Continued neurogenesis in adult Drosophila as a mechanism for recruiting environmental cue-dependent variants.

BACKGROUND: The skills used by winged insects to explore their environment are strongly dependent upon the integration of neurosensory information comprising visual, acoustic and olfactory signals. The neuronal architecture of the wing contains a vast array of different sensors which might convey information to the brain in order to guide the trajectories during flight. In Drosophila, the wing sensory cells are either chemoreceptors or mechanoreceptors and some of these sensors have as yet unknown functions. The axons of these two functionally distinct types of neurons are entangled, generating a single nerve. This simple and accessible coincidental signaling circuitry in Drosophila constitutes an excellent model system to investigate the developmental variability in relation to natural behavioral polymorphisms. METHODOLOGY/PRINCIPAL FINDINGS: A fluorescent marker was generated in neurons at all stages of the Drosophila life cycle using a highly efficient and controlled genetic recombination system that can be induced in dividing precursor cells (MARCM system, flybase web site). It allows fluorescent signals in axons only when the neuroblasts and/or neuronal cell precursors like SOP (sensory organ precursors) undergo division during the precedent steps. We first show that a robust neurogenesis continues in the wing after the adults emerge from the pupae followed by an extensive axonal growth. Arguments are presented to suggest that this wing neurogenesis in the newborn adult flies was influenced by genetic determinants such as the frequency dependent for gene and by environmental cues such as population density. CONCLUSIONS: We demonstrate that the neuronal architecture in the adult Drosophila wing is unfinished when the flies emerge from their pupae. This unexpected developmental step might be crucial for generating non-heritable variants and phenotypic plasticity. This might therefore constitute an advantage in an unstable ecological system and explain much regarding the ability of Drosophila to robustly adapt to their environment.

Exploratory behaviour in NO-dependent cyclase mutants of Drosophila shows defects in coincident neuronal signalling.

BACKGROUND: Drosophila flies explore the environment very efficiently in order to colonize it. They explore collectively, not individually, so that when a few land on a food spot, they attract the others by signs. This behaviour leads to aggregation of individuals and optimizes the screening of mates and egg-laying on the most favourable food spots. RESULTS: Flies perform cycles of exploration/aggregation depending on the resources of the environment. This behavioural ecology constitutes an excellent model for analyzing simultaneous processing of neurosensory information. We reasoned that the decision of flies to land somewhere in order to achieve aggregation is based on simultaneous integration of signals (visual, olfactory, acoustic) during their flight. On the basis of what flies do in nature, we designed laboratory tests to analyze the phenomenon of neuronal coincidence. We screened many mutants of genes involved in neuronal metabolism and the synaptic machinery. CONCLUSION: Mutants of NO-dependent cyclase show a specifically-marked behaviour phenotype, but on the other hand they are associated with moderate biochemical defects. We show that these mutants present errors in integrative and/or coincident processing of signals, which are not reducible to the functions of the peripheral sensory cells.

Approach to systematic analysis of serine/threonine phosphoproteome using Beta elimination and subsequent side effects: intramolecular linkage and/or racemisation.

Complete analysis of the phosphorylation of serine and threonine residues directly from biological extracts is still at an early stage and will remain a challenging goal for many years. Analysis of phosphorylated proteins and identification of the phosphorylated sites in a crude biological extract is a major topic in proteomics, since phosphorylation plays a dominant role in post-translational protein modification. Beta elimination of the serine/threonine-bound phosphate by alkali action generates (methyl)dehydroalanine. The reactivity of this group susceptible of nucleophilic attacks might be used as a tool for phosphoproteome analysis. Most of the known serine/threonine kinases recognize motifs in protein targets that are rich in lysine(s) and/or arginine(s). The (methyl)dehydroalanine resulting from beta elimination of the serine/threonine-bound phosphate by alkali action is likely to react with the amino groups of these neighboring amino acids. Furthermore, the addition reaction of dehydroalanine-peptides with a nucleophilic group more likely generates diastereoisomers derivatives. The internal cyclic bonds and/or the stereoisomer peptide derivatives thus generated confer resistance to trypsin cleavage and/or constitute stop signals for exopeptidases such as carboxypeptidase. This might form the basis of a method to facilitate the systematic identification of phosphorylated peptides.

Simultaneous stimulation of GABA and beta adrenergic receptors stabilizes isotypes of activated adenylyl cyclase heterocomplex.

BACKGROUND: We investigated how the synthesis of cAMP, stimulated by isoproterenol acting through beta-adrenoreceptors and Gs, is strongly amplified by simultaneous incubation with baclofen. Baclofen is an agonist of delta-aminobutyric acid type B receptors [GABAB], known to inhibit adenylyl cyclase via Gi. Because these agents have opposite effects on cAMP levels, the unexpected increase in cAMP synthesis when they are applied simultaneously has been intensively investigated. From previous reports, it appears that cyclase type II contributes most significantly to this phenomenon. RESULTS: We found that simultaneous application of isoproterenol and baclofen specifically influences the association/dissociation of molecules involved in the induction and termination of cyclase activity. Beta/gamma from [GABA]B receptor-coupled Gi has a higher affinity for adenylyl cyclase isoform(s) when these isoforms are co-associated with Gs. Our data also suggest that, when beta/gamma and Galphas are associated with adenylyl cyclase isoform(s), beta/gamma from [GABA]B receptor-coupled Gi retards the GTPase activity of Galphas from adrenergic receptor. These reciprocal regulations of subunits of the adenylyl cyclase complex might be responsible for the drastic increase of cAMP synthesis in response to the simultaneous signals. CONCLUSIONS: Simultaneous signals arriving at a particular synapse converge on molecular detectors of coincidence and trigger specific biochemical events. We hypothesize that this phenomenon comes from the complex molecular architectures involved, including scaffolding proteins that make reciprocal interactions between associated molecules possible. The biochemistry of simultaneous signaling is addressed as a key to synaptic function.

Phosphorylation of proteins in neuron terminals: specificity depends on coincidental signaling.

We investigate the role of neuronal coincidental signaling mediated by the second messengers, on phosphorylation of three major proteins of neurosecretory vesicles. Our data show that different combinations of coincidental signaling generate specific pattern of phosphoproteins and not strictly additional effects. This suggests that an added phosphate on a site might 'mask' or 'unmask' the next sites for specific kinases and phosphatases action by inducing conformation change or protein association. We show that a function of vesicles such as the uptake of glutamate is highly regulated by coincidental signaling.

Drosophila NO-dependent guanylyl cyclase is finely regulated by sequential order of coincidental signaling.

We investigate the mechanism of regulation of Drosophila-soluble guanylate cyclase. Multiple putative sites of phosphorylation for the major kinases are present on both subunits of the heterodimer. We show that NO activation after binding to the heme group, is specifically modulated by sequential phosphorylations. PKA increases the NO stimulation at optimum level when both subunits are phosphorylated. Phosphorylation by CK (casein kinase-like) first, inhibits the PKA phosphorylation of the alpha subunit and limits the PKA upregulation of the cyclase activity. However, PKA phosphorylation first didn't prevent CK phosphorylation of the two subunits and the sequence PKA/CK induces higher level of NO activation than CK/PKA. These phosphorylations occur independently of NO binding and the direct inhibitory effect of calcium is observed for all the sCG forms. These data show that the sGC activity is regulated in a complex way, and the well-known asymmetry of the two subunits appears to cause the reading of the sequence of regulatory signals. This qualifies sGC as molecular detector on which converge coincidental and/or sequential neuronal signals. Furthermore, due to the fact that NO induction is huge (more than 600-fold obtained with the mammal counterpart), we might consider that any variation in kinases activation and/or calcium concentration in micro area of neuronal processes, provokes locally significant quantitative difference of cGMP synthesis in presence of diffusing NO.