Involvement of endocytic organelles in the subcellular trafficking and localization of riboflavin
Involvement of endocytic organelles in the subcellular trafficking and localization of riboflavin
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DOI：
10.1124/jpet.103.051581
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发表时间：
2003-08-01
影响因子：
3.5
通讯作者：
Swaan, PW
中科院分区：
文献类型：
--
作者：
Huang, SN;Phelps, MA;Swaan, PW
我们实验室之前的研究表明，受体介导的内吞作用成分在核黄素（维生素 B-2）的细胞易位中具有潜在作用。为了描绘核黄素内化所涉及的细胞内区室和事件，我们合成了罗丹明标记的核黄素缀合物，以通过荧光显微镜监测其运动。 BeWo 细胞的细胞摄取研究表明，罗丹明-核黄素缀合物对假定的核黄素转运系统表现出与 [H-3] 核黄素相似的配体亲和力，而罗丹明不会显着干扰其内化机制。显微镜分析揭示了罗丹明-核黄素缀合物通过核黄素特异性过程快速内化到整个细胞的酸性囊泡区室中。细胞内点状分布与异硫氰酸荧光素 (FITC) 转铁蛋白（一种已充分表征的受体介导的内吞作用底物）的分布相当。双标记荧光显微镜研究进一步证实，内化 10 分钟后，罗丹明-核黄素缀合物基本上集中在与网格蛋白、rab5、FITC-转铁蛋白和促酸标记物 LysoTracker Blue 相关的囊泡结构内。总之，我们的研究首次提供了内吞机制参与核黄素细胞内运输的直接形态学证据。罗丹明-核黄素缀合物的亚细胞定位表明，在本研究的实验条件下，核黄素的内化遵循经典的受体介导的内吞途径。
Previous studies by our laboratory have suggested the potential role of receptor-mediated endocytosis components in the cellular translocation of riboflavin (vitamin B-2). 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 [H-3] 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.