Localization of puff sites adjacent to the plasma membrane: functional and spatial characterization of Ca2+ signaling in SH-SY5Y cells utilizing membrane-permeant caged IP3.
Localization of puff sites adjacent to the plasma membrane: functional and spatial characterization of Ca2+ signaling in SH-SY5Y cells utilizing membrane-permeant caged IP3.
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DOI：
10.1016/j.ceca.2008.06.001
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发表时间：
2009-01
期刊：
影响因子：
4
通讯作者：
Parker, Ian
中科院分区：
文献类型：
--
作者：
Smith, Ian F.;Wiltgen, Steven M.;Parker, Ian
非洲爪哇卵母细胞一直是研究细胞内钙动态时空机制的理想模型系统，这在很大程度上是因为这个巨大的细胞促进了细胞内钙指示剂、缓冲液和笼状化合物的注入。然而，最近商用的笼状IP3(Ci-IP3)和EGTA的膜透酯形式，现在允许这些化合物轻松地装载到较小的哺乳动物细胞中，允许控制[IP3]i和胞浆钙缓冲。在这里，我们建立了人神经母细胞瘤SH-SY5Y细胞系作为一个有利的实验系统，以成像钙信号，并表征了IP3介导的钙信号机制。不断增加的i-IP3的闪光释放会引起过渡到波的钙离子喷发，但EGTA的细胞内负载会解偶联释放位置，从而允许在广泛的[IP3]范围内研究离散的喷发。泡芙活性在单次光释放后持续几分钟，表明I-IP3在这些细胞中的周转速度很慢，并表明重复的钙尖峰周期为20-30s，不是由[IP3]的振荡驱动的。喷雾的幅度与[IP3]无关，而其频率随着光释放的增加而增加。在SH-SY5Y细胞中，泡芙位置并不优先分布在细胞核附近，而是集中在靠近质膜的地方，在那里它们可以用全内反射显微镜观察到，这为前所未有的钙离子泡芙动力学提供了时空分辨的可能性。
The Xenopus oocyte has been a favored model system in which to study spatio-temporal mechanisms of intracellular Ca2+ dynamics, in large part because this giant cell facilitates intracellular injections of Ca2+ indicator dyes, buffers and caged compounds. However, the recent commercial availability of membrane-permeant ester forms of caged IP3 (ci-IP3) and EGTA, now allows for facile loading of these compounds into smaller mammalian cells, permitting control of [IP3]i and cytosolic Ca2+ buffering. Here, we establish the human neuroblastoma SH-SY5Y cell line as an advantageous experimental system for imaging Ca2+ signaling, and characterize IP3-mediated Ca2+ signaling mechanisms in these cells. Flash photorelease of increasing amounts of i-IP3 evokes Ca2+ puffs that transition to waves, but intracellular loading of EGTA decouples release sites, allowing discrete puffs to be studied over a wide range of [IP3]. Puff activity persists for minutes following a single photorelease, pointing to a slow rate of i-IP3 turnover in these cells and suggesting that repetitive Ca2+ spikes with periods of 20-30s are not driven by oscillations in [IP3]. Puff amplitudes are independent of [IP3], whereas their frequencies increase with increasing photorelease. Puff sites in SH-SY5Y cells are not preferentially localized near the nucleus, but instead are concentrated close to the plasma membrane where they can be visualized by total internal reflection microscopy, offering the potential for unprecedented spatio-temporal resolution of Ca2+ puff kinetics.
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