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. 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 Bianco A, Poukkula M, Cliffe A, Mathieu J, Luque CM, Fulga TA and Rorth P (2007) Two distinct modes of guidance signalling during collective migration of border cells. Nature 448(7151): 362-365. Blanchoin L, Pollard TD and Mullins RD (2000) Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks. Curr Biol 10(20): 1273-1282. Blaser H, Reichman-Fried M, Castanon I, Dumstrei K, Marlow FL, Kawakami K, SolnicaKrezel L, Heisenberg CP and Raz E (2006) Migration of zebrafish primordial germ cells: a role for myosin contraction and cytoplasmic flow. Dev Cell 11(5): 613-627. Block J, Stradal TE, Hanisch J, Geffers R, Kostler SA, Urban E, Small JV, Rottner K and Faix J (2008) Filopodia formation induced by active mDia2/Drf3. J Microsc 231(3): 506-517. Bosticardo M, Marangoni F, Aiuti A, Villa A and Grazia Roncarolo M (2009) Recent advances in understanding the pathophysiology of Wiskott-Aldrich syndrome. Blood 113(25): 6288-6295. Bourne HR and Weiner O (2002) A chemical compass. Nature 419(6902): 21. Bugyi B and Carlier MF (2010) Control of actin filament treadmilling in cell motility. Annu Rev Biophys 39: 449-470. Cai L, Holoweckyj N, Schaller MD and Bear JE (2005) Phosphorylation of coronin 1B by protein kinase C regulates interaction with Arp2/3 and cell motility. J Biol Chem 280(36): 31913-31923. Cai L, Makhov AM, Schafer DA and Bear JE (2008) Coronin 1B antagonizes cortactin and remodels Arp2/3-containing actin branches in lamellipodia. Cell 134(5): 828-842. Chang F, Drubin D and Nurse P (1997) cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin. J Cell Biol 137(1): 169-182. 110 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Charras G and Paluch E (2008) Blebs lead the way: how to migrate without lamellipodia. Nat Rev Mol Cell Biol 9(9): 730-736. Chereau D, Kerff F, Graceffa P, Grabarek Z, Langsetmo K and Dominguez R (2005) Actin-bound structures of Wiskott-Aldrich syndrome protein (WASP)-homology domain and the implications for filament assembly. Proc Natl Acad Sci U S A 102(46): 16644-16649. Chesarone MA and Goode BL (2009) Actin nucleation and elongation factors: mechanisms and interplay. Curr Opin Cell Biol 21(1): 28-37. Copeland JW and Treisman R (2002) The diaphanous-related formin mDia1 controls serum response factor activity through its effects on actin polymerization. Mol Biol Cell 13(11): 4088-4099. Dahl JP, Wang-Dunlop J, Gonzales C, Goad ME, Mark RJ and Kwak SP (2003) Characterization of the WAVE1 knock-out mouse: implications for CNS development. J Neurosci 23(8): 3343-3352. de Belle JS and Heisenberg M (1996) Expression of Drosophila mushroom body mutations in alternative genetic backgrounds: a case study of the mushroom body miniature gene (mbm). Proc Natl Acad Sci U S A 93(18): 9875-9880. Derivery E, Lombard B, Loew D and Gautreau A (2009) The Wave complex is intrinsically inactive. Cell Motil Cytoskeleton 66(10): 777-790. Di Nardo A, Cicchetti G, Falet H, Hartwig JH, Stossel TP and Kwiatkowski DJ (2005) Arp2/3 complex-deficient mouse fibroblasts are viable and have normal leadingedge actin structure and function. Proc Natl Acad Sci U S A 102(45): 16263-16268. Dobereiner HG, Dubin-Thaler BJ, Hofman JM, Xenias HS, Sims TN, Giannone G, Dustin ML, Wiggins CH and Sheetz MP (2006) Lateral membrane waves constitute a universal dynamic pattern of motile cells. Phys Rev Lett 97(3): 038102. 111 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Dramsi S and Cossart P (1998) Intracellular pathogens and the actin cytoskeleton. Annu Rev Cell Dev Biol 14: 137-166. Duchek P and Rorth P (2001) Guidance of cell migration by EGF receptor signaling during Drosophila oogenesis. Science 291(5501): 131-133. Duchek P, Somogyi K, Jekely G, Beccari S and Rorth P (2001) Guidance of cell migration by the Drosophila PDGF/VEGF receptor. Cell 107(1): 17-26. Dumstrei K, Mennecke R and Raz E (2004) Signaling pathways controlling primordial germ cell migration in zebrafish. J Cell Sci 117(Pt 20): 4787-4795. Eden S, Rohatgi R, Podtelejnikov AV, Mann M and Kirschner MW (2002) Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 418(6899): 790-793. Edwards KA and Kiehart DP (1996) Drosophila nonmuscle myosin II has multiple essential roles in imaginal disc and egg chamber morphogenesis. Development 122(5): 14991511. Eisenmann KM, Harris ES, Kitchen SM, Holman HA, Higgs HN and Alberts AS (2007) Dia-interacting protein modulates formin-mediated actin assembly at the cell cortex. Curr Biol 17(7): 579-591. Engqvist-Goldstein AE and Drubin DG (2003) Actin assembly and endocytosis: from yeast to mammals. Annu Rev Cell Dev Biol 19: 287-332. Firtel RA and Chung CY (2000) The molecular genetics of chemotaxis: sensing and responding to chemoattractant gradients. Bioessays 22(7): 603-615. Friedl P and Gilmour D (2009) Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol 10(7): 445-457. Frischknecht F, Moreau V, Rottger S, Gonfloni S, Reckmann I, Superti-Furga G and Way M (1999) Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling. Nature 401(6756): 926-929. 112 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Fulga TA and Rorth P (2002) Invasive cell migration is initiated by guided growth of long cellular extensions. Nat Cell Biol 4(9): 715-719. Galletta BJ and Cooper JA (2009) Actin and endocytosis: mechanisms and phylogeny. Curr Opin Cell Biol 21(1): 20-27. Gao L and Bretscher A (2008) Analysis of unregulated formin activity reveals how yeast can balance F-actin assembly between different microfilament-based organizations. Mol Biol Cell 19(4): 1474-1484. Gasman S, Kalaidzidis Y and Zerial M (2003) RhoD regulates endosome dynamics through Diaphanous-related Formin and Src tyrosine kinase. Nat Cell Biol 5(3): 195-204. Geisbrecht ER and Montell DJ (2002) Myosin VI is required for E-cadherin-mediated border cell migration. Nat Cell Biol 4(8): 616-620. Georgiou M, Marinari E, Burden J and Baum B (2008) Cdc42, Par6, and aPKC regulate Arp2/3-mediated endocytosis to control local adherens junction stability. Curr Biol 18(21): 1631-1638. Goley ED and Welch MD (2006) The ARP2/3 complex: an actin nucleator comes of age. Nat Rev Mol Cell Biol 7(10): 713-726. Goode BL and Eck MJ (2007) Mechanism and function of formins in the control of actin assembly. Annu Rev Biochem 76: 593-627. Groblewska M, Mroczko B and Szmitkowski M (2010) [The role of selected matrix metalloproteinases and their inhibitors in colorectal cancer development]. Postepy Hig Med Dosw (Online) 64: 22-30. Heisenberg M, Borst A, Wagner S and Byers D (1985) Drosophila mushroom body mutants are deficient in olfactory learning. J Neurogenet 2(1): 1-30. Hoeller O and Kay RR (2007) Chemotaxis in the absence of PIP3 gradients. Curr Biol 17(9): 813-817. 113 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Hudson AM and Cooley L (2002) A subset of dynamic actin rearrangements in Drosophila requires the Arp2/3 complex. J Cell Biol 156(4): 677-687. Hulskamp M, Misra S and Jurgens G (1994) Genetic dissection of trichome cell development in Arabidopsis. Cell 76(3): 555-566. Insall R, Muller-Taubenberger A, Machesky L, Kohler J, Simmeth E, Atkinson SJ, Weber I and Gerisch G (2001) Dynamics of the Dictyostelium Arp2/3 complex in endocytosis, cytokinesis, and chemotaxis. Cell Motil Cytoskeleton 50(3): 115-128. Insall RH and Machesky LM (2009) Actin dynamics at the leading edge: from simple machinery to complex networks. Dev Cell 17(3): 310-322. Jekely G and Rorth P (2003) Hrs mediates downregulation of multiple signalling receptors in Drosophila. EMBO Rep 4(12): 1163-1168. Kaksonen M, Toret CP and Drubin DG (2006) Harnessing actin dynamics for clathrinmediated endocytosis. Nat Rev Mol Cell Biol 7(6): 404-414. Kardash E, Reichman-Fried M, Maitre JL, Boldajipour B, Papusheva E, Messerschmidt EM, Heisenberg CP and Raz E (2010) A role for Rho GTPases and cell-cell adhesion in single-cell motility in vivo. Nat Cell Biol 12(1): 47-53; sup pp 41-11. Kessenbrock K, Plaks V and Werb Z (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141(1): 52-67. Kirchhausen T (1998) Wiskott-Aldrich syndrome: a gene, a multifunctional protein and the beginnings of an explanation. Mol Med Today 4(7): 300-304. Korobova F and Svitkina T (2008) Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells. Mol Biol Cell 19(4): 1561-1574. Kovar DR, Harris ES, Mahaffy R, Higgs HN and Pollard TD (2006) Control of the assembly of ATP- and ADP-actin by formins and profilin. Cell 124(2): 423-435. 114 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Kovar DR, Kuhn JR, Tichy AL and Pollard TD (2003) The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin. J Cell Biol 161(5): 875-887. Kunda P, Craig G, Dominguez V and Baum B (2003) Abi, Sra1, and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions. Curr Biol 13(21): 1867-1875. Langridge PD and Kay RR (2006) Blebbing of Dictyostelium cells in response to chemoattractant. Exp Cell Res 312(11): 2009-2017. Lauffenburger DA and Horwitz AF (1996) Cell migration: a physically integrated molecular process. Cell 84(3): 359-369. Lee T and Luo L (2001) Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci 24(5): 251-254. Lewis MW, Robalino IV and Keyhani NO (2009) Uptake of the fluorescent probe FM4-64 by hyphae and haemolymph-derived in vivo hyphal bodies of the entomopathogenic fungus Beauveria bassiana. Microbiology 155(Pt 9): 3110-3120. Li F and Higgs HN (2005) Dissecting requirements for auto-inhibition of actin nucleation by the formin, mDia1. J Biol Chem 280(8): 6986-6992. Liu Y and Montell DJ (1999) Identification of mutations that cause cell migration defects in mosaic clones. Development 126(9): 1869-1878. Llense F and Martin-Blanco E (2008) JNK signaling controls border cell cluster integrity and collective cell migration. Curr Biol 18(7): 538-544. Machacek M, Hodgson L, Welch C, Elliott H, Pertz O, Nalbant P, Abell A, Johnson GL, Hahn KM and Danuser G (2009) Coordination of Rho GTPase activities during cell protrusion. Nature 461(7260): 99-103. Machesky LM, Atkinson SJ, Ampe C, Vandekerckhove J and Pollard TD (1994) Purification of a cortical complex containing two unconventional actins from 115 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Acanthamoeba by affinity chromatography on profilin-agarose. J Cell Biol 127(1): 107-115. Marchand JB, Kaiser DA, Pollard TD and Higgs HN (2001) Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex. Nat Cell Biol 3(1): 76-82. Mathur J (2005) The ARP2/3 complex: giving plant cells a leading edge. Bioessays 27(4): 377-387. Mathur J, Spielhofer P, Kost B and Chua N (1999) The actin cytoskeleton is required to elaborate and maintain spatial patterning during trichome cell morphogenesis in Arabidopsis thaliana. Development 126(24): 5559-5568. McDonald JA, Khodyakova A, Aranjuez G, Dudley C and Montell DJ (2008) PAR-1 kinase regulates epithelial detachment and directional protrusion of migrating border cells. Curr Biol 18(21): 1659-1667. Melani M, Simpson KJ, Brugge JS and Montell D (2008) Regulation of cell adhesion and collective cell migration by hindsight and its human homolog RREB1. Curr Biol 18(7): 532-537. Merrifield CJ, Moss SE, Ballestrem C, Imhof BA, Giese G, Wunderlich I and Almers W (1999) Endocytic vesicles move at the tips of actin tails in cultured mast cells. Nat Cell Biol 1(1): 72-74. Millius A, Dandekar SN, Houk AR and Weiner OD (2009) Neutrophils establish rapid and robust WAVE complex polarity in an actin-dependent fashion. Curr Biol 19(3): 253259. Montell DJ, Rorth P and Spradling AC (1992) slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila C/EBP. Cell 71(1): 51-62. Montero JA and Heisenberg CP (2004) Gastrulation dynamics: cells move into focus. Trends Cell Biol 14(11): 620-627. 116 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Mullins RD, Stafford WF and Pollard TD (1997) Structure, subunit topology, and actinbinding activity of the Arp2/3 complex from Acanthamoeba. J Cell Biol 136(2): 331343. Murdoch C and Finn A (2000) Chemokine receptors and their role in inflammation and infectious diseases. Blood 95(10): 3032-3043. Murphy AM and Montell DJ (1996) Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis. J Cell Biol 133(3): 617-630. Niewiadomska P, Godt D and Tepass U (1999) DE-Cadherin is required for intercellular motility during Drosophila oogenesis. J Cell Biol 144(3): 533-547. Nolen BJ, Littlefield RS and Pollard TD (2004) Crystal structures of actin-related protein 2/3 complex with bound ATP or ADP. Proc Natl Acad Sci U S A 101(44): 1562715632. Oda H, Uemura T and Takeichi M (1997) Phenotypic analysis of null mutants for DEcadherin and Armadillo in Drosophila ovaries reveals distinct aspects of their functions in cell adhesion and cytoskeletal organization. Genes Cells 2(1): 29-40. Oh SW, Kingsley T, Shin HH, Zheng Z, Chen HW, Chen X, Wang H, Ruan P, Moody M and Hou SX (2003) A P-element insertion screen identified mutations in 455 novel essential genes in Drosophila. Genetics 163(1): 195-201. Pacquelet A and Rorth P (2005) Regulatory mechanisms required for DE-cadherin function in cell migration and other types of adhesion. J Cell Biol 170(5): 803-812. Padrick SB, Cheng HC, Ismail AM, Panchal SC, Doolittle LK, Kim S, Skehan BM, Umetani J, Brautigam CA, Leong JM and Rosen MK (2008) Hierarchical regulation of WASP/WAVE proteins. Mol Cell 32(3): 426-438. Palamidessi A, Frittoli E, Garre M, Faretta M, Mione M, Testa I, Diaspro A, Lanzetti L, Scita G and Di Fiore PP (2008) Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration. Cell 134(1): 135-147. 117 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Palazzo AF, Cook TA, Alberts AS and Gundersen GG (2001) mDia mediates Rhoregulated formation and orientation of stable microtubules. Nat Cell Biol 3(8): 723729. Pals ST, Horst E, Scheper RJ and Meijer CJ (1989) Mechanisms of human lymphocyte migration and their role in the pathogenesis of disease. Immunol Rev 108: 111-133. Panchal SC, Kaiser DA, Torres E, Pollard TD and Rosen MK (2003) A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex. Nat Struct Biol 10(8): 591-598. Parent CA and Devreotes PN (1999) A cell's sense of direction. Science 284(5415): 765-770. Penalva MA (2005) Tracing the endocytic pathway of Aspergillus nidulans with FM4-64. Fungal Genet Biol 42(12): 963-975. Peri F (2010) Breaking ranks: how leukocytes react to developmental cues and tissue injury. Curr Opin Genet Dev 20(4): 416-419. Perrais D and Merrifield CJ (2005) Dynamics of endocytic vesicle creation. Dev Cell 9(5): 581-592. Pinheiro EM and Montell DJ (2004) Requirement for Par-6 and Bazooka in Drosophila border cell migration. Development 131(21): 5243-5251. Pollard TD (1986) Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J Cell Biol 103(6 Pt 2): 2747-2754. Pollard TD (2007) Regulation of actin filament assembly by Arp2/3 complex and formins. Annu Rev Biophys Biomol Struct 36: 451-477. Pollard TD, Aebi U, Cooper JA, Fowler WE, Kiehart DP, Smith PR and Tseng PC (1982) Actin and myosin function in acanthamoeba. Philos Trans R Soc Lond B Biol Sci 299(1095): 237-245. Pollard TD and Berro J (2009) Mathematical models and simulations of cellular processes based on actin filaments. J Biol Chem 284(9): 5433-5437. 118 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Pollard TD and Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112(4): 453-465. Pollard TD and Cooper JA (2009) Actin, a central player in cell shape and movement. Science 326(5957): 1208-1212. Prasad M and Montell DJ (2007) Cellular and molecular mechanisms of border cell migration analyzed using time-lapse live-cell imaging. Dev Cell 12(6): 997-1005. Pruyne D, Evangelista M, Yang C, Bi E, Zigmond S, Bretscher A and Boone C (2002) Role of formins in actin assembly: nucleation and barbed-end association. Science 297(5581): 612-615. Raabe T, Clemens-Richter S, Twardzik T, Ebert A, Gramlich G and Heisenberg M (2004) Identification of mushroom body miniature, a zinc-finger protein implicated in brain development of Drosophila. Proc Natl Acad Sci U S A 101(39): 14276-14281. Rasband WS (1997-2009) ImageJ. U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/. Reichman-Fried M, Minina S and Raz E (2004) Autonomous modes of behavior in primordial germ cell migration. Dev Cell 6(4): 589-596. Robertson AS, Smythe E and Ayscough KR (2009) Functions of actin in endocytosis. Cell Mol Life Sci 66(13): 2049-2065. Rocca DL, Martin S, Jenkins EL and Hanley JG (2008) Inhibition of Arp2/3-mediated actin polymerization by PICK1 regulates neuronal morphology and AMPA receptor endocytosis. Nat Cell Biol 10(3): 259-271. Rogers SL, Wiedemann U, Stuurman N and Vale RD (2003) Molecular requirements for actin-based lamella formation in Drosophila S2 cells. J Cell Biol 162(6): 1079-1088. Roh-Johnson M and Goldstein B (2009) In vivo roles for Arp2/3 in cortical actin organization during C. elegans gastrulation. J Cell Sci 122(Pt 21): 3983-3993. 119 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Rohatgi R, Ma L, Miki H, Lopez M, Kirchhausen T, Takenawa T and Kirschner MW (1999) The interaction between N-WASP and the Arp2/3 complex links Cdc42dependent signals to actin assembly. Cell 97(2): 221-231. Rorth P (2002) Initiating and guiding migration: lessons from border cells. Trends Cell Biol 12(7): 325-331. Rorth P (2003) Communication by touch: role of cellular extensions in complex animals. Cell 112(5): 595-598. Rouiller I, Xu XP, Amann KJ, Egile C, Nickell S, Nicastro D, Li R, Pollard TD, Volkmann N and Hanein D (2008) The structural basis of actin filament branching by the Arp2/3 complex. J Cell Biol 180(5): 887-895. Roy R, Yang J and Moses MA (2009) Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer. J Clin Oncol 27(31): 5287-5297. Rozelle AL, Machesky LM, Yamamoto M, Driessens MH, Insall RH, Roth MG, LubyPhelps K, Marriott G, Hall A and Yin HL (2000) Phosphatidylinositol 4,5bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3. Curr Biol 10(6): 311-320. Sahai E and Marshall CJ (2003) Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 5(8): 711-719. Sampath P and Pollard TD (1991) Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments. Biochemistry 30(7): 1973-1980. Sano H, Renault AD and Lehmann R (2005) Control of lateral migration and germ cell elimination by the Drosophila melanogaster lipid phosphate phosphatases Wunen and Wunen 2. J Cell Biol 171(4): 675-683. 120 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Sanz-Moreno V, Gadea G, Ahn J, Paterson H, Marra P, Pinner S, Sahai E and Marshall CJ (2008) Rac activation and inactivation control plasticity of tumor cell movement. Cell 135(3): 510-523. Sawa M, Suetsugu S, Sugimoto A, Miki H, Yamamoto M and Takenawa T (2003) Essential role of the C. elegans Arp2/3 complex in cell migration during ventral enclosure. J Cell Sci 116(Pt 8): 1505-1518. Schirenbeck A, Bretschneider T, Arasada R, Schleicher M and Faix J (2005) The Diaphanous-related formin dDia2 is required for the formation and maintenance of filopodia. Nat Cell Biol 7(6): 619-625. Schmidt KL, Marcus-Gueret N, Adeleye A, Webber J, Baillie D and Stringham EG (2009) The cell migration molecule UNC-53/NAV2 is linked to the ARP2/3 complex by ABI-1. Development 136(4): 563-574. Seth A, Otomo C and Rosen MK (2006) Autoinhibition regulates cellular localization and actin assembly activity of the diaphanous-related formins FRLalpha and mDia1. J Cell Biol 174(5): 701-713. Severson AF, Baillie DL and Bowerman B (2002) A Formin Homology protein and a profilin are required for cytokinesis and Arp2/3-independent assembly of cortical microfilaments in C. elegans. Curr Biol 12(24): 2066-2075. Silva JM, Ezhkova E, Silva J, Heart S, Castillo M, Campos Y, Castro V, Bonilla F, CordonCardo C, Muthuswamy SK, Powers S, Fuchs E and Hannon GJ (2009) Cyfip1 is a putative invasion suppressor in epithelial cancers. Cell 137(6): 1047-1061. Silver DL, Geisbrecht ER and Montell DJ (2005) Requirement for JAK/STAT signaling throughout border cell migration in Drosophila. Development 132(15): 3483-3492. Silver DL and Montell DJ (2001) Paracrine signaling through the JAK/STAT pathway activates invasive behavior of ovarian epithelial cells in Drosophila. Cell 107(7): 831841. 121 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Small JV, Isenberg G and Celis JE (1978) Polarity of actin at the leading edge of cultured cells. Nature 272(5654): 638-639. Snapper SB, Takeshima F, Anton I, Liu CH, Thomas SM, Nguyen D, Dudley D, Fraser H, Purich D, Lopez-Ilasaca M, Klein C, Davidson L, Bronson R, Mulligan RC, Southwick F, Geha R, Goldberg MB, Rosen FS, Hartwig JH and Alt FW (2001) NWASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility. Nat Cell Biol 3(10): 897-904. Soderling SH (2009) Grab your partner with both hands: cytoskeletal remodeling by Arp2/3 signaling. Sci Signal 2(55): pe5. Soderling SH, Langeberg LK, Soderling JA, Davee SM, Simerly R, Raber J and Scott JD (2003) Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice. Proc Natl Acad Sci U S A 100(4): 1723-1728. St Johnston D (2002) The art and design of genetic screens: Drosophila melanogaster. Nat Rev Genet 3(3): 176-188. Stephens L, Milne L and Hawkins P (2008) Moving towards a better understanding of chemotaxis. Curr Biol 18(11): R485-494. Stovold CF, Millard TH and Machesky LM (2005) Inclusion of Scar/WAVE3 in a similar complex to Scar/WAVE1 and 2. BMC Cell Biol 6(1): 11. Strasser GA, Rahim NA, VanderWaal KE, Gertler FB and Lanier LM (2004) Arp2/3 is a negative regulator of growth cone translocation. Neuron 43(1): 81-94. Szymanski DB, Marks MD and Wick SM (1999) Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis. Plant Cell 11(12): 2331-2347. Theveneau E, Marchant L, Kuriyama S, Gull M, Moepps B, Parsons M and Mayor R Collective chemotaxis requires contact-dependent cell polarity. Dev Cell 19(1): 3953. 122 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Theveneau E, Marchant L, Kuriyama S, Gull M, Moepps B, Parsons M and Mayor R (2010) Collective chemotaxis requires contact-dependent cell polarity. Dev Cell 19(1): 39-53. Tilney LG, DeRosier DJ, Weber A and Tilney MS (1992) How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper. J Cell Biol 118(1): 83-93. Tomasevic N, Jia Z, Russell A, Fujii T, Hartman JJ, Clancy S, Wang M, Beraud C, Wood KW and Sakowicz R (2007) Differential regulation of WASP and N-WASP by Cdc42, Rac1, Nck, and PI(4,5)P2. Biochemistry 46(11): 3494-3502. Vartiainen MK and Machesky LM (2004) The WASP-Arp2/3 pathway: genetic insights. Curr Opin Cell Biol 16(2): 174-181. Wallar BJ, Deward AD, Resau JH and Alberts AS (2007) RhoB and the mammalian Diaphanous-related formin mDia2 in endosome trafficking. Exp Cell Res 313(3): 560-571. Wang F, Herzmark P, Weiner OD, Srinivasan S, Servant G and Bourne HR (2002) Lipid products of PI(3)Ks maintain persistent cell polarity and directed motility in neutrophils. Nat Cell Biol 4(7): 513-518. Wang X, He L, Wu YI, Hahn KM and Montell DJ (2010) Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nat Cell Biol 12(6): 591-597. Warren DT, Andrews PD, Gourlay CW and Ayscough KR (2002) Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics. J Cell Sci 115(Pt 8): 1703-1715. Weiner OD, Marganski WA, Wu LF, Altschuler SJ and Kirschner MW (2007) An actinbased wave generator organizes cell motility. PLoS Biol 5(9): e221. 123 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Weiner OD, Neilsen PO, Prestwich GD, Kirschner MW, Cantley LC and Bourne HR (2002) A PtdInsP(3)- and Rho GTPase-mediated positive feedback loop regulates neutrophil polarity. Nat Cell Biol 4(7): 509-513. Xu N, Keung B and Myat MM (2008) Rho GTPase controls invagination and cohesive migration of the Drosophila salivary gland through Crumbs and Rho-kinase. Dev Biol 321(1): 88-100. Yamazaki D, Suetsugu S, Miki H, Kataoka Y, Nishikawa S, Fujiwara T, Yoshida N and Takenawa T (2003) WAVE2 is required for directed cell migration and cardiovascular development. Nature 424(6947): 452-456. Yanai M, Kenyon CM, Butler JP, Macklem PT and Kelly SM (1996) Intracellular pressure is a motive force for cell motion in Amoeba proteus. Cell Motil Cytoskeleton 33(1): 22-29. Yang C, Czech L, Gerboth S, Kojima S, Scita G and Svitkina T (2007) Novel roles of formin mDia2 in lamellipodia and filopodia formation in motile cells. PLoS Biol 5(11): e317. Zallen JA, Cohen Y, Hudson AM, Cooley L, Wieschaus E and Schejter ED (2002) SCAR is a primary regulator of Arp2/3-dependent morphological events in Drosophila. J Cell Biol 156(4): 689-701. Zigmond SH (1977) Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors. J Cell Biol 75(2 Pt 1): 606-616. 124 Arp2/3 complex in border cell migration Lu Ruifeng 2010 Publication Wang C, Lu R, Ouyang X, Ho MW, Chia W, Yu F, Lim KL Drosophila overexpressing parkin R275W mutant exhibits dopaminergic neuron degeneration and mitochondrial abnormalities. 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