Genetic and Cell Biology Methods to Study ESCRTs in Drosophila melanogaster

  • Marco Gualtieri
  • Thomas VaccariEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1998)


Mosaic analysis in Drosophila represents a convenient entry point for studying the role of ESCRT (Endosomal Sorting Complex Required for Transport) genes in multiple cell processes crucial for organ development and homeostasis. Here, we describe the procedure to generate populations of ESCRT-mutant cells within Drosophila larval epithelial organs and to study them in whole-mount preparations using confocal microscopy. The use of antibodies directed to endocytic cargoes, vesicular trafficking, cell proliferation, death, and polarity markers allows one to investigate the consequences of loss of ESCRT activity at the subcellular and tissue level. The protocols described here can be used in fixed tissue as well as in unfixed tissue using endocytic uptake assays.

Key words

ESCRT function Endocytosis Drosophila melanogaster Imaginal discs Whole-mount immunohistochemistry Confocal microscopy 



M.G. and T.V. are supported by AIRC (Associazione Italiana Ricerca contro il Cancro) and WCR (Worldwide Cancer Research).


  1. 1.
    Hurley JH (2015) ESCRTs are everywhere. EMBO J 34(19):2398–2407. Available from: Scholar
  2. 2.
    Horner DS, Pasini ME, Beltrame M, Mastrodonato V, Morelli E, Vaccari T (2018) ESCRT genes and regulation of developmental signaling. Semin Cell Dev Biol 74:29–39. Available from: Scholar
  3. 3.
    Alfred V, Vaccari T (2016) When membranes need an ESCRT: endosomal sorting and membrane remodelling in health and disease. Swiss Med Wkly 146:w14347. Available from: Scholar
  4. 4.
    Jennings BH (2011) Drosophila-a versatile model in biology & medicine. In: Materials today, vol 14. Elsevier, Amsterdam, pp 190–195. Available from: Scholar
  5. 5.
    Vaccari T, Bilder D (2009) At the crossroads of polarity, proliferation and apoptosis: the use of Drosophila to unravel the multifaceted role of endocytosis in tumor suppression. Mol Oncol 3:354–365PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Lloyd TE, Atkinson R, Wu MN, Zhou Y, Pennetta G, Bellen HJ (2002) Hrs regulates endosome membrane invagination and tyrosine kinase receptor signaling in drosophila. Cell 108(2):261–269. Available from: Scholar
  7. 7.
    Yang X, Mao F, Lv X, Zhang Z, Fu L, Lu Y et al (2013) Drosophila Vps36 regulates Smo trafficking in Hedgehog signaling. J Cell Sci 126(18):4230–4238. Available from: Scholar
  8. 8.
    Corrigan L, Redhai S, Leiblich A, Fan S-J, Perera SMW, Patel R et al (2014) BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior. J Cell Biol 206(5):671–688. Available from: Scholar
  9. 9.
    Loncle N, Agromayor M, Martin-Serrano J, Williams DW (2015) An ESCRT module is required for neuron pruning. Sci Rep 5:8461. Available from: Scholar
  10. 10.
    Vaccari T, Bilder D (2005) The Drosophila tumor suppressor vps25 prevents nonautonomous overproliferation by tegulating Notch trafficking. Dev Cell 9(5):687–698. Available from: Scholar
  11. 11.
    Sheng Z, Yu L, Zhang T, Pei X, Li X, Zhang Z et al (2016) ESCRT-0 complex modulates Rbf -mutant cell survival by regulating Rhomboid endosomal trafficking and EGFR signaling. J Cell Sci 129(10):2075–2084. Available from: Scholar
  12. 12.
    Troost T, Jaeckel S, Ohlenhard N, Klein T (2012) The tumour suppressor Lethal (2) giant discs is required for the function of the ESCRT-III component Shrub/CHMP4. J Cell Sci 125(3):763–776. Available from: Scholar
  13. 13.
    Tognon E, Wollscheid N, Cortese K, Tacchetti C, Vaccari T (2014) ESCRT-0 is not required for ectopic notch activation and tumor suppression in drosophila. PLoS One 9(4):e93987. Available from: Scholar
  14. 14.
    Fan J, Jiang K, Liu Y, Jia J, Payre F (2013) Hrs promotes ubiquitination and mediates endosomal trafficking of smoothened in drosophila hedgehog signaling. Xie J, editor. PLoS One 8(11):e79021. Available from: Scholar
  15. 15.
    Sevrioukov EA, Moghrabi N, Kuhn M, Krämer H (2005) A mutation in dVps28 reveals a link between a subunit of the endosomal sorting complex required for transport-I complex and the actin cytoskeleton in Drosophila. Mol Biol Cell 16(5):2301–2312. Available from: Scholar
  16. 16.
    Vaccari T, Rusten TE, Menut L, Nezis IP, Brech A, Stenmark H et al (2009) Comparative analysis of ESCRT-I, ESCRT-II and ESCRT-III function in Drosophila by efficient isolation of ESCRT mutants. J Cell Sci 122(14):2413–2423. Available from: Scholar
  17. 17.
    Aoyama N, Yamakawa T, Sasamura T, Yoshida Y, Ohori M, Okubo H et al (2013) Loss- and gain-of-function analyses of vacuolar protein sorting 2 in Notch signaling of Drosophila melanogaster. Genes Genet Syst, Humana Press 88(1):45–57. Available from:
  18. 18.
    Aradhya R, Zmojdzian M, Da Ponte JP, Jagla K (2015) Muscle niche-driven Insulin-Notch-Myc cascade reactivates dormant adult muscle precursors in drosophila. elife 4:e08497. Available from: Scholar
  19. 19.
    Matias NR, Mathieu J, Huynh J-R, Bellen H, Glover D (2015) Abscission is regulated by the ESCRT-III protein shrub in drosophila germline stem cells. PLoS Genet 11(2):e1004653. Available from: Scholar
  20. 20.
    Sweeney NT, Brenman JE, Jan YN, Gao F-B (2006) The coiled-coil protein shrub controls neuronal morphogenesis in drosophila. Curr Biol 16(10):1006–1011. Available from: Scholar
  21. 21.
    Irion U, St Johnston D (2007) bicoid RNA localization requires specific binding of an endosomal sorting complex. Nature 445(7127):554–558. Available from: Scholar
  22. 22.
    Vaccari T, Lu H, Kanwar R, Fortini ME, Bilder D (2008) Endosomal entry regulates Notch receptor activation in Drosophila melanogaster. J Cell Biol 180(4):755–762. Available from: Scholar
  23. 23.
    Valentine M, Hogan J, Collier S (2014) The drosophila Chmp1 protein determines wing cell fate through regulation of epidermal growth factor receptor signaling. Dev Dyn 243(8):977–987. Available from: Scholar
  24. 24.
    Jia T, Macare C, Desrivières S, Gonzalez DA, Tao C, Ji X et al (2016) Neural basis of reward anticipation and its genetic determinants. Proc Natl Acad Sci 113(14):3879–3884. Available from: Scholar
  25. 25.
    Legent K, Liu HH, Treisman JE (2015) Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation. Development 142(8):1480–1491. Available from: Scholar
  26. 26.
    Theodosiou NA, Xu T (1998) Use of FLP/FRT system to studydrosophiladevelopment. Methods 14(4):355–365. Available from: Scholar
  27. 27.
    Schulz JG, David G, Hassan BA (2009) A novel method for tissue-specific RNAi rescue in Drosophila. Nucleic Acids Res 37(13):e93. Available from: Scholar
  28. 28.
    Michaud S, Marin R, Tanguay RM (1997) Regulation of heat shock gene induction and expression during Drosophila development. Cell Mol Life Sci 53(1):104–113. Available from: Scholar
  29. 29.
    Lee T, Luo L (2001) Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci 24(5):251–254. Available from: Scholar
  30. 30.
    Suster ML, Seugnet L, Bate M, Sokolowski MB (2004) Refining GAL4-driven transgene expression inDrosophila with a GAL80 enhancer-trap. Genesis 39(4):240–245. Available from: Scholar
  31. 31.
    Roote J, Prokop A (2013) How to design a genetic mating scheme: a basic training package for Drosophila genetics. G3 (Bethesda) 3(2):353–358CrossRefGoogle Scholar
  32. 32.
    Thompson BJ, Mathieu J, Sung HH, Loeser E, Rørth P, Cohen SM (2005) Tumor suppressor properties of the ESCRT-II complex component Vps25 in Drosophila. Dev Cell 9(5):711–720PubMedCrossRefGoogle Scholar
  33. 33.
    Moberg KH, Schelble S, Burdick SK, Hariharan IK (2005) Mutations in erupted, the drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev Cell 9(5):699–710PubMedCrossRefGoogle Scholar
  34. 34.
    Le Borgne R, Bardin A, Schweisguth F (2005) The roles of receptor and ligand endocytosis in regulating Notch signaling. Development 132(8):1751–1762. Available from: Scholar
  35. 35.
    Harrison DA, McCoon PE, Binari R, Gilman M, Perrimon N (1998) Drosophila unpaired encodes a secreted protein that activates the JAK signaling pathway. Genes Dev 12(20):3252. Available from: Scholar
  36. 36.
    Teleman AA, Cohen SM (2000) Dpp gradient formation in the drosophila wing imaginal disc. Cell 103(6):971–980. Available from: Scholar
  37. 37.
    Lu H, Bilder D (2005) Endocytic control of epithelial polarity and proliferation in Drosophila. Nat Cell Biol 7(12):1232–1239. Available from: Scholar
  38. 38.
    Wucherpfennig T, Wilsch-Bräuninger M, González-Gaitán M (2003) Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release. J Cell Biol 161(3):609–624. Available from: Scholar
  39. 39.
    Bökel C, Schwabedissen A, Entchev E, Renaud O, González-Gaitán M (2006) Sara endosomes and the maintenance of dpp signaling levels across mitosis. Science 314(5802):1135–1139. Available from: Scholar
  40. 40.
    Daskalaki A, Shalaby NA, Kux K, Tsoumpekos G, Tsibidis GD, Muskavitch MAT et al (2011) Distinct intracellular motifs of Delta mediate its ubiquitylation and activation by Mindbomb1 and Neuralized. J Cell Biol 195(6):1017–1031. Available from: Scholar
  41. 41.
    Tanaka T, Nakamura A (2008) The endocytic pathway acts downstream of Oskar in Drosophila germ plasm assembly. Development 135(6):1107–1117. Available from: Scholar
  42. 42.
    Dollar G, Struckhoff E, Michaud J, Cohen RS (2002) Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation. Development 129(2):517–526. Available from: Scholar
  43. 43.
    Jafar-Nejad H, Andrews HK, Acar M, Bayat V, Wirtz-Peitz F, Mehta SQ et al (2005) Sec15, a component of the exocyst, promotes notch signaling during the asymmetric division of drosophila sensory organ precursors. Dev Cell 9(3):351–363. Available from: Scholar
  44. 44.
    Wang W, Li Y, Zhou L, Yue H, Luo H (2011) Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe. Biochem Biophys Res Commun 410(4):714–720. Available from: Scholar
  45. 45.
    Petzoldt AG, Gleixner EM, Fumagalli A, Vaccari T, Simons M (2013) Elevated expression of the V-ATPase C subunit triggers JNK-dependent cell invasion and overgrowth in a Drosophila epithelium. Dis Model Mech 6(3):689–700. Available from: Scholar
  46. 46.
    Markstein M, Dettorre S, Cho J, Neumüller RA, Craig-Müller S, Perrimon N (2014) Systematic screen of chemotherapeutics in Drosophila stem cell tumors. Proc Natl Acad Sci U S A 111(12):4530–4535. Available from: Scholar
  47. 47.
    Sisson JC, Field C, Ventura R, Royou A, Sullivan W (2000) Lava lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization. J Cell Biol 151(4):905–918. Available from: Scholar
  48. 48.
    Emery G, Hutterer A, Berdnik D, Mayer B, Wirtz-Peitz F, Gaitan MG et al (2005) Asymmetric Rab11 endosomes regulate delta recycling and specify cell fate in the drosophila nervous system. Cell 122(5):763–773. Available from: Scholar
  49. 49.
    Mehta SQ, Hiesinger PR, Beronja S, Zhai RG, Schulze KL, Verstreken P et al (2005) Mutations in Drosophila sec15 reveal a function in neuronal targeting for a subset of exocyst components. Neuron 46(2):219–232. Available from: Scholar
  50. 50.
    Blair SS (2007) Dissection of imaginal discs in drosophila. Cold Spring Harb Protoc 2007(24):pdb.prot4794–pdb.prot4794Google Scholar
  51. 51.
    Stocker H, Gallant P (2008) Getting started. In: Methods in molecular biology, Clifton, NJ, pp 27–44. Available from:
  52. 52.
    Spratford CM, Kumar JP (2014) Dissection and immunostaining of imaginal discs from Drosophila melanogaster. J Vis Exp 91:51792. Available from: Scholar
  53. 53.
    Kyriacou CP (2000) Drosophila protocols edited by William Sullivan, Michael Ashburner and R. Scott Hawley. Trends Genet 16(11):524. Available from: Scholar
  54. 54.
    Chassefeyre R, Martínez-Hernández J, Bertaso F, Bouquier N, Blot B, Laporte M et al (2015 Feb) Regulation of postsynaptic function by the dementia-related ESCRT-III subunit CHMP2B. J Neurosci 35(7):3155–3173PubMedPubMedCentralCrossRefGoogle Scholar
  55. 55.
    Daul AL, Komori H, Lee CY (2010) Immunofluorescent staining of Drosophila larval brain tissue. Cold Spring Harb Protoc 5:7Google Scholar
  56. 56.
    Egger B, Gold KS, Brand AH (2010) Notch regulates the switch from symmetric to asymmetric neural stem cell division in the Drosophila optic lobe. Development 137(18):2981–2987PubMedPubMedCentralCrossRefGoogle Scholar
  57. 57.
    Kennison JA (2008) Dissection of larval salivary glands and polytene chromosomes preparation. Cold Spring Harb Protoc 3(5):pdb.prot4708Google Scholar
  58. 58.
    Lebestky T, Jung SH, Banerjee U (2003) A Serrate-expressing signaling center controls Drosophila hematopoiesis. Genes Dev 17(3):348–353PubMedPubMedCentralCrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Dipartimento di BioscienzeUniversità degli Studi di MilanoMilanItaly

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