2A independent functions of PTPA and mutating the isomerase active site would provide insights in how PP2A acquires its substrate specificity. In summary, PTPA as a novel gene involved in asymmetric cell division mediates Mira localization in both metaphase and interphase during neuroblast asymmetric division. 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Jiang, 2005, The yeast phosphotyrosyl phosphatase activator is part of the Tap42-phosphatase complexes.: Mol Biol Cell, v. 16, p. 2119-27. 96 [...]... 3 Figure 6 lesion in ptpa mutant Figure 7 Disruption of ptpa causes a mislocalization of Mira complex 53 during metaphase Figure 8 Subcellular localization of PTPA 55 Figure 9 Exogenous PTPA can recapitulate endogenous PTPA 57 subcellular localization and restore wild type Mira localization in ptpa mutant Figure 10 ptpa mutant exhibits ectopic nuclear Pros/ Mira in 61 interphase neuroblasts and premature... in the asymmetric cell division of Drosophila neuroblasts PTPA mediates localization of Miranda (Mira) and its associated cell fate determinants during neuroblast asymmetric division in both interphase and metaphase Mira is xii    an obligatory adaptor protein of two cell fate determinants, Prospero (Pros) and Brain tumor (Brat), for their asymmetric localization In ptpa mutant neuroblasts, Mira and... 16 Nuclear localization of Flfl and PTPA in ptpa and flflN42 77 mutant respectively Figure 17 PTPA is not required in Flfl -Mira complex formation or 79 formed a stable complex with Mira LIST OF TABLES Chapter 2 Table 1 Primers used for genomic fragment sequencing 33 Table 2 Mutant primers used for site-directed mutagenesis 35 xi    SUMMARY How a cell divides is one of the most important issues in developmental... in ptpa mutant neuroblasts cannot restore Mira cortical association in metaphase Therefore, PTPA acts downstream from or in parallel to aPKC pathway in asymmetric localization of Mira in metaphase Genetics and biochemistry results suggest that loss of specific activity of Protein phosphatase 2A (PP2A) in ptpa mutant is, at least partially, the cause of Mira mislocalization Together, PTPA mediates Mira. .. organisms, proper asymmetric cell divisions are required in maintaining progenitor cell pools and generating cell diversity for different biological functions In adult organisms, asymmetric divisions are also important in tissue homeostasis and damage repair Any defects in asymmetric cell division may result in tissue degeneration or tumor growth (Wodarz and Gonzalez, 2006) How does a cell division... (adapted from Egger et al., 2008) 7 Introduction 1.4 Asymmetric cell division in Drosophila neuroblasts As a model system, Drosophila has been a workhorse in asymmetric cell division research for more than a decade Many key players that facilitate asymmetric division have been discovered from studying Drosophila neuroblasts All neuroblasts divide asymmetrically into a larger and a smaller daughter cell... governs the mitotic spindle orientation and localization of cell fate determinants, is predetermined before mitosis This polarity information is carried by a well conserved protein complex, Par complex, which was first identified in C elegans The Par complex in Drosophila consists of Drosophila Par-3, also known as Bazooka (Baz), Drosophila Par6 as well as Drosophila atypical protein kinase C (aPKC)... releases Pros and Brat upon the completion of cytokinesis In either pros or brat mutant, Mira is normally localized to the basal cortex Thus basal localization of Mira is independent from its cargo proteins However, in insc mutant Mira crescent 17 Introduction disappears or randomly forms on the cell cortex Therefore, Insc is required for Mira basal localization Another cell fate determinant Numb also... brain Figure 11 PTPA acts downstream from or in parallel to Grh 64 Figure 12 ptpa mutant neuroblasts produce fewer progenies 66 Figure 13 ptpa is epistatic to aPKC and Lgl in localizing Mira complex 68 to the basal cortex x    Figure 14 Mts level increases in ptpa mutant larval brain neuroblasts 71 Figure 15 Neither reducing nor overexpressing Mts in ptpa mutant can 75 reverse the Mira mislocalization... 1999; Wodarz et al., 2000; Wodarz et al., 1999) During the asymmetric cell division, the Par complex is the first protein complex that asymmetrically localizes to the apical cortex The role of the Par complex during the asymmetric division is mainly to exclude basally-localized proteins from apical cortex The most upstream protein of the Par complex is Baz In baz mutants, neither Par6 or aPKC can be . PHOSPHATASE ACTIVATOR IS REQUIRED FOR MIRANDA LOCALIZATION DURING DROSOPHILA NEUROBLASTS ASYMMETRIC DIVISIONS HUANG ZHENXING (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF. this study 42 2.4.2 Generation of positively labeled neuroblast MARCM clones 43 iv  CHAPTER 3: PTPA IS REQUIRED FOR MIRA COMPLEX LOCALIZATION DURING DROSOPHILA NEUROBLAST ASYMMETRIC DIVISION. localization of Flfl and PTPA in ptpa and flfl N42 77 mutant respectively Figure 17 PTPA is not required in Flfl -Mira complex formation or 79 formed a stable complex with Mira LIST OF TABLES