Abstract
Collective migration of loosely or closely associated cell groups is prevalent in animal development, physiological events, and cancer metastasis. However, our understanding of the mechanisms of collective cell migration is incomplete. Drosophila border cells provide a powerful in vivo genetic model to study collective migration and identify essential genes for this process. Using border cell-specific RNAi-silencing in Drosophila, we knocked down 360 conserved signaling transduction genes in adult flies to identify essential pathways and genes for border cell migration. We uncovered a plethora of signaling genes, a large proportion of which had not been reported for border cells, including Rack1 (Receptor of activated C kinase) and brk (brinker), mad (mother against dpp), and sax (saxophone), which encode three components of TGF-β signaling. The RNAi knock down phenotype was validated by clonal analysis of Rack1 mutants. Our data suggest that inhibition of Src activity by Rack1 may be important for border cell migration and cluster cohesion maintenance. Lastly, results from our screen not only would shed light on signaling pathways involved in collective migration during embryogenesis and organogenesis in general, but also could help our understanding for the functions of conserved human genes involved in cancer metastasis.
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Friedl P, Gilmour D. Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol, 2009, 10: 445–457
Yilmaz M, Christofori G. Mechanisms of motility in metastasizing cells. Mol Cancer Res, 2010, 8: 629–642
Friedl P, Locker J, Sahai E, Segall JE. Classifying collective cancer cell invasion. Nat Cell Biol, 2012, 14: 777–783
He L, Wang X, Montell DJ. Shining light on Drosophila oogenesis: live imaging of egg development. Curr Opin Genet Dev, 2011, 21: 612–619
Montell DJ, Yoon WH, Starz-Gaiano M. Group choreography: mechanisms orchestrating the collective movement of border cells. Nat Rev Mol Cell Biol, 2012, 13: 631–645
Spradling AC. Germline cysts: communes that work. Cell, 1993, 72: 649–651
Montell DJ, Rorth P, Spradling AC. Slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila C/EBP. Cell, 1992, 71: 51–62
Duchek P, Rorth P. Guidance of cell migration by EGF receptor signaling during Drosophila oogenesis. Science, 2001, 291: 131–133
Duchek P, Somogyi K, Jekely G, Beccari S, Rorth P. Guidance of cell migration by the Drosophila PDGF/VEGF receptor. Cell, 2001, 107: 17–26
McDonald JA, Pinheiro EM, Montell DJ. PVF1, a PDGF/VEGF homolog, is sufficient to guide border cells and interacts genetically with Taiman. Development, 2003, 130: 3469–3478
Murphy AM, Montell DJ. Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis. J Cell Biol, 1996, 133: 617–630
Bai J, Uehara Y, Montell DJ. Regulation of invasive cell behavior by taiman, a Drosophila protein related to AIB1, a steroid receptor coactivator amplified in breast cancer. Cell, 2000, 103: 1047–1058
Jang AC, Chang YC, Bai J, Montell D. Border-cell migration requires integration of spatial and temporal signals by the BTB protein Abrupt. Nat Cell Biol, 2009, 11: 569–579
Silver DL, Montell DJ. Paracrine signaling through the JAK/STAT pathway activates invasive behavior of ovarian epithelial cells in Drosophila. Cell, 2001, 107: 831–841
Beccari S, Teixeira L, Rorth P. The JAK/STAT pathway is required for border cell migration during Drosophila oogenesis. Mech Dev, 2002, 111: 115–123
Ghiglione C, Devergne O, Georgenthum E, Carballes F, Medioni C, Cerezo D, Noselli S. The Drosophila cytokine receptor Domeless controls border cell migration and epithelial polarization during oogenesis. Development, 2002, 129: 5437–5447
Silver DL, Geisbrecht ER, Montell DJ. Requirement for JAK/STAT signaling throughout border cell migration in Drosophila. Development, 2005, 132: 3483–3492
McDonald JA, Pinheiro EM, Kadlec L, Schupbach T, Montell DJ. Multiple EGFR ligands participate in guiding migrating border cells. Dev Biol, 2006, 296: 94–103
Llense F, Martin-Blanco E. JNK signaling controls border cell cluster integrity and collective cell migration. Curr Biol, 2008, 18: 538–544
Melani M, Simpson KJ, Brugge JS, Montell D. Regulation of cell adhesion and collective cell migration by hindsight and its human homolog RREB1. Curr Biol, 2008, 18: 532–537
Geisbrecht ER, Sawant K, Su Y, Liu ZC, Silver DL, Burtscher A, Wang X, Zhu AJ, McDonald JA. Genetic interaction screens identify a role for hedgehog signaling in Drosophila border cell migration. Dev Dyn, 2013, 242: 414–431
Padgett RW, St Johnston RD, Gelbart WM. A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-beta family. Nature, 1987, 325: 81–84
Twombly V, Blackman RK, Jin H, Graff JM, Padgett RW, Gelbart WM. The TGF-beta signaling pathway is essential for Drosophila oogenesis. Development, 1996, 122: 1555–1565
Wisotzkey RG, Mehra A, Sutherland DJ, Dobens LL, Liu X, Dohrmann C, Attisano L, Raftery LA. Medea is a Drosophila Smad4 homolog that is differentially required to potentiate DPP responses. Development, 1998, 125: 1433–1445
Campbell G, Tomlinson A. Transducing the Dpp morphogen gradient in the wing of Drosophila: regulation of Dpp targets by brinker. Cell, 1999, 96: 553–562
Jazwinska A, Kirov N, Wieschaus E, Roth S, Rushlow C. The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation. Cell, 1999, 96: 563–573
Minami M, Kinoshita N, Kamoshida Y, Tanimoto H, Tabata T. brinker is a target of Dpp in Drosophila that negatively regulates Dpp-dependent genes. Nature, 1999, 398: 242–246
Muller B, Hartmann B, Pyrowolakis G, Affolter M, Basler K. Conversion of an extracellular Dpp/BMP morphogen gradient into an inverse transcriptional gradient. Cell, 2003, 113: 221–233
Saller E, Bienz M. Direct competition between Brinker and Drosophila Mad in Dpp target gene transcription. EMBO Rep, 2001, 2: 298–305
Kirkpatrick H, Johnson K, Laughon A. Repression of dpp targets by binding of brinker to mad sites. J Biol Chem, 2001, 276: 18216–18222
Upadhyai P, Campbell G. Brinker possesses multiple mechanisms for repression because its primary co-repressor, Groucho, may be unavailable in some cell types. Development, 2013, 140: 4256–4265
Hasson P, Muller B, Basler K, Paroush Z. Brinker requires two corepressors for maximal and versatile repression in Dpp signalling. EMBO J, 2001, 20: 5725–5736
Spitaler M, Cantrell DA. Protein kinase C and beyond. Nat Immunol, 2004, 5: 785–790
Buensuceso CS, Woodside D, Huff JL, Plopper GE, O’Toole TE. The WD protein Rack1 mediates protein kinase C and integrin-dependent cell migration. J Cell Sci, 2001, 114: 1691–1698
Besson A, Wilson TL, Yong VW. The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility. J Biol Chem, 2002, 277: 22073–22084
Cox EA, Bennin D, Doan AT, O’Toole T, Huttenlocher A. RACK1 regulates integrin-mediated adhesion, protrusion, and chemotactic cell migration via its Src-binding site. Mol Biol Cell, 2003, 14: 658–669
Kiely PA, Leahy M, O’Gorman D, O’Connor R. RACK1-mediated integration of adhesion and insulin-like growth factor I (IGF-I) signaling and cell migration are defective in cells expressing an IGF-I receptor mutated at tyrosines 1250 and 1251. J Biol Chem, 2005, 280: 7624–7633
Kiely PA, O’Gorman D, Luong K, Ron D, O’Connor R. Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and beta1 integrin to promote cell migration. Mol Cell Biol, 2006, 26: 4041–4051
Sang N, Severino A, Russo P, Baldi A, Giordano A, Mileo AM, Paggi MG, De Luca A. RACK1 interacts with E1A and rescues E1A-induced yeast growth inhibition and mammalian cell apoptosis. J Biol Chem, 2001, 276: 27026–27033
Choi DS, Young H, McMahon T, Wang D, Messing RO. The mouse RACK1 gene is regulated by nuclear factor-kappa B and contributes to cell survival. Mol Pharmacol, 2003, 64: 1541–1548
Mourtada-Maarabouni M, Kirkham L, Farzaneh F, Williams GT. Functional expression cloning reveals a central role for the receptor for activated protein kinase C 1 (RACK1) in T cell apoptosis. J Leukoc Biol, 2005, 78: 503–514
Mamidipudi V, Cartwright CA. A novel pro-apoptotic function of RACK1: suppression of Src activity in the intrinsic and Akt pathways. Oncogene, 2009, 28: 4421–4433
Mamidipudi V, Dhillon NK, Parman T, Miller LD, Lee KC, Cartwright CA. RACK1 inhibits colonic cell growth by regulating Src activity at cell cycle checkpoints. Oncogene, 2007, 26: 2914–2924
Shor B, Calaycay J, Rushbrook J, McLeod M. Cpc2/RACK1 is a ribosome-associated protein that promotes efficient translation in Schizosaccharomyces pombe. J Biol Chem, 2003, 278: 49119–49128
Nilsson J, Sengupta J, Frank J, Nissen P. Regulation of eukaryotic translation by the RACK1 protein: a platform for signalling molecules on the ribosome. EMBO Rep, 2004, 5: 1137–1141
Gerbasi VR, Weaver CM, Hill S, Friedman DB, Link AJ. Yeast Asc1p and mammalian RACK1 are functionally orthologous core 40S ribosomal proteins that repress gene expression. Mol Cell Biol, 2004, 24: 8276–8287
Sengupta J, Nilsson J, Gursky R, Spahn CM, Nissen P, Frank J. Identification of the versatile scaffold protein RACK1 on the eukaryotic ribosome by cryo-EM. Nat Struct Mol Biol, 2004, 11: 957–962
Stebbins EG, Mochly-Rosen D. Binding specificity for RACK1 resides in the V5 region of beta II protein kinase C. J Biol Chem, 2001, 276: 29644–29650
Steele MR, McCahill A, Thompson DS, MacKenzie C, Isaacs NW, Houslay MD, Bolger GB. Identification of a surface on the beta-propeller protein RACK1 that interacts with the cAMP-specific phosphodiesterase PDE4D5. Cell Signal, 2001, 13: 507–513
Chang BY, Chiang M, Cartwright CA. The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1. J Biol Chem, 2001, 276: 20346–20356
Kiely PA, Sant A, O’Connor R. RACK1 is an insulin-like growth factor 1 (IGF-1) receptor-interacting protein that can regulate IGF-1-mediated Akt activation and protection from cell death. J Biol Chem, 2002, 277: 22581–22589
Kiely PA, Baillie GS, Barrett R, Buckley DA, Adams DR, Houslay MD, O’Connor R. Phosphorylation of RACK1 on tyrosine 52 by c-Abl is required for insulin-like growth factor I-mediated regulation of focal adhesion kinase. J Biol Chem, 2009, 284: 20263–20274
Kiely PA, Baillie GS, Lynch MJ, Houslay MD, O’Connor R. Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and beta1 integrin and for tumor cell proliferation and migration. J Biol Chem, 2008, 283: 22952–22961
Liliental J, Chang DD. Rack1, a receptor for activated protein kinase C, interacts with integrin beta subunit. J Biol Chem, 1998, 273: 2379–2383
Kadrmas JL, Smith MA, Pronovost SM, Beckerle MC. Characterization of RACK1 function in Drosophila development. Dev Dyn, 2007, 236: 2207–2215
Courtneidge SA. Role of Src in signal transduction pathways. The Jubilee Lecture. Biochem Soc Trans, 2002, 30: 11–17
Chang BY, Conroy KB, Machleder EM, Cartwright CA. RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, inhibits activity of src tyrosine kinases and growth of NIH 3T3 cells. Mol Cell Biol, 1998, 18: 3245–3256
Chang BY, Harte RA, Cartwright CA. RACK1: a novel substrate for the Src protein-tyrosine kinase. Oncogene, 2002, 21: 7619–7629
Mamidipudi V, Chang BY, Harte RA, Lee KC, Cartwright CA. RACK1 inhibits the serum- and anchorage-independent growth of v-Src transformed cells. FEBS Lett, 2004, 567: 321–326
Abram CL, Courtneidge SA. Src family tyrosine kinases and growth factor signaling. Exp Cell Res, 2000, 254: 1–13
Frame MC. Newest findings on the oldest oncogene; how activated src does it. J Cell Sci, 2004, 117: 989–998
Dodson GS, Guarnieri DJ, Simon MA. Src64 is required for ovarian ring canal morphogenesis during Drosophila oogenesis. Development, 1998, 125: 2883–2892
Takahashi F, Endo S, Kojima T, Saigo K. Regulation of cell-cell contacts in developing Drosophila eyes by Dsrc41, a new, close relative of vertebrate c-src. Genes Dev, 1996, 10: 1645–1656
Takahashi M, Takahashi F, Ui-Tei K, Kojima T, Saigo K. Requirements of genetic interactions between Src42A, armadillo and shotgun, a gene encoding E-cadherin, for normal development in Drosophila. Development, 2005, 132: 2547–2559
Liu Y, Montell DJ. Identification of mutations that cause cell migration defects in mosaic clones. Development, 1999, 126: 1869–1878
Mathieu J, Sung HH, Pugieux C, Soetaert J, Rorth P. A sensitized PiggyBac-based screen for regulators of border cell migration in Drosophila. Genetics, 2007, 176: 1579–1590
Wan P, Wang D, Luo J, Chu D, Wang H, Zhang L, Chen J. Guidance receptor promotes the asymmetric distribution of exocyst and recycling endosome during collective cell migration. Development, 2013, 140: 4797–4806
Starz-Gaiano M, Melani M, Wang X, Meinhardt H, Montell DJ. Feedback inhibition of Jak/STAT signaling by apontic is required to limit an invasive cell population. Dev Cell, 2008, 14: 726–738
McDonald JA, Khodyakova A, Aranjuez G, Dudley C, Montell DJ. PAR-1 kinase regulates epithelial detachment and directional protrusion of migrating border cells. Curr Biol, 2008, 18: 1659–1667
Geisbrecht ER, Montell DJ. Myosin VI is required for E-cadherin-mediated border cell migration. Nat Cell Biol, 2002, 4: 616–620
Lee HH, Frasch M. Survey of forkhead domain encoding genes in the Drosophila genome: classification and embryonic expression patterns. Dev Dyn, 2004, 229: 357–366
Baig J, Chanut F, Kornberg TB, Klebes A. The chromatin-remodeling protein Osa interacts with CyclinE in Drosophila eye imaginal discs. Genetics, 2010, 184: 731–744
Zavadil J, Bottinger EP. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene, 2005, 24: 5764–5774
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Luo, J., Zuo, J., Wu, J. et al. In vivo RNAi screen identifies candidate signaling genes required for collective cell migration in Drosophila ovary. Sci. China Life Sci. 58, 379–389 (2015). https://doi.org/10.1007/s11427-014-4786-z
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DOI: https://doi.org/10.1007/s11427-014-4786-z