Genetic analysis of the role of ARP2 3 complex in border cell migration in drosophila melanogaster

GENETIC ANALYSIS OF THE ROLE OF ARP2/3 COMPLEX IN BORDER CELL MIGRATION IN DROSOPHILA MELANOGASTER LU RUIFENG (Ph.D of Science), NUS A THESIS SUBMITTED FOR THE DEGREE OF PhD OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCE NATIONAL UNIVERSITY OF SINGAPORE 2010 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Acknowledgement First of all, I’d like to take this opportunity to express my gratitude to Dr. Pernille Rørth for her supervision and guidance on the project. Her enthusiasm on science has been affecting the group every day. Help from many people contribute to the work. I give my warmest thanks to Dr. Hsin-Ho Sung. Hsin-Ho designed the scheme of the screens and generated fly stock for the screens but he was glad to pass the project to me. I also highly appreciate the daily help from Dr. Adam Cliffe. He has written thousands lines of macros for ImageJ, which liberates us from hours of routine works on movie processing and data sorting. My thanks also go to Dr. Minna Poukkula. She and Dr. Cliffe developed and optimized the protocol for live imaging. Minna taught me live imaging technique and quality control of movies. All the best for her future academic career. Thank Dr. Inaki Mikiko for sharing of stocks and movies on cytochalasin-D drug treated border cells. Thank Dr. Cliffe and Dr. Smitha Vishnu for critical reading and suggestions on the manuscript. Thank Rishta Changede for her discussion on ELMO RNAi data. Thank Nachen Yang for exchanging ideas and discussion on experiments. Thanks also go to Dr. He Yuehui for his continuous encouragement and caring for my research progress. Last but not least, I thank Dr. Stephen Cohen for funding me to finish the thesis at the last year of my PhD study. a Arp2/3 complex in border cell migration Lu Ruifeng 2010 Table of contents Summary·······························································································i List of figures·························································································ii List of symbols·······················································································iv I. Introduction······································································1 1.1 Cell migration········ ·······································································2 1.2 Chemotaxis···················································································4 1.3 Protrusions in migrating cells······························································5 1.4 Actin biochemistry··········································································6 1.5 Biological processes that depend on actin···············································7 1.6 Regulation of actin filament remodeling··················································9 1.7 Arp2/3 complex and formins are actin filament nucleators···························11 1.7.1 Arp2/3 complex······································································11 1.7.2 Formins···············································································13 1.7.3 Arp2/3 complex is essential for many cellular processes······················14 1.8 Regulation of the Arp2/3 complex and Diaphanous···································17 1.9 Regulation of WASP and WAVEs·······················································21 1.10 Regulation of formins·····································································23 1.11 Dendritic nucleation model of actin filament network······························23 1.12 Collective cell migration·································································24 1.13 Border cell provides a good model to study collective migration in vivo········25 1.13.1 Drosophila oogenesis·····························································25 1.13.2 Molecular requirement for border cell migration in Drosophila oogenesis ···········································································28 1.13.3 Live imaging opens doors for studying the dynamics of cell migration in vivo···················································································31 II. Results ···········································································33 2.1 FLP/FRT mosaic screen in border cell··················································33 2.2 mbm germline mutants showed strong border cell migration delay················36 2.3 FRT-l(2)SH 1750 homozygous border cells show migration defect···············39 2.4 MARCM clone analysis of Arp2/3 subunits············································44 2.5 AFG RNAi of Arp2/3 subunits and SCAR·············································49 2.6 Live image analysis of Arp2/3 and SCAR RNAi······································52 2.7 Arp2/3 depleted border cells move slowly·············································54 2.8 Cell motility is intact in Arp2/3 depleted border cells································57 2.9 Extensions formed less in Arp2/3 and SCAR reduction border cells···············58 2.10 Extension lifetime is less affected by Arp2/3 complex······························60 2.11 The productivity of extensions in Arp2/3 depleted border cells···················61 2.12 Early phase and late phase are guided by different mechanisms···················62 2.13 Diaphanous is also important for border cell migration·····························69 b Arp2/3 complex in border cell migration Lu Ruifeng 2010 2.14 Arp2/3 does not affect E-cadherin adhesion··········································73 2.15 Arp2/3 is not required for internalization of membrane material··················75 III. Discussion·······································································78 3.1 FLP/FRT screen············································································79 3.2 Arp2/3 affects border cell migration in cell-autonomous and non cell-autonomous patterns······················································································79 3.3 Both Arp2/3 and Dia are required for border cell to initiate migration·············80 3.4 Arp2/3 is required for cluster movement but not cell motility·······················82 3.5 Arp2/3 and Dia control protrusion formation··········································83 3.6 Arp2/3 complex is required differently for early phase and late phase of migration····················································································83 3.7 Physiological function of protrusions in border cells·································88 3.8 Physiological function of actin dynamics in border cell migration·················91 3.9 Arp2/3 and Dia act in different ways in border cell migration······················93 3.10 Mesanchymal movement and amoeboid movement·································95 IV. Material and methods······················································100 4.1 Fly husbandry·············································································101 4.2 X-Gal staining············································································101 4.3 Somatic mutant clone generation······················································102 4.4 Germ line mutant clone generation····················································103 4.5 Immunofluorescence·····································································104 4.6 Additional clonal analysis·······························································105 4.7 MARCM clone generation······························································105 4.8 Flipout Expression of UAS-driven Genetic Constructs·····························105 4.9 Preparation of egg chambers for live imaging········································106 4.10 Confocal microscopy imaging························································107 4.11 Determination of migration speed ···················································108 References·······················································································109 Publication······················································································125 c Arp2/3 complex in border cell migration Lu Ruifeng 2010 Summary During oogenesis of Drosophila, one group of cells called border cells delaminate from the anterior epithelium and migrate to the oocyte in a stereotypic way. Border cells provide a good system to study cell migration in vivo due to their genetic tractability. arc-p34 was isolated from a border cell mutant clone screen due to its strong effect on border cell migration. arc-p34 encodes the Drosophila homolog of mammalian ARPC2, a component of Arp2/3 complex. When the level of various Arp2/3 components is reduced by RNAi, many border cell clusters fail to initiate the migration. If they initiate migration, these border cell clusters move much slower at first, but migrate normally later, suggesting distinct mechanisms differentially depend on Arp2/3. Single cell tracking shows that Arp2/3-impaired border cells are still motile, but show less directional movement. Thus Arp2/3 may be acting upstream or downstream of guidance cues to steer border cell migration. i Arp2/3 complex in border cell migration Lu Ruifeng 2010 List of Figures Figure 1.1 Function of chemokines is to induce cell migration. Figure 1.2 Migrating cell send out lamellipodia and filopodia. Figure 1.3 Actin is important for various biological processes. Figure 1.4 Models of actin filament nucleation by Arp2/3 and formins Figure 1.5 Arp2/3 is important for the expansion of trichome in Arabidopsis thaliana Figure 1.6 Domains organization in WASPs and WAVEs and the regulation mechanisms. Figure 1.7 Dendritic nucleation model of actin assembly Figure 1.8 Border cells are specified in stage of oogenesis. Figure 1.9 RTK signaling guides border cell migration. Figure 2.1 Germ line mutant for mbm1819 affects oocyte patterning. Figure 2.2 The gene disrupted in FRT-l(2)SH1750 is important for border cell migration. Figure 2.3 Figure 2.3 Border cell migration shows delayed phenotype in FRT-l(2) SH1750 germline mutant clones. Figure 2.4 Figure 2.4 Complementation test between FRT-l(2)SH1750 and small deletions uncovering 38A-38D. Figure 2.5 Abnormal oogenesis in FRT-l(2) SH1750 mutants. Figure 2.6 MARCM clonal analysis of Arc-p34. Figure 2.7 Reduction of Arp2/3 complex subunits or SCAR protein level delayed border cell migration Figure 2.8 AFG RNAi of Arc-p34 showed initiation defect and dramatic migration defect. Figure 2.9 Arp2/3 activity affect border cell migration via reducing directionality. ii Arp2/3 complex in border cell migration Lu Ruifeng 2010 Figure 2.10 The number, size and life time of extensions. Figure 2.11 The productivity of cellular protrusions is reduced if Arp2/3 is depleted from border cells. Figure 2.12 Knocking down Arp2/3 complex causes migration phenotype. Figure 2.13 Knocking down Arp2/3 complex decreased border cell migration speed. Figure 2.14 Analysis of number, length, size and life time of cellular extensions in slbo-Gal4 RNAi border cells. Figure 2.15 Diaphanous affects border cell migration independent of its role in cytokinesis. Figure 2.16 Diaphanous exerts distinct function in early phase and late phase of the migration. Figure 2.17 Quantification of Arp2/3 RNAi or Dia RNAi border cell migration in background of one copy of E-Cadherin. Figure 2.18 Arp2/3 is not essential for internalization of FM4-64 in border cells. Figure 4.1 Scheme of generation of mutant clone with labeled marker. Figure 4.2 Scheme of germline mutant screen. iii Arp2/3 complex in border cell migration Lu Ruifeng 2010 List of Symbols ARP2/3, actin related protein 2/3 EGFR, epidermal growth factor receptor ELMO, engulfment and cell motility EMT, epithelium to mesenchymal transition Ena/WASP, Enabled/vasodilator-stimulated phosphoprotein Mbc, myoblast city Mbm, mushroom body miniature PICK1, protein interacting with Cα-kinase PVR, PDGF and VEGF receptors SCAR, suppressor of cyclic AMP receptor Slbo, slow border cell SOP2, suppressor of profilin WASP, Wiskott-Aldrich syndrome family proteins WAVE, Wiskott-Aldrich verprolin homologous protein iv Arp2/3 complex in border cell migration Lu Ruifeng 2010 I. Introduction Arp2/3 complex in border cell migration Lu Ruifeng 2010 1.1 Cell migration Cell migration is a defining feature of animal cells (Pollard and Cooper 2009), which is crucial for both single cellular organisms and multicellular organisms. Single cell organisms migrate to reach nutrients and to escape from dangers, as well as to facilitate dispersal. In multicellular organism, cell migration is required for embryonic morphogenesis, wound healing and immune surveillance (Pollard and Borisy 2003). One of the earliest examples of migration in development is gastrulation (Montero and Heisenberg 2004). During gastrulation, large groups of cells migrate collectively as sheets to form three embryonic layers: ectoderm, mesoderm, and endoderm. Subsequently, cells migrate out from various epithelial layers to specific location. Interactions with new microenvironment induce them to differentiate to form the specialized cells that make up different tissues and organs. In vertebrates, after gastrulation, neural crest cells are specified at the border of the neural plate and the non-neural ectoderm. The neural crest is a population of migrating, pluripotent cells which appears transiently in the dorsal neuroectoderm. During neurulation, the borders of the neural plate converge at the dorsal midline to form the neural tube. Subsequently, neural crest cells from the roof plate of the neural tube undergo an epithelial to mesenchymal transition (EMT), delaminating from the neuroepithelium and migrating as loosely associated strands or streams throughout the entire embryo and give rise to different tissues, including craniofacial bones and cartilage, the enteric and peripheral nervous systems and pigment cells. Migration is also a prominent component of tissue repair and immune surveillance. 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J Neurosci. 2007 Aug 8;27(32):8563-70. 125 [...]... et al 1997) Since profilin binds to actin monomer, FH1 domains can bind the profilin-G-actin 13 Arp2/ 3 complex in border cell migration Lu Ruifeng 2010 complex near the barbed end of actin filaments It was postulated that formin nucleates new filaments by binding and stabilizing the intermediate actin dimer and trimer (Pruyne et al 2002) Drosophila Diaphanous belongs to the formin protein family, which... investigating its function in vivo Knockout and knockdown experiments showed that the Arp2/ 3 complex is essential for the viability of many cell types The Arp2/ 3 complex appears important in a variety of specialized cell functions that involve the actin cytoskeleton Arp2/ 3 mutant mammalian cells often have lower levels of actin filaments, consistent with the role of Arp2/ 3 in actin filament nucleation Arp2/ 3. .. loss of migration in the epidermal cells (Sawa et al 20 03) The leading edge of the migrating epidermis in Arp2/ 3 depleted C elegans embryos shows a lack of filamentous actin, and the finger like protrusions that normally form are absent (Sawa et al 20 03) One report has revealed the involvement of Arp2/ 3 in guiding longitudinal migration of excretory cells in C elegans (Sanz-Moreno et al 2008) Arp2/ 3 is... These small GTPases in turn activate the Arp2/ 3 complex which initiates a novel F-actin branch at the side of existing filament However the mechanism 23 Arp2/ 3 complex in border cell migration Lu Ruifeng 2010 of how Arp2/ 3 crosslinks actin filaments is still not clear A complex lacking Arp2 can bind to actin filament but loses its nucleation function, supporting the idea that Arp2 and Arp3 form dimer which... the barbed end of actin filaments and inhibit elongation to maintain the G-actin pool Therefore, actin 10 Arp2/ 3 complex in border cell migration Lu Ruifeng 2010 filament growth depends on the competition between nucleators and capping factors High-affinity binding of capping factors determines the length of F-actin and limits the number of free barbed ends, which reduces the rate of G-actin monomer depletion... Goldstein 2009) Roles of Arp2/ 3 complex in Drosophila Rogers et al used RNAi to systematically study the molecules required for lamella formation in Drosophila S2 cells They found that RNAi knockdown of components of the Arp2/ 3 complex or SCAR/WACE impaired the formation of lamella (Rogers et al 20 03) It has been found that the role of Arp2/ 3 in endocytosis is important in the remodeling of epithelia adhesion... structural base of Arp2/ 3 complex, which suggests that ARPC2 and ARPC4 form the structural core of the complex, with the remaining subunits surrounding them ARPC2 and ARPC4 contact the mother filament, whereas ARP2 and ARP3 associated with the pointed end of the nascent filament (Rouiller et al 2008) The structural organization of Arp2 and Arp3 are similar 12 Arp2/ 3 complex in border cell migration Lu... (Perinuclear binding protein and substrate for protein kinase C) is a neuronal BAR domain-containing protein and regulates postsynaptic trafficking of glutamate receptors A VCA-like domain was identified in PICK1 it was initially suspected to be an activator of Arp2/ 3 Surprisingly, PICK1 inhibited the activity of Arp2/ 3 complex Further analysis indicated that PICK1 competed with VCA domains for binding... fertilizes the oocyte If border cells are absent or the migration of the border cells fails, the micropile lacks entry pore for sperm, leading to failure of fertilization (Montell et al 1992) 27 Arp2/ 3 complex in border cell migration Lu Ruifeng 2010 1. 13. 2 Molecular requirement for border cell migration in Drosophila oogenesis Drosophila border cells provide a powerful model to study cell migration in vivo... form of attachment Generally, the net dynamics of actin filament are determined by nucleation, branching, elongating at one hand and severing and capping at the other 1.7 Arp2/ 3 complex and formins are actin filament nucleators 1.7.1 Arp2/ 3 complex Arp2/ 3 was first isolated from Acanthamoeba castellanii based on its affinity for the actin binding protein profilin (Machesky et al 1994) Soon after, the . GENETIC ANALYSIS OF THE ROLE OF ARP2/ 3 COMPLEX IN BORDER CELL MIGRATION IN DROSOPHILA MELANOGASTER LU RUIFENG (Ph.D of Science), NUS A THESIS SUBMITTED FOR THE DEGREE. surveillance. In the renewal of skin and intestine, fresh epithelial cells migrate up from the basal layer Arp2/ 3 complex in border cell migration Lu Ruifeng 2010 3 and the crypts, respectively Quantification of Arp2/ 3 RNAi or Dia RNAi border cell migration in background of one copy of E-Cadherin. Figure 2.18 Arp2/ 3 is not essential for internalization of FM4-64 in border cells. Figure