Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.

Central neural circuitry mediating courtship song perception in male Drosophila.

Animals use acoustic signals across a variety of social behaviors, particularly courtship. In Drosophila, song is detected by antennal mechanosensory neurons and further processed by second-order aPN1/aLN(al) neurons. However, little is known about the central pathways mediating courtship hearing. In this study, we identified a male-specific pathway for courtship hearing via third-order ventrolateral protocerebrum Projection Neuron 1 (vPN1) neurons and fourth-order pC1 neurons. Genetic inactivation of vPN1 or pC1 disrupts song-induced male-chaining behavior. Calcium imaging reveals that vPN1 responds preferentially to pulse song with long inter-pulse intervals (IPIs), while pC1 responses to pulse song closely match the behavioral chaining responses at different IPIs. Moreover, genetic activation of either vPN1 or pC1 induced courtship chaining, mimicking the behavioral response to song. These results outline the aPN1-vPN1-pC1 pathway as a labeled line for the processing and transformation of courtship song in males.

Central brain neurons expressing doublesex regulate female receptivity in Drosophila.

Drosophila melanogaster females respond to male courtship by either rejecting the male or allowing copulation. The neural mechanisms underlying these female behaviors likely involve the integration of sensory information in the brain. Because doublesex (dsx) controls other aspects of female differentiation, we asked whether dsx-expressing neurons mediate virgin female receptivity to courting males. Using intersectional techniques to manipulate the activities of defined subsets of dsx-expressing neurons, we found that activation of neurons in either the pCd or pC1 clusters promotes receptivity, while silencing these neurons makes females unreceptive. Furthermore, pCd and pC1 neurons physiologically respond to the male-specific pheromone cis-vaccenyl acetate (cVA), while pC1 neurons also respond to male courtship song. The pCd and pC1 neurons expressing dsx in females do not express transcripts from the fruitless (fru) P1 promoter. Thus, virgin female receptivity is controlled at least in part by neurons that are distinct from those governing male courtship.

Biochemical membrane lipidomics during Drosophila development.

Lipids play critical roles in energy homeostasis, membrane structure, and signaling. Using liquid chromatography and mass spectrometry, we provide a comprehensive semiquantification of lipids during the life cycle of Drosophila melanogaster (230 glycerophospholipids, 210 sphingolipids, 6 sterols and sterol esters, and 60 glycerolipids) and obtain biological insights through this biochemical resource. First, we find a high and constant triacylglycerol-to-membrane lipid ratio during pupal stage, which is nonobvious in the absence of nutrient uptake and tissue remodeling. Second, sphingolipids undergo specific changes in headgroup (glycosylation) and tail configurations (unsaturation and hydroxylation on sphingoid base and fatty acyls, respectively), which correlate with gene expression of known (GlcT/CG6437; FA2H/ CG30502) and putative (Cyt-b5-r/CG13279) enzymes. Third, we identify a gender bias in phosphoethanolamine-ceramides as a lead for future investigation into sexual maturation. Finally, we partially characterize ghiberti, required for male meiotic cytokinesis, as a homolog of mammalian serine palmitoyltransferase.

doublesex functions early and late in gustatory sense organ development.

Somatic sexual dimorphisms outside of the nervous system in Drosophila melanogaster are largely controlled by the male- and female-specific Doublesex transcription factors (DSX(M) and DSX(F), respectively). The DSX proteins must act at the right times and places in development to regulate the diverse array of genes that sculpt male and female characteristics across a variety of tissues. To explore how cellular and developmental contexts integrate with doublesex (dsx) gene function, we focused on the sexually dimorphic number of gustatory sense organs (GSOs) in the foreleg. We show that DSX(M) and DSX(F) promote and repress GSO formation, respectively, and that their relative contribution to this dimorphism varies along the proximodistal axis of the foreleg. Our results suggest that the DSX proteins impact specification of the gustatory sensory organ precursors (SOPs). DSX(F) then acts later in the foreleg to regulate gustatory receptor neuron axon guidance. These results suggest that the foreleg provides a unique opportunity for examining the context-dependent functions of DSX.

Turning males on: activation of male courtship behavior in Drosophila melanogaster.

The innate sexual behaviors of Drosophila melanogaster males are an attractive system for elucidating how complex behavior patterns are generated. The potential for male sexual behavior in D. melanogaster is specified by the fruitless (fru) and doublesex (dsx) sex regulatory genes. We used the temperature-sensitive activator dTRPA1 to probe the roles of fru(M)- and dsx-expressing neurons in male courtship behaviors. Almost all steps of courtship, from courtship song to ejaculation, can be induced at very high levels through activation of either all fru(M) or all dsx neurons in solitary males. Detailed characterizations reveal different roles for fru(M) and dsx in male courtship. Surprisingly, the system for mate discrimination still works well when all dsx neurons are activated, but is impaired when all fru(M) neurons are activated. Most strikingly, we provide evidence for a fru(M)-independent courtship pathway that is primarily vision dependent.

Sex and the single cell. II. There is a time and place for sex.

The Drosophila melanogaster sex hierarchy controls sexual differentiation of somatic cells via the activities of the terminal genes in the hierarchy, doublesex (dsx) and fruitless (fru). We have targeted an insertion of GAL4 into the dsx gene, allowing us to visualize dsx-expressing cells in both sexes. Developmentally and as adults, we find that both XX and XY individuals are fine mosaics of cells and tissues that express dsx and/or fruitless (fru(M)), and hence have the potential to sexually differentiate, and those that don't. Evolutionary considerations suggest such a mosaic expression of sexuality is likely to be a property of other animal species having two sexes. These results have also led to a major revision of our view of how sex-specific functions are regulated by the sex hierarchy in flies. Rather than there being a single regulatory event that governs the activities of all downstream sex determination regulatory genes-turning on Sex lethal (Sxl) RNA splicing activity in females while leaving it turned off in males-there are, in addition, elaborate temporal and spatial transcriptional controls on the expression of the terminal regulatory genes, dsx and fru. Thus tissue-specific aspects of sexual development are jointly specified by post-transcriptional control by Sxl and by the transcriptional controls of dsx and fru expression.

TRAPPII is required for cleavage furrow ingression and localization of Rab11 in dividing male meiotic cells of Drosophila.

Although membrane addition is crucial for cytokinesis in many animal cell types, the specific mechanisms supporting cleavage furrow ingression are not yet understood. Mutations in the gene brunelleschi (bru), which encodes the Drosophila ortholog of the yeast Trs120p subunit of TRAPPII, cause failure of furrow ingression in male meiotic cells. In non-dividing cells, Brunelleschi protein fused to GFP is dispersed throughout the cytoplasm and enriched at Golgi organelles, similarly to another Drosophila TRAPPII subunit, dBet3. Localization of the membrane-trafficking GTPase Rab11 to the cleavage furrow requires wild-type function of bru, and genetic interactions between bru and Rab11 increase the failure of meiotic cytokinesis and cause synthetic lethality. bru also genetically interacts with four wheel drive (fwd), which encodes a PI4Kbeta, such that double mutants exhibit enhanced failure of male meiotic cytokinesis. These results suggest that Bru cooperates with Rab11 and PI4Kbeta to regulate the efficiency of membrane addition to the cleavage furrow, thus promoting cytokinesis in Drosophila male meiotic cells.