ABSTRACT
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.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Animals , Animals, Genetically Modified , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Gene Knockdown Techniques/methods , Imaginal Discs/diagnostic imaging , Imaginal Discs/metabolism , Immunohistochemistry/methods , Larva , Microscopy, Confocal/methods , Mutation , Staining and Labeling/methods , Tissue Fixation/methodsABSTRACT
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.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Extracellular Vesicles/metabolism , Hedgehog Proteins/metabolism , Intravital Microscopy/methods , Animals , Animals, Genetically Modified , Drosophila melanogaster/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Imaginal Discs/diagnostic imaging , Imaginal Discs/metabolism , Larva , Ligands , Microscopy, Fluorescence , Protein Binding , Tissue Fixation/methods , Wings, Animal/diagnostic imaging , Wings, Animal/metabolismABSTRACT
SNAP-29 is expressed throughout the life cycle of fruit fly and exhibits wide tissue distribution patterns. Unlike other SNAP-25-like proteins (i.e., SNAP-25, SNAP-23/24, and SNAP-47) which primarily support exocytosis at the plasma membrane, SNAP-29 regulates various intracellular trafficking events, by partnering with proteins active in both exocytosis and endocytosis. Here we describe the protocol to localize SNAP-29 in early embryos, imaginal discs from third instar larva, and immortalized S2 cells via immunofluorescence microscopy.
