A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein.
A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein (LDL) particles in living cells has been developed. The method is based on the fluorescence resonance energy transfer (RET) between two fluorescent molecules incorporated into LDL particles. NBD-cholesterol linoleate (NBD-CL) and octadecyl rhodamine B (R18) were incorporated simultaneously into LDL, as a RET donor and a RET acceptor, respectively. In this preparation of LDL (RET-LDL), efficient RET was observed, and after the disruption of the LDL particle by a Triton X-100 treatment, the relief of the RET was observed. RET-LDL was endocytosed by CHO cells via LDL receptors, and the RET-LDL particles were disintegrated after the uptake. The resultant relief of the RET upon the disintegration of the LDL was monitored by flow cytometry, and the amount of intact LDL in cells was estimated by calculation. The disintegration occurred with an about 25 min lag, and was inhibited by several lysosomal inhibitors. These results indicate that the disintegration was not a nonspecific event, but took place at the level of lysosomes. Since living cells can be analyzed by the present method, when coupled to flow sorting, it would permit the isolation of cells having different properties in the endocytic pathway of LDL.
Ohashi
M
M
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Murata
M
M
Ohnishi
S
S
D000818:Animals; D002469:Cell Separation / Q000379:methods; D002738:Chloroquine / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects*; D008985:Monensin / Q000494:pharmacology; D015739:Nocodazole / Q000494:pharmacology
D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; D002738:Chloroquine; D008985:Monensin; D015739:Nocodazole