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

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

Abstract

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 

Notes

Acknowledgments

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.

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Copyright information

© 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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