Three odorant-binding proteins are co-expressed in sensilla trichodea of Drosophila melanogaster
Introduction
Drosophila melanogaster is proving to be a valuable model system for understanding the molecular basis of chemoreception, in particular after the discovery of the first insect olfactory receptor proteins (Clyne et al., 1999, Gao and Chess, 1999, Vosshall et al., 1999). The molecular and genetic tools available in this system have opened alleys for the experimental manipulation of chemosensory components which are still not accessible in most other species (e.g. Elmore et al., 2003, Hallem et al., 2004). Even electrophysiological investigation of the function of single receptor neurones has now become an established routine in these flies, despite of their minute size (De Bruyne et al., 1999, De Bruyne et al., 2001).
Drosophila detects odorants with olfactory sensilla located on the third antennal segment (funiculus) and on the maxillary palps (reviewed by Stocker, 1994). Sensilla are organules consisting usually of a cuticular hair, innervated by only few receptor neurones surrounded by three auxiliary cells. The total number of receptor neurones is thus segregated into many different units (‘compound nose’) which allows for their divergent differentiation and functional adaptation. In Drosophila, these sensilla fall into three main morphological categories, the sensilla (s.) trichodea, s. basiconica, and s. coeloconica, which are further classified into several sub-types. A detailed account of the fine structure and maps of the distribution of these sub-types are found in the atlas published by Shanbhag et al., 1999, Shanbhag et al., 2000.
Odour molecules are adsorbed on the cuticular surface of sensilla, pass through pores in the cuticle and enter the sensillum lymph bathing the olfactory neurone dendrites (for review see Steinbrecht, 1999). The question of how the lipophilic odour molecules cross the aqueous sensillum lymph in order to reach the membrane of the receptor neurones has puzzled scientists for a long time. It now appears that odorant-binding proteins (OBPs), which occur in high concentration in the sensillum lymph of insects, are crucial for this task. The first members of this family to be discovered were pheromone-binding proteins (PBPs), which are predominantly expressed in antennae of male moths and bind the female pheromone in vitro (Vogt and Riddiford, 1981). Meanwhile a great number of these soluble proteins with six cysteins at conserved positions have been described in a great variety of insect species (for review see Vogt et al., 2002). The genome of Drosophila carries at least 35—according to other reports even 50—members of the OBP-gene family (Galindo and Smith, 2001, Graham and Davies, 2002, Hekmat-Scafe et al., 2002), this is comparable to the number of its odorant-receptor genes. Meanwhile, not only the sequence but also the 3-D structure of some of these OBPs is known and there are hypotheses about the binding mechanism (Wojtasek and Leal, 1999, Sandler et al., 2000, Horst et al., 2001, Kruse et al., 2003). Analogous proteins, though structurally different, exist in the mucus of vertebrate noses (for review see Tegoni et al., 2000, Pelosi, 2001.
In insects, the expression of different OBPs in different subsets of sensilla has been taken as a hint that OBPs not only solubilise and transport odorants, but also might contribute to the discrimination between odorants or classes of odorants (e.g. Vogt et al., 1991, Steinbrecht et al., 1995, Vogt et al., 2002). Nevertheless, direct evidence for such a contribution has been provided only recently by biochemical and electrophysiological experiments (Maida et al., 2003, Pophof, 2002, Pophof, 2004). Among other, not mutually exclusive functions of OBPs, a role in stimulus termination and inactivation, as well as in general detoxification is discussed (Ziegelberger, 1996). Quantitative models illustrate the role that OBPs may play in determining the time course of receptor activation and odorant deactivation in moths (Kaissling, 2001).
In this paper, we report that three putative OBPs (LUSH=OBP76a, OS-E=OBP83b and OS-F=OBP83a) co-express in one of the three main morphological types of olfactory sensilla of Drosophila, the s. trichodea. Double immunolabelling procedures permit a direct localisation of two OBPs in identical sensilla and cells. Thus, we could ask the question, whether these three OBPs are also intracellularly co-localised or whether they occur in separate compartments, e.g. in different secretory granules. Finally, we establish for the first time which sensillar cells are responsible for the biosynthesis and turnover of an OBP. These studies provide new insight into the biology of these interesting proteins.
Section snippets
Fly stocks, transgenes
D. melanogaster wild-type, stock Canton S (CS), were obtained from stock cultures in Germany and the USA. Flies were further cultured in Seewiesen in a cornmeal-sugar-agar medium supplemented with dry yeast and kept at room temperature under a 24 h day/night cycle. Lush− mutants were prepared as described in Kim et al. (1998). LacZ was expressed in transgenic animals using the three kilobase lush promoter described previously (Kim et al., 1998). Briefly, three kilobases of upstream genomic
LUSH is co-localised with OS-E and OS-F in s. trichodea
LUSH is co-localised with OS-E and OS-F in all morphological subtypes of s. trichodea (T-1, T-2, T-3), which are structurally identical except for the numbers of olfactory neurons they contain (Shanbhag et al., 1999). Thus, also subtype T-1, which houses only one receptor neurone, expresses all three OBPs (Fig. 1). The binding proteins are detected in the extracellular sensillum lymph, but also in intracellular granules in all sensillar cells (Fig. 1, Fig. 3, Fig. 4, Fig. 7, Fig. 8).
Biosynthesis and turnover of OBPs
Most immunolocalisation studies on OBPs in insects so far attempted to correlate the expression of certain OBPs with certain morphological and—if possible—functional sensillum types, while questions about the pathways of biosynthesis and turnover had rarely been asked.
Nevertheless, already the first study localising an OBP in the electron microscope presented data suggesting that this OBP is biosynthesized only in two auxiliary cells (Steinbrecht et al., 1992): labelling for the
Acknowledgements
S.R. Shanbhag was supported by an Alexander-von-Humboldt fellowship during her stay in Seewiesen. We are grateful to Drs D. Hekmat-Scafe and J.R. Carlson from Yale Univeristy for the subtracted antibodies against OS-E and OS-F and to Barbara Müller for expert technical assistance. We thank Drs K.-E. Kaissling and B. Pophof for valuable comments on the manuscript.
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2020, Methods in EnzymologyCitation Excerpt :Of particular interest was the discovery of several OBPs co-localized in a sensillum that houses only a single receptor cell in Drosophila (Fig. 6). Using double labeling on the same section it could also be demonstrated that these different OBPs are present in the same secretory granules (Fig. 7), i.e., that their synthesis does not follow completely separate pathways (Shanbhag et al., 2005). The general localization of OBPs in the sensillum lymph of insect olfactory sensilla has been taken as an indicator of their participating in stimulus conduction and/or stimulus inactivation (see Kaissling, 2001, for a detailed discussion), which has been also suggested by electrophysiological experiments (Van den Berg & Ziegelberger, 1991) and ligand binding assays (Maida, Krieger, Gebauer, Lange, & Ziegelberger, 2000).
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2015, Insect Biochemistry and Molecular BiologyCitation Excerpt :Some studies have demonstrated that reduced expression or genetic alteration of an OBP can lower the sensitivity of a sensory neuron to its odor ligand and/or alter chemosensory behavioral phenotypes (e.g. Syed et al., 2006; Pelletier et al., 2010; Wang et al., 2010; Swarup et al., 2011); other studies have demonstrated promising methodologies that reduce OBP expression but have not characterized resulting physiological or behavioral phenotypes (e.g. Sengul and Tu, 2010). In D. melanogaster, the pheromone receptor OR67d was shown to require the OBP LUSH in order to respond to the pheromone cis-vaccenyl acetate (cVA), even in the presence of two other OBPs (Xu et al., 2005; Shanbhag et al., 2005; Laughlin et al., 2008); a recent study (Gomez-Diaz et al., 2013) has clarified that this action of LUSH is not directly on OR67d, but is rather more consistent with the transport and other activities originally suggested (Vogt et al., 1985; Vogt and Riddiford, 1986). LUSH protein has been shown to interact with behavioral ligands in a manner consistent with interactions described for other OBPs (e.g. Zhou et al., 2004).
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2011, Journal of Biological ChemistryCitation Excerpt :This was based on observations that (a) OBP4 has a higher affinity for indole than OBP1, (b) OBP1 and OBP4 co-localize within sensilla, and (c) OBP4 can cooperatively increase the binding of a fluorescent dye (1-NPN) to OBP1 or OBP3 (33). OBP4, OBP1, and OBP3 are the orthologues of the D. melanogaster OBPs LUSH, OS-E, and OS-F respectively, which are known to co-localize within the same sensilla (34). To understand the molecular events that occur in response to indole, we solved the crystal structure of An.