[
International Worm Meeting,
2011]
Although marking the endocytic route with gold tracer has been successfully demonstrated in many mammalian cell types, its application to the intestinal cells of C. elegans has yet to be realized. In this study we fed the worm with 6 nm cationic gold particles in conjunction with OP50 for 30 minutes to two hours. Ultrathin sections of resin embedded as well as cryoprotected samples were prepared and examined with electron microscopy. The majority of gold tracer remained in the lumen of the gut in association with degrading bacteria. Rarely were gold particles found among the microvilli and much less in membrane-bound vesicles inside the cell. The apparent inaccessibility of gold tracer to endosomes from the apical pole of the intestinal cells may be inherent to this particular cell type, or our tracer may be too large. We are redoing the experiments with a smaller tracer. However, to rule out the possibility of an initial aldehyde fixation artifact, high pressure freeze fixation is also being tested. Here we report two advances in the handling of C. elegans for high pressure freezing: 1. Single worm confinement in cellulose capillary tubes for oriented frozen hydrated sectioning. 2. Rehydration of high pressure frozen/freeze substituted sample amenable for subsequent immunolocalization studies. In addition, we are testing standard post-embedding immunolocalizations on plastic-embedded tissues to identify the precise positions of actors in endocytosis.