Cholesterol is stored in adrenal cells as ester in lipid droplets, which are transported to mitochondria to provide a substrate for steroid hormone synthesis. Using mouse adrenal tumour cells (Y-1), we show here that approximately 33% of the adrenal cell cholesterol ester is bound tightly to intermediate filaments while the rest is either loosely attached or free in the cytosol. Specific binding of droplets to intermediate filaments was demonstrated by immunofluorescence and electron microscopy. Immunofluorescence was based upon Nile Red to stain lipid and antibodies to vimentin, actin and tubulin. Electron microscopy, including immunoelectron microscopy with protein A conjugated to gold particles (5 nm), was used to examine whole mounts of cytoskeletons and intermediate filaments. Immunofluorescence reveals that bound droplets are surrounded by a capsule containing vimentin and can be removed from the filaments by extraction with ethanol or 6 M urea. Negative staining of the urea extracts revealed isolated droplets. To the extent that cholesterol ester is the storage form of steroidogenic cholesterol, the knowledge that lipid droplets containing such esters are attached to intermediate filaments may prove important in unravelling the complex process of the transport of cholesterol to mitochondria.

This content is only available via PDF.