ESCRT Mutant Analysis and Imaging of ESCRT Components in the Model Fungus Ustilago maydis

  • Carl Haag
  • Thomas Klein
  • Michael FeldbrüggeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1998)


The ESCRT machinery (endosomal sorting complex required for transport) is an evolutionarily highly conserved multiprotein complex involved in numerous cellular processes like endocytosis, membrane repair, or endosomal long-distance transport. In fungal hyphae, endocytosis and long-distance mRNA transport are tightly linked, as endocytotic vesicles are also the key carrier vehicles for mRNAs. Studying the regulatory component Did2 (CHMP1) in the plant pathogen Ustilago maydis revealed that loss of Did2 resulted in disturbed endosomal maturation, thereby causing defects in microtubule-dependent transport of early endosomes. Here, we describe methods and protocols that allow studying the role of ESCRT components during endosomal transport. We present experimental strategies to analyze U. maydis ESCRT mutant phenotypes and test complementation with heterologous components, such as ESCRT regulators from Drosophila melanogaster.

Key words

CHMP Early endosomes ESCRT regulator Microtubules mRNA transport Small Rab-type GTPases 



We thank lab members for critical comments on the manuscript. Our research was in part financed by grants from the German Science Foundation through DFG-FOR2333, DFG-EXC1024 CEPLAS, and DFG-CRC1208.


  1. 1.
    Christ L, Raiborg C, Wenzel EM, Campsteijn C, Stenmark H (2017) Cellular functions and molecular mechanisms of the ESCRT membrane-scission machinery. Trends Biochem Sci 42:42–56. Scholar
  2. 2.
    Campsteijn C, Vietri M, Stenmark H (2016) Novel ESCRT functions in cell biology: spiraling out of control? Curr Opin Cell Biol 41:1–8. Scholar
  3. 3.
    Hanson PI, Cashikar A (2012) Multivesicular body morphogenesis. Annu Rev Cell Dev Biol 28:337–362. Scholar
  4. 4.
    McCullough J, Clippinger AK, Talledge N, Skowyra ML, Saunders MG, Naismith TV, Colf LA, Afonine P, Arthur C, Sundquist WI, Hanson PI, Frost A (2015) Structure and membrane remodeling activity of ESCRT-III helical polymers. Science 350:1548–1551. Scholar
  5. 5.
    Babst M, Katzmann DJ, Snyder WB, Wendland B, Emr SD (2002) Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body. Dev Cell 3:283–289CrossRefGoogle Scholar
  6. 6.
    Kranz A, Kinner A, Kolling R (2001) A family of small coiled-coil-forming proteins functioning at the late endosome in yeast. Mol Biol Cell 12:711–723CrossRefGoogle Scholar
  7. 7.
    Azmi IF, Davies BA, Xiao J, Babst M, Xu Z, Katzmann DJ (2008) ESCRT-III family members stimulate Vps4 ATPase activity directly or via Vta1. Dev Cell 14:50–61. Scholar
  8. 8.
    Bankaitis VA, Johnson LM, Emr SD (1986) Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci U S A 83:9075–9079CrossRefGoogle Scholar
  9. 9.
    Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH (1992) Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell 3:1389–1402CrossRefGoogle Scholar
  10. 10.
    Hecht KA, O'Donnell AF, Brodsky JL (2014) The proteolytic landscape of the yeast vacuole. Cell Logist 4:e28023. Scholar
  11. 11.
    Gabriely G, Kama R, Gerst JE (2007) Involvement of specific COPI subunits in protein sorting from the late endosome to the vacuole in yeast. Mol Cell Biol 27:526–540. Scholar
  12. 12.
    Penalva MA (2010) Endocytosis in filamentous fungi: cinderella gets her reward. Curr Opin Microbiol 13:684–692. Scholar
  13. 13.
    Haag C, Pohlmann T, Feldbrugge M (2017) The ESCRT regulator Did2 maintains the balance between long-distance endosomal transport and endocytic trafficking. PLoS Genet 13:e1006734. Scholar
  14. 14.
    Lanver D, Tollot M, Schweizer G, Lo Presti L, Reissmann S, Ma LS, Schuster M, Tanaka S, Liang L, Ludwig N, Kahmann R (2017) Ustilago maydis effectors and their impact on virulence. Nat Rev Microbiol 15:409–421. Scholar
  15. 15.
    Steinberg G, Penalva MA, Riquelme M, Wosten HA, Harris SD (2017) Cell biology of hyphal growth. Microbiol Spectr 5.
  16. 16.
    Vollmeister E, Schipper K, Feldbrügge M (2012) Microtubule-dependent mRNA transport in the model microorganism Ustilago maydis. RNA Biol 9:1–8CrossRefGoogle Scholar
  17. 17.
    Haag C, Steuten B, Feldbrügge M (2015) Membrane-coupled mRNA trafficking in fungi. Annu Rev Microbiol 69:265–281. Scholar
  18. 18.
    Schuster M, Kilaru S, Fink G, Collemare J, Roger Y, Steinberg G (2011) Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a non-uniform microtubule array. Mol Biol Cell 22:3645–3657. Scholar
  19. 19.
    Baumann S, Pohlmann T, Jungbluth M, Brachmann A, Feldbrügge M (2012) Kinesin-3 and dynein mediate microtubule-dependent co-transport of mRNPs and endosomes. J Cell Sci 125:2740–2752. Scholar
  20. 20.
    Steinberg G (2012) The transport machinery for motility of fungal endosomes. Fungal Genet Biol 49:675–676. Scholar
  21. 21.
    König J, Baumann S, Koepke J, Pohlmann T, Zarnack K, Feldbrügge M (2009) The fungal RNA-binding protein Rrm4 mediates long-distance transport of ubi1 and rho3 mRNAs. EMBO J 28:1855–1866. Scholar
  22. 22.
    Baumann S, König J, Koepke J, Feldbrügge M (2014) Endosomal transport of septin mRNA and protein indicates local translation on endosomes and is required for correct septin filamentation. EMBO Rep 15:94–102. Scholar
  23. 23.
    Guimaraes SC, Schuster M, Bielska E, Dagdas G, Kilaru S, Meadows BR, Schrader M, Steinberg G (2015) Peroxisomes, lipid droplets, and endoplasmic reticulum "hitchhike" on motile early endosomes. J Cell Biol 211:945–954. Scholar
  24. 24.
    Olgeiser L, Haag C, Boerner S, Ule J, Busch A, Koepke J, König J, Feldbrügge M, Zarnack K (2019) The key protein of endosomal mRNP transport Rrm4 binds translational landmark sites of cargo mRNAs. EMBO reports 20(1), e46588. Scholar
  25. 25.
    Zander S, Baumann S, Weidtkamp-Peters S, Feldbrügge M (2016) Endosomal assembly and transport of heteromeric septin complexes promote septin cytoskeleton formation. J Cell Sci 129:2778–2792. Scholar
  26. 26.
    Hunn BH, Cragg SJ, Bolam JP, Spillantini MG, Wade-Martins R (2015) Impaired intracellular trafficking defines early Parkinson’s disease. Trends Neurosci 38:178–188. Scholar
  27. 27.
    Lee JA, Gao FB (2012) Neuronal functions of ESCRTs. Exp Neurobiol 21:9–15. Scholar
  28. 28.
    Falk J, Konopacki FA, Zivraj KH, Holt CE (2014) Rab5 and Rab4 regulate axon elongation in the Xenopus visual system. J Neurosci 34:373–391. Scholar
  29. 29.
    Konopacki FA, Wong HH, Dwivedy A, Bellon A, Blower MD, Holt CE (2016) ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis. Open Biol 6:150218. Scholar
  30. 30.
    Baumann S, Takeshita N, Grün N, Fischer R, Feldbrügge M (2015) Live cell imaging of endosomal trafficking in fungi. In: Tang BL (ed) Methods in molecular biology: membrane trafficking, 2nd edn. Springer, New York, pp 347–363Google Scholar
  31. 31.
    Hervas-Aguilar A, Rodriguez-Galan O, Galindo A, Abenza JF, Arst HN Jr, Penalva MA (2010) Characterization of Aspergillus nidulans DidB Did2, a non-essential component of the multivesicular body pathway. Fungal Genet Biol 47:636–646. Scholar
  32. 32.
    Bösch K, Frantzeskakis L, Vranes M, Kamper J, Schipper K, Göhre V (2016) Genetic manipulation of the plant pathogen Ustilago maydis to study fungal biology and plant microbe interactions. J Vis Exp.
  33. 33.
    Brachmann A, König J, Julius C, Feldbrügge M (2004) A reverse genetic approach for generating gene replacement mutants in Ustilago maydis. Mol Genet Genomics 272:216–226CrossRefGoogle Scholar
  34. 34.
    Terfrüchte M, Joehnk B, Fajardo-Somera R, Braus G, Riquelme M, Schipper K, Feldbrügge M (2014) Establishing a versatile Golden Gate cloning system for genetic engineering in fungi. Fungal Genet Biol 62:1–10. Scholar
  35. 35.
    Loubradou G, Brachmann A, Feldbrügge M, Kahmann R (2001) A homologue of the transcriptional repressor Ssn6p antagonizes cAMP signalling in Ustilago maydis. Mol Microbiol 40:719–730CrossRefGoogle Scholar
  36. 36.
    Stock J, Sarkari P, Kreibich S, Brefort T, Feldbrügge M, Schipper K (2012) Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis. J Biotechnol 161:80–91. Scholar
  37. 37.
    Nickerson DP, West M, Odorizzi G (2006) Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes. J Cell Biol 175:715–720. Scholar
  38. 38.
    Henne WM, Stenmark H, Emr SD (2013) Molecular mechanisms of the membrane sculpting ESCRT pathway. Cold Spring Harb Perspect Biol 5:a016766. Scholar
  39. 39.
    Zarnack K, Maurer S, Kaffarnik F, Ladendorf O, Brachmann A, Kämper J, Feldbrügge M (2006) Tetracycline-regulated gene expression in the pathogen Ustilago maydis. Fungal Genet Biol 43:727–738CrossRefGoogle Scholar
  40. 40.
    Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128:779–792CrossRefGoogle Scholar
  41. 41.
    Sarkari P, Reindl M, Stock J, Müller O, Kahmann R, Feldbrügge M, Schipper K (2014) Improved expression of single-chain antibodies in Ustilago maydis. J Biotechnol 191:165–175. Scholar
  42. 42.
    Brachmann A, Weinzierl G, Kämper J, Kahmann R (2001) Identification of genes in the bW/bE regulatory cascade in Ustilago maydis. Mol Microbiol 42:1047–1063CrossRefGoogle Scholar

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

Authors and Affiliations

  1. 1.Cluster of Excellence on Plant Sciences (CEPLAS), Institute for MicrobiologyHeinrich-Heine University DüsseldorfDüsseldorfGermany
  2. 2.Institute of GeneticsHeinrich-Heine University DüsseldorfDüsseldorfGermany

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