Attachment of steroidogenic lipid droplets to intermediate filaments in adrenal cells

Light microscopy of living and extracted adrenal cells (Y-1 mouse adrenal tumour cells and cultured bovine fasciculata cells), using Nomarski optics and fluorescence with nile red to stain lipid, revealed in both cell types that lipid droplets remain attached to intermediate filaments when the cells are extracted to prepare these structures. Electron microscopy of thin sections shows the presence of lipid droplets in both cell types. The droplets differ in appearance but are, in both cases, surrounded by a complete capsule 5 nm wide. The droplets in Y-1 cells include those associated with lysosomes and crystalline structures in addition to typical rounded forms. Only the latter type is seen in bovine fasciculata. Intermediate filaments apparently ending in droplets can also be seen. Immunoelectron microscopy with anti-vimentin and Protein A conjugated to gold particles together with measurement of the diameter of these structures identifies them as intermediate filaments. When adrenal cells are permeabilised and extracted under mild or severe conditions using Triton X-100, thin sections showed that lipid droplets remain associated with the cytoskeleton and in particular intermediate filaments. Extraction under mild and severe conditions cleared the cell contents, revealing attachment of intermediate filaments to lipid droplets with greater clarity than in unextracted cells, i.e. homogenised cells or cells subjected to lysis. Such attachment was unequivocally demonstrated in stereo pairs. These observations support our earlier studies showing attachment of droplets to intermediate filaments, which suggests a role for these filaments in intracellular transport of cholesterol.