Fluorescent annexin A1 reveals dynamics of ceramide platforms in living cells
Fluorescent annexin A1 reveals dynamics of ceramide platforms in living cells
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DOI：
10.1111/j.1600-0854.2008.00800.x
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发表时间：
2008-10-01
期刊：
影响因子：
4.5
通讯作者：
Draeger, Annette
中科院分区：
文献类型：
--
作者：
Babiychuk, Eduard B.;Monastyrskaya, Katia;Draeger, Annette
神经酰胺在细胞凋亡过程中发生后，促进质膜结构的总体重组，涉及信号分子的聚集和囊泡形成、融合和运输的放大。膜联蛋白是一个蛋白质家族，在Ca 2+存在下，与含有带负电荷磷脂的膜结合。在这里，我们表明，神经酰胺增加膜联蛋白A1膜相互作用的亲和力。在生理相关的范围内的Ca 2+浓度，这导致膜联蛋白A1-膜相互作用的Ca 2+的敏感性增加。在固定细胞中，使用神经酰胺特异性抗体，我们建立了膜联蛋白A1与富含神经酰胺的质膜区域（神经酰胺平台）的直接相互作用。在活细胞中，膜联蛋白A1的细胞内动力学与质膜神经酰胺的动力学相匹配。在膜联蛋白家族的蛋白质中，与神经酰胺平台的相互作用仅限于膜联蛋白A1，并通过其独特的N-末端结构域传递。我们表明，细胞内钙超载发生在细胞应激的条件下诱导神经酰胺的生产。使用荧光标记的膜联蛋白A1作为神经酰胺平台和膜联蛋白A6作为非选择性膜标记的报告，我们可视化神经酰胺平台的第一次在活细胞中，并提供证据的神经酰胺驱动的分离和内化的膜相关蛋白。
Upon its genesis during apoptosis, ceramide promotes gross reorganization of the plasma membrane structure involving clustering of signalling molecules and an amplification of vesicle formation, fusion and trafficking. The annexins are a family of proteins, which in the presence of Ca2+, bind to membranes containing negatively charged phospholipids. Here, we show that ceramide increases affinity of annexin A1-membrane interaction. In the physiologically relevant range of Ca2+ concentrations, this leads to an increase in the Ca2+ sensitivity of annexin A1-membrane interaction. In fixed cells, using a ceramide-specific antibody, we establish a direct interaction of annexin A1 with areas of the plasma membrane enriched in ceramide (ceramide platforms). In living cells, the intracellular dynamics of annexin A1 match those of plasmalemmal ceramide. Among proteins of the annexin family, the interaction with ceramide platforms is restricted to annexin A1 and is conveyed by its unique N-terminal domain. We demonstrate that intracellular Ca2+ overload occurring at the conditions of cellular stress induces ceramide production. Using fluorescently tagged annexin A1 as a reporter for ceramide platforms and annexin A6 as a non-selective membrane marker, we visualize ceramide platforms for the first time in living cells and provide evidence for a ceramide-driven segregation and internalization of membrane-associated proteins.