lush

DEVELOPMENTAL BIOLOGY

Adult

Rabbit polyclonal antiserum was raised to bacterially expressed Lush protein for direct examination of the expression of this protein in wild-type and lush flies. Affinity-purified anti-Lush antibodies recognize protein in accessory cells of trichoid sensilla on the ventral-lateral portion of the third-antennal segment in wild-type males and females, in a pattern identical to LacZ expression in ET249. In contrast to the LacZ that is localized to the support cell cytoplasm in ET249 flies, Lush protein is clearly present within the shafts of the trichoid sensilla, confirming that it is secreted into the sensillum lymph. No labeling of olfactory neurons is observed. Western blots of antennal extracts from wild-type and lush mutant flies probed with anti-Lush antiserum reveal that the mutants are completely defective for Lush expression. Southern blot analysis of lush mutant DNA confirms that the 3-kb deletion removes the entire protein-coding region of the lush gene. This suggests that loss of this odorant-binding protein gene is responsible for the chemosensory defects in lush mutants (Kim, 1998).

Effects of Mutation or Deletion

Odorant binding proteins (OBPs) are extracellular proteins localized to the chemosensory systems of most terrestrial species. OBPs are expressed by nonneuronal cells and secreted into the fluid bathing olfactory neuron dendrites. Several members have been shown to interact directly with odorants, but the significance of this is not clear. Mutants of the Drosophila OBP lush are completely devoid of evoked activity to the pheromone 11-cis vaccenyl acetate (VA), revealing that this binding protein is absolutely required for activation of pheromone-sensitive chemosensory neurons. lush mutants are also defective for pheromone-evoked behavior. Importantly, a genetic effect of lush mutants on spontaneous activity in VA-sensitive neurons in the absence of pheromone is described. The defects in spontaneous activity and VA sensitivity are reversed by germline transformation with a lush transgene or by introducing recombinant Lush protein into mutant sensilla. These studies directly link pheromone-induced behavior with OBP-dependent activation of a subset of olfactory neurons (Xu, 2005).

Mutants lacking Lush are insensitive to 11-cis vaccenyl acetate, both at the level of olfactory neuron activation and at the level of aggregation behavior normally elicited by this pheromone. These defects are due specifically to loss of Lush expression in T1 sensilla, because transgenic expression of Lush protein restores function. Other members of the OBP family (OBP83a, or OS-F and OBP83b, or OS-E and moth APO3, or unfolded Lush) do not functionally compensate for the loss of Lush. Therefore, Lush is absolutely and specifically required in the sensillum lymph for VA pheromone signal transduction by T1 neurons, providing a clear demonstration that an OBP member functions in olfaction and mediates activation of olfactory neurons (Xu, 2005).

lush mutants are also defective for spontaneous activity in T1 neurons. This unexpected pheromone-independent phenotype reveals a genetic interaction between Lush and T1 neuron activation mechanisms. T1 neurons do not produce Lush and do not require it for cell fate determination or general health, since direct introduction of recombinant Lush restores function as fast as the protein can diffuse into the sensillum lymph. This time frame would seem too short for any growth factor-like signal requiring transcription and translation. Individual action potentials have normal shape and kinetics in lush mutants, further suggesting there is no intrinsic defect in the T1 neurons. Even relatively dilute preparations of exogenous Lush restore spontaneous activity and VA sensitivity, while expression of other OBPs at high levels does not. This eliminates any nonspecific osmotic effects resulting from absence of the abundant Lush protein in the sensillum lymph producing the observed defects. Finally, VA activates wild-type T1 but not T2 sensilla, though both sensilla types express Lush. This demonstrates a requirement for both Lush and a T1 neuron-specific factor. The simplest explanation consistent with these findings is that extracellular Lush protein can stimulate, directly or indirectly, T1 neurons to produce action potentials through an unknown T1-specific receptor (Xu, 2005).

Members of the OBP family interact directly with odorant ligands, leading to several proposals for OBP function. For example, OBPs may function by removing or inactivating odorants from the sensillum lymph, by solubilizing hydrophobic pheromone ligands, by concentrating pheromone molecules in the lymph, by acting as filters to screen out subsets of odorants, by functioning as buffers to prevent saturation of the responses during high stimulus intensities, or by transporting odorants to the olfactory neurons or to act as coreceptors with odorants to activate olfactory neurons. Recent structural studies have led to the proposal that local pH changes near dendrites might induce unloading of pheromone that subsequently proceeds to activate neuronal receptors. However, there has been little direct in vivo evidence to support or refute these models. Diffusion of antiserum to an OBP in taste sensilla reduced activity, suggesting that the binding protein might facilitate activation of a chemosensory neuron. A role for binding proteins has been implicated in the specificity of neuronal activation in moths. This work showed that the wrong pheromone could activate a pheromone-sensitive neuron when prebound to an OBP that normally binds the activating pheromone (Pophof, 2002). Lush OBP is absolutely required for activation of T1 neurons by VA. This finding is not consistent with odorant removal as a sole function for Lush. Similarly, a role in activation of pheromone-sensitive neurons indicates that Lush is not a buffer or filter for VA. The data suggest that Lush activates T1 neuronal surface receptors responsible for action potential generation. Therefore, while Lush may bind and transport pheromone, it is not a simple carrier or solubilizing factor for pheromone but instead has a more specific role as a signal transduction component. This model would be consistent with the findings of Pophof in the moth system (Pophof, 2002) and may reflect a general mechanism through which OBPs function in insects (Xu, 2005).

A working model is proposed in which Lush functions as an adaptor to bridge the presence of gaseous pheromone molecules to activation of specific neuronal receptors expressed on T1 olfactory neurons. VA may induce a specific conformational change in Lush protein that in turn activates T1 receptors. If such a conformational change occurs spontaneously at low frequency, this would explain the observed loss of spontaneous activity in lush mutants. Ligand-induced conformational changes have been reported previously in pheromone binding proteins from Mamestra brassicae and Bombyx mori upon pheromone binding (Campanacci, 2001; Horst, 2001; Wojtasek, 1999). An important test of this model will be to show that VA pheromone itself is not a direct activator of T1 olfactory neurons, but triggers neuronal activity indirectly through conformational changes in Lush. Consistent with this idea, even 100% VA is incapable of producing activity in T1 neurons in lush mutants. If Lush is the ligand for the T1 receptors, this would refute the pH release model that posits that pheromone release from the OBP mediating activation of neuronal receptors. Alternatively, components of both Lush and an exposed portion of the bound pheromone may activate neuronal receptors (Kaissling, 2001). Lush could also act indirectly by recruiting other factors in the sensillum lymph that ultimately activate T1 neurons. Solving the X-ray crystal structure of the Lush-VA complex and identifying the neuronal receptors that mediate VA sensitivity will allow this model to be corroborated or refuted. Finally, Lush also influences the alcohol responses of T2B neurons. lush mutants are defective for avoidance of concentrated alcohols, and lush mutant T2B neurons fail to show inhibition by concentrated alcohol. Both defects are reversed by expression of a wild-type lush transgene, suggesting the Lush-dependent inhibition of T2B neurons results in behavioral avoidance. This finding would be consistent with the model if a Lush-alcohol complex resulted in activation of T2B receptors and activation of these receptors inhibits these neurons. Inhibition of Drosophila olfactory neurons by odorants has been reported in the literature. Lush binds ethanol (Kruse, 2003) and several phthalate compounds in vitro (Zhou, 2004). Phthalates, even at full strength, do not influence activity in trichoid neurons in the current study. Ethanol influences the responses of T2B neurons but not T2A or T1 neurons, and VA has no effect on T2 neurons. This suggests that ligand binding to a binding protein does not confer universal biological activity in vivo. Instead, different ligands may induce distinct conformations in the binding protein, or perhaps form part of a receptor interaction domain that is discriminated by different neuronal receptors. Structural analysis of these complexes with receptors will allow these interactions to be defined (Xu, 2005).

Social aggregation behavior is induced by activation of T1 neurons, revealing the first stage of a neuronal circuit mediating aggregation in this insect. It is not clear why an aggregation pheromone would be produced only in males. Perhaps this aids roaming flies to identify a safe environment to mate and lay eggs. VA appears to act synergistically with food odorants, consistent with this notion. In Drosophila, at least 35 genes encode OBPs expressed in virtually every Drosophila olfactory and gustatory organ. Recently, a putative taste receptor was implicated in detecting contact pheromone during mating in Drosophila. It will be interesting to determine if OBPs are required for this behavior and if other members of the chemosensory gene family are required for VA sensitivity in T1 neurons. Similarly, it would be of great importance to determine if other OBPs mediate additional behaviors in this animal. In fire ants, the number of egg-laying queens determines the size of the colony. Queen number is determined by workers that kill extra queens depending on the allele of Gp-9 the workers carry. Gp-9 has been identified as a member of the odorant binding protein family (Krieger, 2002). Therefore, it is possible that binding proteins mediate a diverse array of pheromone-mediated social interactions in insects. It should be feasible to design synthetic ligands capable of interacting and inducing appropriate conformational changes with various binding proteins, permitting the manipulation of these signaling pathways. Such compounds could be used to control any number of insect behaviors including aggregation, mating, and colony size (Xu, 2005).

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date revised: 15 March 2005

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