Probing the mechanism of transport and compartmentalisation of polyamines in mammalian cells
Probing the mechanism of transport and compartmentalisation of polyamines in mammalian cells
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DOI：
10.1016/s1074-5521(00)80019-8
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发表时间：
1999-10-01
影响因子：
--
通讯作者：
Travis, N
中科院分区：
文献类型：
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作者：
Cullis, PM;Green, RE;Travis, N
背景：许多哺乳动物细胞具有主动摄取多胺的系统，但对这种转运蛋白的分子机制知之甚少。多胺的命运采取了从媒体和多胺稳态的关系仍有待充分建立。本研究的目的是开发一系列的改性多胺，特别是配体纳入荧光团，探索的多胺运输系统的结构公差和探针的多胺从medium.Results获得的细胞内位置：我们合成了广泛的多胺类似物纳入细胞毒性剂，荧光发色团和庞大的取代基。所有这些类似物已被证明是很好的竞争性抑制剂的亚精胺摄取在一系列哺乳动物细胞，直接证据摄取的荧光多胺类似物和它们的亚细胞分布从共聚焦激光扫描荧光显微镜，这表明它们积累在颗粒状结构内的细胞质，而不是在细胞核中。我们表明，他们的摄取是通过多胺运输系统，通过显示，预处理DFMO，多胺生物合成的一种有效的抑制剂，导致增强的摄取，和细胞缺乏多胺运输系统没有积累这些多胺analogs.Conclusions：多胺运输系统具有令人惊讶的广泛的结构耐受性。衍生自细胞外池的含荧光团的多胺类似物位于细胞质内的颗粒状结构中，而不是在哺乳动物细胞的细胞核中的任何很大程度上。这些观察结果可能是一致的机制，涉及受体介导的内吞作用，颗粒状的“结构”可能反映多胺囊泡内的区室化。
Background: Many mammalian cells possess an active polyamine uptake system but little is known about the molecular mechanism of this transporter. The fate of polyamines taken up from the medium and the relationship to polyamine homeostasis remains to be fully established. The aim of this study was to develop a range of modified polyamines, particularly ligands incorporating a fluorophore, to explore the structural tolerances of the polyamine transport system and to probe the intracellular location of polyamines acquired from the medium.Results: We synthesised a wide range of polyamine analogues incorporating cytotoxic agents, fluorescent chromophores and bulky substituents. All of these analogues have been shown to be good competitive inhibitors of spermidine uptake in a range of mammalian cells, Direct evidence for uptake of the fluorescent polyamine analogues and their subcellular distribution was obtained from confocal laser scanning fluorescence microscopy, which showed that they accumulated in granular structures within the cytoplasm and not in the nucleus. We demonstrated that their uptake is through the polyamine transport system by showing that pretreatment with DFMO, a potent inhibitor of polyamine biosynthesis, led to enhanced uptake, and cells deficient in the polyamine transport system did not accumulate these polyamine analogues.Conclusions: The polyamine transport system has a surprisingly broad structural tolerance. Fluorophore-containing polyamine analogues derived from the extracellular pool are located in granular structures within the cytoplasm and not to any great extent in the nuclei of mammalian cells. These observations might be consistent with a mechanism involving receptor-mediated endocytosis, and the granular 'structures' seen might reflect polyamine compartmentalisation within vesicles.