Raft and cytoskeleton associations of an ABC transporter:: P-glycoprotein
Raft and cytoskeleton associations of an ABC transporter:: P-glycoprotein
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DOI：
10.1002/cyto.a.20081
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发表时间：
2004-10-01
期刊：
影响因子：
3.7
通讯作者：
Szabó, G
中科院分区：
文献类型：
--
作者：
Bacso, Z;Nagy, H;Szabó, G
背景：一项新的流式细胞分析已经在相应的报告中描述（Gombos等）。方法：在加入筏子保存的Triton X-100或Nonidet P-40后，分析了免疫荧光强度下降的动力学，两者都破坏了整个膜。两种洗涤剂的温和处理使细胞只附着在那些通过筏固定在细胞骨架上或独立于筏的蛋白质上。通过环糊精影响微丝和调节胆固醇膜水平的药物被用来区分筏介导的和非筏相关的Pgp关联。使用共聚焦显微镜和流式细胞荧光能量转移测量来确认Pgp与筏成分的共定位。结果：该方法被证明足够敏感，可以分辨uic2标记的细胞表面Pgps对Triton X-100和Nonidet P-40的抗性差异。大约34%的UIC2 fab标记的Pgp分子通过抗洗涤剂、胆固醇敏感微域或直接与细胞骨架相关，而大约15%的Pgp分子被发现与细胞骨架直接相关。因此，共聚焦显微镜显示Pgps与筏标记共定位，主要在微绒毛中。荧光共振能量转移效率数据表明pgp与筏标记CD44、CD59和G(M1)-神经节苷类之间的分子接近性也表明pgp的很大一部分存在于筏微域中。Pgp的Raft association似乎具有重要的功能意义，因为它的调节明显影响药物泵送。结论：通过在动力学模式下使用流式细胞术抗洗涤剂试验，我们能够评估Pgp在生理相关水平上表达的筏关联程度和肌动蛋白细胞骨架锚定。我们证明了Pgp的很大一部分与LS-174-T人结肠癌细胞密切相关，并且这种定位可能影响其转运蛋白功能。本报告中提出的动态流式细胞术洗涤剂抗性分析被认为普遍适用于低水平表达膜蛋白的分子相互作用分析。(C) 2004 Wiley-Liss, Inc。
Background: A novel flow cytometric assay has been described in an accompanying report (Gombos et al.,Methods: The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft-preserving Triton X-100 or Nonidet P-40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft-mediated and non-raft-related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents.Results: The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2-labeled cell-surface Pgps to Triton X-100 versus Nonidet P-40. Approximately 34% of the UIC2 Fab-labeled Pgp molecules were associated with the cytoskeleton through detergent-resistant, cholesterol-sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgpand the raft markers CD44, CD59, and G(M1)-gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping.Conclusions: By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS-174-T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levels. (C) 2004 Wiley-Liss, Inc.