Institute for Clinical Neurobiology, University of Würzburg, Würzburg, Germany.
Eur J Cell Biol. 2011 Aug;90(8):613-9. doi: 10.1016/j.ejcb.2011.02.010. Epub 2011 May 10.
The lumen of the endoplasmic reticulum (ER) contributes to the dynamics of Ca(2+) signaling by acting as a source or sink of signal Ca(2+). Despite its relevance for the understanding of the cell biology and pathophysiology of the luminal calcium store, the direct measurement of luminal Ca(2+) release and uptake is still critical when Ca(2+) homeostasis is analyzed in neural cells. For the analysis of Ca(2+)-dependent signaling, synthetic Ca(2+) indicators have become popular. The properties of these indicators allow only limited targeting to subcellular structures such as the ER. Recently, we introduced a new strategy for the targeting of synthetic Ca(2+) indicators to the lumen of the ER. The method, termed Targeted-Esterase-induced Dye loading (TED) is based on the targeted recombinant expression of a high carboxylesterase (CES) activity in the lumen of the ER, which is needed to trap synthetic indicators. The method combines the selectivity of protein targeting with the biochemical advantages of low-affinity synthetic Ca(2+) indicators. TED permits direct and non-disruptive measurement and imaging of Ca(2+)-store dynamics. Here, we summarize major topics in the cell biology of ER Ca(2+) signaling and discuss the perspectives of the TED method for the morphological and physiological analysis of temporal and spatial Ca(2+)-dynamics in neural cells.
内质网（ER）的腔作为信号 Ca(2+) 的源或汇，有助于 Ca(2+) 信号的动态变化。尽管它对于理解腔钙库的细胞生物学和病理生理学具有重要意义，但当分析神经细胞中的 Ca(2+) 稳态时，直接测量腔 Ca(2+) 的释放和摄取仍然至关重要。对于 Ca(2+)-依赖性信号的分析，合成 Ca(2+) 指示剂已变得流行。这些指示剂的特性仅允许有限地靶向细胞器，如内质网。最近，我们引入了一种将合成 Ca(2+) 指示剂靶向内质网腔的新策略。该方法称为靶向酯酶诱导的染料加载（TED），基于内质网腔中高羧酸酯酶（CES）活性的靶向重组表达，这是捕获合成指示剂所必需的。该方法将蛋白靶向的选择性与低亲和力合成 Ca(2+) 指示剂的生化优势相结合。TED 允许直接和非侵入性地测量和成像 Ca(2+) 储存动力学。在这里，我们总结了内质网 Ca(2+) 信号转导的细胞生物学中的主要主题，并讨论了 TED 方法在神经细胞中时空 Ca(2+) 动力学的形态和生理分析中的应用前景。
