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Loss of the Y chromosome in Drosophila has no impact on cell viability and therefore allows us to assay the impact of environmental agents and genetic alterations on chromosomal loss. To detect in vivo chromosome loss in cells of the developing Drosophila wing primordia, we first engineered a Y chromosome with an attP docking site. By making use of the ΦC31 integrase system, we site-specifically integrated a genomic transgene encompassing the multiple wing hair (mwh) locus into this attP site, leading to a mwh(+)Y chromosome. This chromosome fully rescues the mwh mutant phenotype, an excellent recessive wing cell marker mutation. Loss of this mwh(+)Y chromosome in wing primordial cells then leads to manifestation of the mwh mutant phenotype in mwh-homozygous cells. The forming mwh clones permit us to quantify the effect of agents and genetic alterations by assaying frequency and size of the mwh mosaic spots. To illustrate the use of the mwh(+)Y loss system, the effects of four known mutagens (X-rays, colchicine, ethyl methanesulfonate, and formaldehyde) and two genetic conditions (loss- and gain-of-function lodestar mutant alleles) are documented. The procedure is simple, sensitive, and inexpensive.

In Drosophila collections of green fluorescent protein (GFP) trap lines have been used to probe the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we describe a method, based on nonoverlapping, highly specific, shRNA transgenes directed against GFP, that extends the utility of these collections to loss-of-function studies. Furthermore, we used a MiMIC transposon to generate GFP traps in Drosophila cell lines with distinct subcellular localization patterns, which will permit high-throughput screens using fluorescently tagged proteins. Finally, we show that fluorescent traps, paired with recombinant nanobodies and mass spectrometry, allow the study of endogenous protein complexes in Drosophila.

In a forward screen for genes affecting neurotransmission in Drosophila, we identified mutations in dynamin-related protein (drp1). DRP1 is required for proper cellular distribution of mitochondria, and in mutant neurons, mitochondria are largely absent from synapses, thus providing a genetic tool to assess the role of mitochondria at synapses. Although resting Ca2+ is elevated at drp1 NMJs, basal synaptic properties are barely affected. However, during intense stimulation, mutants fail to maintain normal neurotransmission. Surprisingly, FM1-43 labeling indicates normal exo- and endocytosis, but a specific inability to mobilize reserve pool vesicles, which is partially rescued by exogenous ATP. Using a variety of drugs, we provide evidence that reserve pool recruitment depends on mitochondrial ATP production downstream of PKA signaling and that mitochondrial ATP limits myosin-propelled mobilization of reserve pool vesicles. Our data suggest a specific role for mitochondria in regulating synaptic strength.