Figure 1. Mutation of acs-3, a long chain acyl-CoA synthase, results in altered fat storage.

From a mutagenesis screen we identified a line, acs-3(ft5), that exhibits elevated staining when fed bacteria labeled both with Nile Red (50nM) and BODIPY-labeled fatty acid (A). Further analysis of acs-3(ft5) mutants revealed the presence of large refractile droplets visible using DIC microscopy, which were not seen in wild type animals. These large droplets stain with BODIPY-labeled fatty acid (B). The lipase ATGL, a lipid droplet associated protein, appears as rings associated with the perimeter of these large droplets in acs-3(ft5) mutant animals (C). When acs-3(ft5) mutant animals are labeled with 5μM (100x) Nile Red, these droplets stain yellow-gold, while intestinal cells of wild type animals lack these large droplets (D). To measure emission spectra, we utilized a spectral confocal microscope, exciting with a 488nm laser, and collecting emitted light at 5nm intervals between 500nm and 660nm. The emission peak for an individual droplet is between 470-480nm, indicating that these large droplets contain neutral lipids (E). When fixed and stained with the lipid dye Sudan Black, these large droplets are clearly labeled (F). In all panels, arrows indicate the position of abnormally large lipid droplets. In (C), the scale bar represents 5μM and N denotes the position of an intestinal cell nucleus.