Créton R, Steele M E, Jaffe L F
Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA.
Cell Calcium. 1997 Dec;22(6):439-46. doi: 10.1016/s0143-4160(97)90071-3.
Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 microM in sea urchin eggs, 2 microM in Drosophila eggs, and only 0.11 microM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 microM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 microM apo-aequorin would take 50 days in zebrafish eggs, 2 days [corrected] in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 microm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.
水母发光蛋白是一种生物发光钙指示剂，由一个21 kDa的蛋白质（脱辅基水母发光蛋白）和一个亲脂性辅因子（腔肠素）共价连接而成。水母发光蛋白基因可以在多种细胞系和组织中表达，从而实现对特定细胞类型的非侵入性钙成像。在本文中，我们描述了在胚胎发育过程中利用基因导入的脱辅基水母发光蛋白进行钙成像的可能性和局限性。通过将水母发光蛋白注射到海胆、果蝇和斑马鱼的卵中，我们发现较小的卵需要更高浓度的水母发光蛋白。我们的结果表明，为了测量游离胞质钙的静息水平，海胆卵中需要至少40 μM的水母发光蛋白浓度，果蝇卵中需要2 μM，而斑马鱼卵中仅需要0.11 μM。我们使用一种简单的测定方法来确定体内表达的脱辅基水母发光蛋白的绝对浓度以及水母发光蛋白形成的百分比。通过在果蝇卵母细胞中表达水母发光蛋白基因来说明该测定方法的应用。这些卵母细胞可形成高达1 μM的脱辅基水母发光蛋白。在我们的实验中，只有0.3%的这种脱辅基水母发光蛋白与从培养基中进入的腔肠素结合形成水母发光蛋白，这对于卵母细胞的钙成像来说是不够的，但确实能够对卵巢进行体内成像。从这些研究中，我们得出结论，腔肠素进入细胞是水母发光蛋白形成的限速步骤。根据果蝇中腔肠素的摄取速率，我们估计在斑马鱼卵中1 μM脱辅基水母发光蛋白完全转化需要50天，在果蝇卵中需要2天[已修正]，在海胆卵中需要7天，在10微米的组织培养细胞中需要18小时。我们的结果表明，当大量小细胞能够在腔肠素中孵育时，基于基因导入脱辅基水母发光蛋白的工作将最为成功。在胚胎发育过程中，这将涉及将腔肠素引入晚期胚胎的循环系统。早期胚胎的钙成像可能最好通过注射水母发光蛋白来完成，这可以避免腔肠素进入这一缓慢过程。
