Figure 5. ODR-4 Is Expressed on Intracellular Membranes in Chemosensory Neurons. (A) GFP-tagged ODR-4 (pODR-4-4 in Figure 4B) is expressed in ten types of amphid neurons: AWA, AWC, AWB, ADF, ADL, ASG, ASH, ASI, ASJ, and ASK, and in the two types of phasmid neurons, PHA and PHB. Expression is seen from the 3-fold embryonic stage onward. The amphid neurons are shown in the head of an adult (lateral view, large arrow points to ADL). The phasmid neurons are visible in the tail of an adjacent larva (small arrow, dorsal view). Scale bar, 30 μm.(B) GFP-tagged ODR-4 appears punctate in the dendrites (arrowhead) and axons but is not seen in the cilia. This C-terminal tag rescues the
odr-4 olfactory defect (pODR-4-4, Figure 4B). Similar localization was observed in animals stained with anti-GFP antisera to enhance ODR-4 detection (data not shown). The expression pattern was not altered in
odr-4(
n2144) or
odr-8(
ky31) mutants.(C) GFP-tagged ODR-4 labels intracellular membranes in the cell body reminiscent of endoplasmic reticulum. Wide-field deconvolution image of a neuronal cell body expressing this protein shows fluorescence on intracellular structures. Three-dimensional rendering and rotation of a stack of images revealed interconnected sheets and tubules; surface invaginations suggested that the labeled membranes are cytoplasmic, not on the surface. Scale bar, 2 μm.(D) Expressing ODR-4 only in AWA neurons was sufficient to rescue diacetyl chemotaxis (see Figure 4B). GFP-tagged ODR-4 expressed from the
odr-10 promoter was expressed only in the AWA neuron (arrow). The arrowhead points to a puncta in the dendrite.(E-H) Double fusions of ODR-4 to GFP and beta-galactosidase, which is functional only in the cytosolic compartment. When the transmembrane domain of ODR-4 was deleted and replaced by GFP-LacZ, robust beta-galactosidase activity was observed (E), but olfaction was not rescued. Conversely, when GFP-lacZ was fused after the transmembrane domain, olfactory function was restored, but no beta-galactosidase activity was observed (F). GFP was expressed well in both fusion proteins (G and H). See Figure 4B for constructs and rescue data. Scale bar, 30 μm.