Three odorant-binding proteins are co-expressed in sensilla trichodea of Drosophila melanogaster

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Abstract

Odorant-binding proteins (OBPs) are small soluble proteins present in the aqueous medium surrounding olfactory receptor neurones. In this study we examine the expression patterns of three Drosophila OBPs (LUSH=OBP76a, OS-E=OBP83b and OS-F=OBP83a), using post-embedding immunocytochemistry. All three OBPs are co-expressed in sensilla trichodea whereas sensilla intermedia show co-expression of OS-E and OS-F only, but not of LUSH. Thus, it is confirmed that an individual sensillum can contain more than one OBP, even if it comprises only a single receptor neurone, such as the subtype T-1. In s. trichodea of lush mutants, expression of OS-E and OS-F is not impaired. No other sensillum type on antenna or maxillary palp (e.g. sensilla basiconica, sensilla coeloconica) expresses LUSH, OS-E or OS-F. Within the s. trichodea the three OBPs show the same labelling pattern: the extracellular sensillum lymph in the hair lumen and the sensillum-lymph cavities are heavily labelled. Intracellularly, the three OBPs are co-localised in a variety of dense granules in all auxiliary cells, and also in the receptor neurones. Immunocytochemical data from antennal sections of flies where lush gene expression has been tagged with the reporter gene lacZ suggest that LUSH is synthesised only in the trichogen and the thecogen cells. Thus, LUSH OBP is produced and secreted by two auxiliary cells, whereas its turnover and decomposition does not appear to be restricted to these auxiliary cells but may also occur in the tormogen and receptor cells. The immunocytochemical results are discussed with respect to current concepts of the function of odorant-binding proteins.

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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