应用重组表达钙离子敏感蛋白YC2.1研究粟酒裂殖酵母细胞内钙离子浓度的分布

The Intracellular Distribution of Ca^(2+) in Schizosaccharomyces pombe Shown by Cameleon Calcium Indicator YC2.1

把重组表达钙离子敏感蛋白的YC2.1基因(yellowcameleon2.1)导入了粟酒裂殖酵母中,观察了粟酒裂殖酵母细胞内钙离子浓度的分布。结果发现,钙离子敏感蛋白所指示的钙离子呈细胞周缘胞质较高浓度分布,而在细胞胞质中部的钙离子浓度相对低一些。通过DAPI染色实验证实这是由于胞质中部细胞核的填充而形成。fluo-3染色的裂殖酵母细胞,由于fluo-3进入到细胞器(房室化现象),所以出现胞质的内部区域高的荧光信号,而在周缘的胞质区相对弱,不能真实反应胞质钙离子的分布。因此重组表达钙离子敏感蛋白测定钙离子的方法优于fluo-3荧光探针的方法,对于裂殖酵母细胞胞内钙离子的研究具有良好的应用前景。

We transformed the recombined yellow cameleon 2.1 （YC2.1） gene into the Schizosaccharomyces pombe and observed the distribution of free calcium concentration in the cytoplasm shown by expressed YC2.1. The results showed that the calcium fluorescence ratio of YC2.1 appeared mainly around the peripheral area of the cytoplasm in the fission yeast cell, less in the center area of the cytoplasm in which occupied by the organelles and nuclear. In contrast, the calcium fluorescence of fluo-3 was higher in the center of the cytoplasm not than around the peripheral area of the cytoplasm because fluo-3 was sequestered into organelles in the cell. Therefore, fluo-3 could not reflect the true distribution of calcium in the cytoplasm. Our data provided that the recombinant yellow cameleon calcium indicator is better than the traditional chemical fluorescence indicator fluo-3 for measuring of free calcium in cytoplasm, and will promote studying of distribution and role of intracellular calcium in yeast cell proliferation.