Molecular probes for sensing the cholesterol composition of subcellular organelle membranes.
Molecular probes for sensing the cholesterol composition of subcellular organelle membranes.
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DOI：
10.1016/j.bbalip.2006.06.016
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发表时间：
2006-10
期刊：
影响因子：
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通讯作者：
Rong Wang;M. Hosaka;Lu Han;Hiromi Yokota-Hashimoto;M. Suda;D. Mitsushima;S. Torii;T. Takeuchi
中科院分区：
文献类型：
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作者：
Rong Wang;M. Hosaka;Lu Han;Hiromi Yokota-Hashimoto;M. Suda;D. Mitsushima;S. Torii;T. Takeuchi
神经内分泌细胞含有两种类型的促分泌剂调节的酸性隔膜：分泌颗粒(SGS)和突触样微泡(SLMV)，它们可以通过吖啶橙(AO)和DAMP等嗜酸性探针进行识别。我们使用胰腺β细胞系MIN6研究了这些探针在SGS和SLMV中的积累与培养液中葡萄糖水平的函数关系。AO在低糖条件下积累，而在高糖条件下不积累。Ao的积聚与葡萄糖引起的SLMV动态变化有很好的相关性，而潮湿则定位于SGS。因为据报道SG膜的胆固醇含量很高，所以我们制备了胆固醇水平升高的脂质体。AO很好地结合到胆固醇组成为20%至40%的脂质体中，而DUMP在胆固醇水平超过40%的脂质体中也是如此。事实上，当胆固醇从MIN6 SG膜中被耗尽时，DAMP掺入减少，相反，AO被掺入。在PC12细胞中，AO对SGS的掺入显著，而DAMP掺入有限。在PC12细胞的SG膜中，胆固醇组分始终存在37-39%。我们认为，胆固醇敏感探针，AO和DAMP，是研究酸性细胞器膜中胆固醇组成的有用工具。
Neuroendocrine cells contain two types of secretagogue-regulated acidic compartments: secretory granules (SGs) and synaptic-like microvesicles (SLMVs), which can be identified by acidotropic probes such as acridine orange (AO) and DAMP. We investigated the accumulation of these probes in SGs and SLMVs as a function of glucose levels in the culture media using a pancreatic β-cell line MIN6. AO was accumulated in the low-glucose condition, but not in the high-glucose condition. The AO accumulation correlated well with the SLMV dynamics by glucose and DAMP was localized in the SGs. Because SG membranes are reportedly high in cholesterol, we prepared liposomes with increasing cholesterol levels. AO is well incorporated into liposomes having a 20 to 40 mol% cholesterol composition, whereas DAMP was so in those having over 40 mol% cholesterol levels. Indeed, when cholesterol was depleted from MIN6 SG membranes, DAMP incorporation decreased, instead AO was incorporated. In PC12 cells, AO incorporation into SGs was significant but DAMP incorporation was limited. Consistently, the cholesterol composition was found 37 to 39 mol% in the SG membrane of PC12 cells. We suggest that cholesterol-sensing probes, AO and DAMP, are useful tools for investigating cholesterol compositions in acidic organelle membranes.