Functional Analysis of ESCRT-Positive Extracellular Vesicles in the Drosophila Wing Imaginal Disc.
Animals
Animals, Genetically Modified
Drosophila Proteins
/ metabolism
Drosophila melanogaster
/ genetics
Endosomal Sorting Complexes Required for Transport
/ genetics
Extracellular Vesicles
/ metabolism
Hedgehog Proteins
/ metabolism
Imaginal Discs
/ diagnostic imaging
Intravital Microscopy
/ methods
Larva
Ligands
Microscopy, Fluorescence
Protein Binding
Tissue Fixation
/ methods
Wings, Animal
/ diagnostic imaging
Drosophila
ESCRT
Ectosomes
Exosomes
Extracellular vesicles
Hedgehog
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2019
2019
Historique:
entrez:
29
6
2019
pubmed:
30
6
2019
medline:
3
3
2020
Statut:
ppublish
Résumé
A large number of studies have shown that proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) can trigger the biogenesis of different types of Extracellular Vesicles (EV). The functions that these vesicular carriers exert in vivo remain, however, poorly understood. In this chapter, we describe a series of experimental approaches that we established in the Drosophila wing imaginal disc to study the importance of ESCRT-positive EVs for the extracellular transport of signaling molecules, as exemplified by a functional analysis of the mechanism of secretion and propagation of the major developmental morphogen Hedgehog (Hh).Through the combined use of genetic, cell biological, and imaging approaches, we investigate four important aspects of exovesicle biology: (1) The genetic identification of ESCRT proteins that are specifically required for Hh secretion. (2) The imaging of ESCRT and Hh-positive EVs in the lumenal space of both living and fixed wing imaginal discs. (3) The receptor-mediated capture of Hh-containing EVs on the surface of Hh-receiving cells. (4) The effect of manipulations of ESCRT function on the extracellular pool of Hh ligands.
Identifiants
pubmed: 31250292
doi: 10.1007/978-1-4939-9492-2_3
doi:
Substances chimiques
Drosophila Proteins
0
Endosomal Sorting Complexes Required for Transport
0
Hedgehog Proteins
0
Ligands
0
hh protein, Drosophila
149291-21-4
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
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