Confocal imaging and tracking of the exocytotic routes for D-serine-mediated gliotransmission
Confocal imaging and tracking of the exocytotic routes for D-serine-mediated gliotransmission
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DOI：
10.1002/glia.20696
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发表时间：
2008-09-01
期刊：
影响因子：
6.2
通讯作者：
Mothet, Jean-Pierre
中科院分区：
文献类型：
--
作者：
Martineau, Magalie;Galli, Thierry;Mothet, Jean-Pierre
D-丝氨酸是星形胶质细胞衍生的 N-甲基-D-天冬氨酸受体调节剂，但其细胞内运输途径仍然很大程度上未知。在这里，我们将共聚焦显微镜与共定位量化相结合来追踪储存 D-丝氨酸的星形细胞器。我们报告，D-丝氨酸与转染的 eGFP-synaptobrevin/VAMP2 和 eGFP-cellubrevin/VAMP3 共定位，这两个 v-SNARE 是调节分泌途径的。未发现与内体分选和回收系统标记物的显着共定位：EEA1、eGFP-endobrevin/VAMP8、eGFP-TI-VAMP/VAMP7、LAMP1 和 CD63。用秋水仙碱阻断囊泡出芽表明分泌囊泡将 D-丝氨酸下游输入高尔基体。最后，用 Ca2+-离子载体 A23187、谷氨酸激动剂或缓激肽处理星形胶质细胞会触发突触短蛋白/VAMP2 易位至质膜，同时 D-丝氨酸从受调节的分泌途径中消失。我们的结果为 D-丝氨酸在受调节的分泌途径中的囊泡储存以及可能通过 Ca2+ 动员来招募这些储存以释放 D-丝氨酸提供了形态学证据。 (C) 2008 Wiley-Liss, Inc.
D-Serine is an astrocyte-derived regulator for N-methyl-D-aspartate receptors, but the intracellular routes of its trafficking are still largely unknown. Here, we combined confocal microscopy with colocalization quantification to track the astrocytic organelles that store D-serine. We report that D-serine colocalizes with the transfected eGFP-synaptobrevin/VAMP2 and eGFP-cellubrevin/VAMP3, two v-SNAREs of the regulated secretory pathway. No significant colocalization was found with markers of the endosomal sorting and recycling system: EEA1, eGFP-endobrevin/VAMP8, eGFP-TI-VAMP/VAMP7, LAMP1, and CD63. Blockade of vesicular budding with colchicine shows that secretory vesicles import D-serine downstream to the Golgi apparatus. Finally, treatment of astrocytes with the Ca2+-ionophore A23187, glutamate agonists, or bradykinin trigger translocation of synaptobrevin/VAMP2 to the plasma membrane with a concomitant disappearance of D-serine from the regulated secretory pathway. Our results provide morphological evidence for a vesicular storage of D-serine in the regulated secretory pathway and the possible recruitment of these stores by Ca2+ mobilization to release D-serine. (C) 2008 Wiley-Liss, Inc.