Genetic and Cell Biology Methods to Study ESCRTs in Drosophila melanogaster

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.