Intracellular transport of high-density lipoprotein 3 in intestinal epithelial cells (Caco-2) is tubulin associated.
BACKGROUND: A retroendocytotic pathway for high-density lipoprotein 3 (HDL(3)) in cultured intestinal epithelial cell lines has been described. In small intestinal crypt cells and Caco-2, HDL(3) is internalized, transported to lipid droplets and, after solubilization of these lipid droplets, resecreted. In the present study we examined the mechanisms of intracellular transport of HDL(3) in the Caco-2 cell line. METHODS: Apolipoprotein E free HDL(3 )was gold-labeled for transmission electron microscopy and 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine iodide [DiI(3)] labeled for fluorescence and confocal laser scanning microscopy. For tubulin desintegration Caco-2 cells were incubated with taxol, colchicine and beta- and gamma-lumicolchicine. Tubulin staining was performed using a FITC labeled antibody. Uptake of HDL(3) was quantified by FACS analysis. RESULTS: HDL(3) was rapidly internalized and found to be in contact with lipid droplets in the perinuclear region after 10 min. By transmission electron microscopy a frequent colocalization of HDL(3)-containing vesicles and tubular structures was demonstrated. The close association of HDL(3)-containing vesicles with fluorescence stained tubulin could be confirmed by confocal laser scanning microscopy. Preincubation of the cells with taxol and colchicine did not completely prevent internalization but reduced it during a 2-hour incubation period to less than 50% of the control cells. The transport of DiI(3)-labeled HDL(3) to the lipid droplets in the perinuclear region was almost completely blocked by taxol and colchicine. CONCLUSION: Internalization and intracellular transport of HDL(3) in intestinal epithelial cells (Caco-2) is dependent on a tubulin-mediated mechanism.