Uptake and transport of fluorescent derivatives of dolichol in human fibroblasts.
We are using fluorescent derivatives to visualize the endocytic transport of dolichol intermediates from the cell surface to the lysosome, and to estimate their rate of turnover within the lysosome. Anthroyl dolichol and anthroyl [1-14C]dolichol were synthesized and purified by chromatography on silica and C18 Sep-Paks followed by high-performance liquid chromatography on C18. The successful synthesis of anthroyl polyisoprenoid alcohols was confirmed by the use of uv-visible spectrometry and by fluorescence spectrometry. The purified esters were taken up into Ham's media containing 10-30% fetal calf serum or alternatively reconstituted into phospholipid liposomes for delivery to human fibroblasts in culture. The uptake of fluorescent dolichol esters into the cells and into lysosomes was demonstrated using fluorescence microscopy. The localization of anthroyl dolichol in lysosomes was further documented by simultaneously labeling fibroblasts with anthroyl dolichol and FITC-dextran a recognized lysosomal marker. Fibroblasts generally showed several groupings (domains) of lysosomes, some were dually labeled while others were labeled exclusively with either anthroyl dolichol or FITC-dextran. Labeling with anthroyl dolichol was very slow relative to labeling of the same fibroblasts with FITC-dextran suggesting that anthroyl dolichol acts as a labeling agent for intracellular membranes, particularly those of the lysosome while the dextran fluorescence is presumably of lysosolic origin. Several types of experiments were done with anthroyl [1-14C]dolichol to establish that the fluorescence seen in lysosomes represents anthroyl dolichol. Anthroyl dolichol appears to enter fibroblasts intact, since we were unable to recover any free [1-14C]dolichol from total lipid extracts of (i) media used for the uptake of anthroyl dolichol or (ii) the media removed from cells labelled for 42 h. In addition, attempts to hydrolyze anthroyl [1-14C]dolichol in vitro using whole fibroblast homogenates at pH 4.0 and 7.5 were unsuccessful, even though the fibroblasts expressed acid lipase activity using 4-methylumbelliferyl palmitate as substrate.
Jiang
L W
LW
Department of Biochemistry, University of Western Ontario, Canada.
Mitchell
B A
BA
Teodoro
J G
JG
Rip
J W
JW
D001692:Biological Transport; D002240:Carbohydrate Sequence; D004286:Dolichols / Q000031:analogs & derivatives* / Q000378:metabolism; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data
D004286:Dolichols; D016650:Fluorescein-5-isothiocyanate