EZ:
NR:
CD:
Effects of expression of
Accumulation of ODR-10 depends on the sex and fed state of
We investigated a potential role for VPS-4 in ODR-10 regulation using fluorescence microscopy and an attraction chemotaxis assay. We expressed VPS-4 DN under the
Starvation of animals has a subtle effect on the abundance of ODR-10 that accumulates in the cilia of AWA neurons, but that accumulation does not affect the efficiency of chemotaxis in wild type animals (Figure 1B and C). However, when food stress was paired with a disruption of the ESCRT pathway the aberrant accumulation of ODR-10::GFP correlated with a modest but significant decrease in chemotaxis index (Figure 1C). It is still unknown whether ODR-10 is a direct target of the ESCRT machinery or other ubiquitin-related pathways. These data could also represent indirect effects due to changes in availability or localization of cofactors that are required for normal receptor processing and/or localization within the AWA neuron.
Animals were reared on lawns of OP50 on NGM plates according to established protocols. Well-fed, uncrowded young adult hermaphrodites were used for both imaging and chemotaxis assays. For assays using “starved” animals, L4 larvae were moved to NGM plates lacking bacteria 18-24 hours prior to assay. For fluorescence microscopy, young adult hermaphrodites were placed in 3µl M9 on coverslip, which was then inverted onto 3µl of solidified 2% agarose with 0.5% Levamisole (MP Biomedicals, Inc.) on the microscope slide. Animals were imaged on a Leica DMi6000 inverted microscope equipped with a Leica DFC3000G cooled CCD camera and GFP filter, at 200X total magnification (20X objective, NA 0.4). Animals were imaged using 150 ms exposures and gain of 10. Animals were described as having “no fluorescence” if no cells could be detected using these imaging parameters. Chemotaxis was assayed as previously published (Sengupta et al. 1996) using young adult hermaphrodites, 0.1% (v/v) diacetyl in ethanol was used as the attractant and ethanol as a negative control, with 0.02% (w/v) sodium azide as a paralytic at the site of the chemoattractant and control. Animals used for chemotaxis assays did not express ODR-10::GFP.
Strains: N2 (Bristol)
CX3344
This work was supported by Research and Creative Opportunities for Undergraduates grants awarded to Ellen Zocher and Nelson Ruth by Western Washington University.