Multimodal stimulus coding by a gustatory sensory neuron in Drosophila larvae.

Accurate perception of taste information is crucial for animal survival. In adult Drosophila, gustatory receptor neurons (GRNs) perceive chemical stimuli of one specific gustatory modality associated with a stereotyped behavioural response, such as aversion or attraction. We show that GRNs of Drosophila larvae employ a surprisingly different mode of gustatory information coding. Using a novel method for calcium imaging in the larval gustatory system, we identify a multimodal GRN that responds to chemicals of different taste modalities with opposing valence, such as sweet sucrose and bitter denatonium, reliant on different sensory receptors. This multimodal neuron is essential for bitter compound avoidance, and its artificial activation is sufficient to mediate aversion. However, the neuron is also essential for the integration of taste blends. Our findings support a model for taste coding in larvae, in which distinct receptor proteins mediate different responses within the same, multimodal GRN.

Genomics of clinal variation in Drosophila: disentangling the interactions of selection and demography.

Clines in phenotypes and genotype frequencies across environmental gradients are commonly taken as evidence for spatially varying selection. Classical examples include the latitudinal clines in various species of Drosophila, which often occur in parallel fashion on multiple continents. Today, genomewide analysis of such clinal systems provides a fantastic opportunity for unravelling the genetics of adaptation, yet major challenges remain. A well-known but often neglected problem is that demographic processes can also generate clinality, independent of or coincident with selection. A closely related issue is how to identify true genic targets of clinal selection. In this issue of Molecular Ecology, three studies illustrate these challenges and how they might be met. Bergland et al. report evidence suggesting that the well-known parallel latitudinal clines in North American and Australian D. melanogaster are confounded by admixture from Africa and Europe, highlighting the importance of distinguishing demographic from adaptive clines. In a companion study, Machado et al. provide the first genomic comparison of latitudinal differentiation in D. melanogaster and its sister species D. simulans. While D. simulans is less clinal than D. melanogaster, a significant fraction of clinal genes is shared between both species, suggesting the existence of convergent adaptation to clinaly varying selection pressures. Finally, by drawing on several independent sources of evidence, Bo?ičević et al. identify a functional network of eight clinal genes that are likely involved in cold adaptation. Together, these studies remind us that clinality does not necessarily imply selection and that separating adaptive signal from demographic noise requires great effort and care.

Postmating-prezygotic isolation between two allopatric populations of Drosophila montana: fertilisation success differs under sperm competition

Postmating but prezygotic (PMPZ) interactions are increasingly recognized as a potentially important early-stage barrier in the evolution of reproductive isolation. A recent study described a potential example between populations of the same species: single matings between Drosophila montana populations resulted in differential fertilisation success because of the inability of sperm from one population (Vancouver) to penetrate the eggs of the other population (Colorado). As the natural mating system of D. montana is polyandrous (females remate rapidly), we set up double matings of all possible crosses between the same populations to test whether competitive effects between ejaculates influence this PMPZ isolation. We measured premating isolation in no-choice tests, female fecundity, fertility and egg-to-adult viability after single and double matings as well as second-male paternity success (P-2). Surprisingly, we found no PMPZ reproductive isolation between the two populations under a competitive setting, indicating no difficulty of sperm from Vancouver males to fertilize Colorado eggs after double matings. While there were subtle differences in how P-2 changed over time, suggesting that Vancouver males' sperm are somewhat less competitive in a first-male role within Colorado females, these effects did not translate into differences in overall P-2. Fertilisation success can thus differ dramatically between competitive and noncompetitive conditions, perhaps because the males that mate second produce higher quality ejaculates in response to sperm competition. We suggest that unlike in more divergent species comparisons, where sperm competition typically increases reproductive isolation, ejaculate tailoring can reduce the potential for PMPZ isolation when recently diverged populations interbreed.

Mode of interaction of the Gαo subunit of heterotrimeric G proteins with the GoLoco1 motif of Drosophila Pins is determined by guanine nucleotides.

Drosophila GoLoco motif-containing protein Pins is unusual in its highly efficient interaction with both GDP- and the GTP-loaded forms of the α-subunit of the heterotrimeric Go protein. We analysed the interactions of Gαo in its two nucleotide forms with GoLoco1-the first of the three GoLoco domains of Pins-and the possible structures of the resulting complexes, through combination of conventional fluorescence and FRET measurements as well as through molecular modelling. Our data suggest that the orientation of the GoLoco1 motif on Gαo significantly differs between the two nucleotide states of the latter. In other words, a rotation of the GoLoco1 peptide in respect with Gαo must accompany the nucleotide exchange in Gαo. The sterical hindrance requiring such a rotation probably contributes to the guanine nucleotide exchange inhibitor activity of GoLoco1 and Pins as a whole. Our data have important implications for the mechanisms of Pins regulation in the process of asymmetric cell divisions.

The Drosophila TNF Eiger Is an Adipokine that Acts on Insulin-Producing Cells to Mediate Nutrient Response.

Adaptation of organisms to ever-changing nutritional environments relies on sensor tissues and systemic signals. Identification of these signals would help understand the physiological crosstalk between organs contributing to growth and metabolic homeostasis. Here we show that Eiger, the Drosophila TNF-α, is a metabolic hormone that mediates nutrient response by remotely acting on insulin-producing cells (IPCs). In the condition of nutrient shortage, a metalloprotease of the TNF-α converting enzyme (TACE) family is active in fat body (adipose-like) cells, allowing the cleavage and release of adipose Eiger in the hemolymph. In the brain IPCs, Eiger activates its receptor Grindelwald, leading to JNK-dependent inhibition of insulin production. Therefore, we have identified a humoral connexion between the fat body and the brain insulin-producing cells relying on TNF-α that mediates adaptive response to nutrient deprivation.

The Ionotropic receptors IR21a and IR25a mediate cool sensing in Drosophila.

Animals rely on highly sensitive thermoreceptors to seek out optimal temperatures, but the molecular mechanisms of thermosensing are not well understood. The Dorsal Organ Cool Cells (DOCCs) of the Drosophila larva are a set of exceptionally thermosensitive neurons critical for larval cool avoidance. Here, we show that DOCC cool-sensing is mediated by Ionotropic Receptors (IRs), a family of sensory receptors widely studied in invertebrate chemical sensing. We find that two IRs, IR21a and IR25a, are required to mediate DOCC responses to cooling and are required for cool avoidance behavior. Furthermore, we find that ectopic expression of IR21a can confer cool-responsiveness in an Ir25a-dependent manner, suggesting an instructive role for IR21a in thermosensing. Together, these data show that IR family receptors can function together to mediate thermosensation of exquisite sensitivity.

Genomic evidence for adaptive inversion clines in Drosophila melanogaster.

Clines in chromosomal inversion polymorphisms-presumably driven by climatic gradients-are common but there is surprisingly little evidence for selection acting on them. Here we address this long-standing issue in Drosophila melanogaster by using diagnostic single nucleotide polymorphism (SNP) markers to estimate inversion frequencies from 28 whole-genome Pool-seq samples collected from 10 populations along the North American east coast. Inversions In(3L)P, In(3R)Mo, and In(3R)Payne showed clear latitudinal clines, and for In(2L)t, In(2R)NS, and In(3R)Payne the steepness of the clinal slopes changed between summer and fall. Consistent with an effect of seasonality on inversion frequencies, we detected small but stable seasonal fluctuations of In(2R)NS and In(3R)Payne in a temperate Pennsylvanian population over 4 years. In support of spatially varying selection, we observed that the cline in In(3R)Payne has remained stable for >40 years and that the frequencies of In(2L)t and In(3R)Payne are strongly correlated with climatic factors that vary latitudinally, independent of population structure. To test whether these patterns are adaptive, we compared the amount of genetic differentiation of inversions versus neutral SNPs and found that the clines in In(2L)t and In(3R)Payne are maintained nonneutrally and independent of admixture. We also identified numerous clinal inversion-associated SNPs, many of which exhibit parallel differentiation along the Australian cline and reside in genes known to affect fitness-related traits. Together, our results provide strong evidence that inversion clines are maintained by spatially-and perhaps also temporally-varying selection. We interpret our data in light of current hypotheses about how inversions are established and maintained.

The role of the TRAF-interacting protein in proliferation and differentiation.

Ubiquitination of proteins is a post-translational modification, which decides on the cellular fate of the protein. Addition of ubiquitin moieties to proteins is carried out by the sequential action of three enzymes: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; and E3, ubiquitin ligase. The TRAF-interacting protein (TRAIP, TRIP, RNF206) functions as Really Interesting New Gene (RING)-type E3 ubiquitin ligase, but its physiological substrates are not yet known. TRAIP was reported to interact with TRAF [tumor necrosis factor (TNF) receptor-associated factors] and the two tumor suppressors CYLD and Syk (spleen tyrosine kinase). Ectopically expressed TRAIP was shown to inhibit nuclear factor-kappa B (NF-κB) signalling. However, recent results suggested a role for TRAIP in biological processes other than NF-κB regulation. Knock-down of TRAIP in human epidermal keratinocytes repressed cellular proliferation and induced a block in the G1/S phase of the cell cycle without affecting NF-κB signalling. TRAIP is necessary for embryonal development as mutations affecting the Drosophila homologue of TRAIP are maternal effect-lethal mutants, and TRAIP knock-out mice die in utero because of aberrant regulation of cell proliferation and apoptosis. These findings underline the tight link between TRAIP and cell proliferation. In this review, we summarize the data on TRAIP and put them into a larger perspective regarding the role of TRAIP in the control of tissue homeostasis.

Molecular mechanisms for the regulation of skin homeostasis

L'ubiquitination est une modification des protéines conservée, consistant en l'addition de résidus « ubiquitine » et régulant le destin cellulaire des protéines. La protéine « TRAF-interacting protein » TRAIP (ou TRIP) est une ligase E3 qui catalyse l'étape finale de l'ubiquitination. TRAIP est conservé dans l'évolution et est nécessaire au développement des organismes puisque l'ablation de TRAIP conduit à la mort embryonnaire aussi bien de la drosophile que de la souris. De plus, la réduction de l'expression de TRAIP dans des kératinocytes épidermiques humains réprime la prolifération cellulaire et induit un arrêt du cycle cellulaire en phase Gl, soulignant le lien étroit entre TRAIP et la prolifération cellulaire. Comme les mécanismes de régulation de la prolifération jouent un rôle majeur dans l'homéostasie de la peau, il est important de caractériser la fonction de TRAIP dans ces mécanismes. En utilisant des approches in vitro, nous avons déterminé que la protéine TRAIP est instable, modifiée par l'addition d'ubiquitine et ayant une demi-vie d'environ 4 heures. Nos analyses ont également révélé que l'expression de TRAIP est dépendante du cycle cellulaire, atteignant un pic d'expression en phase G2/M et que l'induction de son expression s'effectue principalement au cours de la transition Gl/S. Nous avons identifié le facteur de transcription E2F1 comme en étant le responsable, en régulant directement le promoteur de TRAIP. Aussi, TRAIP endogène ou surexprimée est surtout localisée au niveau du nucléole, une organelle nucléaire qui est désassemblée pendant la division cellulaire. Pour examiner la localisation subcellulaire de TRAIP pendant la mitose, nous avons imagé la protéine TRAIP fusionnée à une protéine fluorescente, à l'intérieur de cellules vivantes nommées HeLa, à l'aide d'un microscope confocal. Dans ces conditions, TRAIP est majoritairement localisée autour des chromosomes en début de mitose, puis est arrangée au niveau de l'ADN chromosomique en fin de mitose. La détection de TRAIP endogène à l'aide d'un anticorps spécifique a confirmé cette localisation. Enfin, l'inactivation de TRAIP dans les cellules HeLa par interférence ARN a inhibé leur capacité à s'arrêter en milieu de mitose. Nos résultats suggèrent que le mécanisme sous-jacent peut être lié au point de contrôle de l'assemblage du fuseau mitotique. - Ubiquitination of proteins is a post-translational modification which decides the cellular fate of the protein. The TRAF-interacting protein (TRAIP, TRIP) functions as an E3 ubiquitin ligase mediating addition of ubiquitin moieties to proteins. TRAIP interacts with the deubiquitinase CYLD, a tumor suppressor whose functional inactivation leads to skin appendage tumors. TRAIP is required for early embryonic development since removal of TRAIP either in Drosophila or mice by mutations or knock¬out is lethal due to aberrant regulation of cell proliferation and apoptosis. Furthermore, shRNA- mediated knock-down of TRAIP in human epidermal keratinocytes (HEK) repressed cell proliferation and induced a Gl/S phase block in the cell cycle. Additionally, TRAIP expression is strongly down- regulated during keratinocyte differentiation supporting the notion of a tight link between TRAIP and cell proliferation. We thus examined the biological functions of TRAIP in epithelial cell proliferation. Using an in vitro approach, we could determine that the TRAIP protein is unstable, modified by addition of ubiquitin moieties after translation and exhibits a half-life of 3.7+/-1-6 hours. Our analysis revealed that the TRAIP expression is modulated in a cell-cycle dependent manner, reaching a maximum expression level in G2/M phases. In addition, the expression of TRAIP was particularly activated during Gl/S phase transition and we could identify the transcription factor E2F1 as an activator of the TRAIP gene promoter. Both endogenous and over-expressed TRAIP mainly localized to the nucleolus, a nuclear organelle which is disassembled during cell division. To examine the subcellular localization of TRAIP during M phase, we performed confocal live-cell imaging of a functional fluorescent protein TRAIP-GFP in HeLa cells. TRAIP was distributed in the cytoplasm and accumulated around mitotic chromosomes in pro- and meta-phasic cells. TRAIP was then confined to chromosomal DNA location in anaphase and later phases of mitosis. Immune-detection of endogenous TRAIP protein confirmed its particular localization in mitosis. Finally, inactivating TRAIP expression in HeLa cells using RNA interference abrogated the cells ability to stop or delay mitosis progression. Our results suggested that TRAIP may involve the spindle assembly checkpoint.

Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

Why do females mate with multiple males ? The sexually selected sperm hypothesis

One debated issues in evolutionary biology is, why in many species females mate with multiple males. Several hypotheses have been put forward, yet the benefits of multiple mating (here defined as mating with several males) remain unclear in many cases. The sperm sexual selection (SSS) hypothesis has been developed to account for the widespread occurrence of multiple mating in females. It argues that multiple mating by females may rapidly spread, when initially a small fraction of the females mate multiply, and if there is a heritable difference among males in one or several of the four characteristics: (1) the quantity of sperm they produce; (2) the success of their sperm in reaching and fertilizing an egg; (3) their ability to displace the sperm that females stored during previous mating; and (4) their ability to prevent any other male from subsequently introducing sperm (e.g., differential efficiency of mating plugs).

Association of hemolytic activity of Pseudomonas entomophila, a versatile soil bacterium, with cyclic lipopeptide production.

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C(10)OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.

Normal hemopoiesis and lymphopoiesis in the combined absence of numb and numblike.

The mammalian ortholog of the conserved Drosophila adaptor protein Numb (Nb) and its homolog Numblike (Nbl) modulate neuronal cell fate determination at least in part by antagonizing Notch signaling. Because the Notch pathway has been implicated in regulating hemopoietic stem cell self-renewal and T cell fate specification in mammals, we investigated the role of Nb and Nbl in hemopoiesis using conditional gene targeting. Surprisingly simultaneous deletion of both Nb and Nbl in murine bone marrow precursors did not affect the ability of stem cells to self-renew or to give rise to differentiated myeloid or lymphoid progeny, even under competitive conditions in mixed chimeras. Furthermore, T cell fate specification and intrathymic T cell development were unaffected in the combined absence of Nb and Nbl. Collectively our data indicate that the Nb family of adaptor proteins is dispensable for hemopoiesis and lymphopoiesis in mice, despite their proposed role in neuronal stem cell development.

Can MMTV exploit TLR4?

The recognition of microbial pathogens based on their molecular patterns is essential for host defense. Recently, Toll-like receptors have been shown not only to recognize viruses as well as bacteria and fungi, but also to trigger an efficient immune response. A recent publication proposed that the retrovirus mouse mammary tumor virus exploits the pattern-recognition receptor Toll-like receptor 4 to achieve more efficient infection.