Fig. 2

The IR8a CREL functions in subcellular trafficking. a Immunofluorescence with antibodies against GFP (green), IR8a (blue) and IR64a (red) on antennal sections of animals expressing the indicated transgenes in Ir8a neurons. Genotypes are of the form Ir8a-Gal4/UAS-EGFP:Ir8a^x. The white asterisks (in this and other panels) indicate the central cavity of sacculus chamber 3, into which the IR64a+IR8a-expressing OSN ciliated dendrites project (see also the merged panels, in which bright-field images are overlaid to provide anatomical landmarks). In the top left panel, the arrowhead marks the ciliated ending of one neuron; the soma and inner segment of this neuron are also indicated (the outer segment—before the cilium—is difficult to see because only trace levels of receptors are detected in this region). Scale bar (for all panels in this figure): 10 μm. For each genotype, the phenotype was assessed in multiple sections of antennae from at least 20 animals from two independent genetic crosses, allowing observation of several hundred different neurons. We quantified the localisation properties by counting the number of sensory cilia with detectable EGFP signal as a percentage of the total number of cell bodies in the imaged samples; this is not expected to be 100% because sensory endings for each OSN soma are not necessarily present in the thin tissue sections (see ‘Methods’ section on imaging): EGFP:IR8a^wt = 75% (83 labelled cilia/111 soma), EGFP:IR8a^∆CREL = 0% (0/93), EGFP:IR8a^N669Q = 61% (65/106). b Immunofluorescence with antibodies against GFP (green), IR8a (blue) and IR64a (red) on antennal sections of animals expressing the indicated transgenes in Ir8a neurons in an Ir8a mutant background. Genotypes are of the form Ir8a¹/Y;Ir8a-Gal4/UAS-EGFP:Ir8a^x. EGFP:IR8a^∆CREL and EGFP:IR8a^N669Qare impaired in localisation to the cilia in the absence of endogenous IR8a (the occasional projections from the soma represent protein within the inner segment only). In addition, both proteins appear to be destabilised; consequently, endogenous IR64a is also detected at substantially lower levels in these two genotypes (but see Additional file 4: Figure S4). OSNs that express EGFP:IR8a^∆CREL also display signs of sickness (e.g. smaller soma). For each genotype, the phenotype was assessed in multiple sections of antennae from at least 20 animals from two independent genetic crosses. Quantifications: EGFP:IR8a^wt = 79% (177/225), EGFP:IR8a^∆CREL = 0% (0/198), EGFP:IR8a^N669Q = 35% (78/220)