Abstract
Previous studies by our laboratory have suggested the potential role of receptor-mediated endocytosis components in the cellular translocation of riboflavin (vitamin B2). To delineate the intracellular compartments and events involved in the internalization of riboflavin, we synthesized a rhodamine-labeled riboflavin conjugate to monitor its movement via fluorescent microscopy. Cellular uptake studies in BeWo cells show that rhodamine-riboflavin conjugate exhibits similar ligand affinity toward the putative riboflavin transport system as [3H]riboflavin, whereas rhodamine does not significantly interfere with its internalization mechanism. Microscope analysis reveals rapid internalization of the rhodamine-riboflavin conjugate via a riboflavin-specific process into acidic vesicular compartments throughout the cells. The intracellular punctate distribution is comparable with that of fluorescein isothiocyanate (FITC)-transferrin, a well characterized receptor-mediated endocytosis substrate. Double-labeling fluorescence microscopy studies further confirm that with 10 min of internalization, rhodamine-riboflavin conjugate substantially concentrates within vesicular structures associated with clathrin, rab5, FITC-transferrin, and the acidotropic marker LysoTracker Blue. In summary, our studies provide, for the first time, direct morphological evidence of the involvement of endocytosis machinery in the intracellular trafficking of riboflavin. The subcellular localization of rhodamine-riboflavin conjugate suggests that, under the experimental conditions in this study, the internalization of riboflavin follows a classical receptor-mediated endocytosis pathway.
Footnotes
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This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health (NIDDK R01-DK56631).
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DOI: 10.1124/jpet.103.051581.
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ABBREVIATIONS: BSA, bovine serum albumin; DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline.
- Received March 13, 2003.
- Accepted April 17, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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