Miyawaki A, Llopis J, Heim R, McCaffery J M, Adams J A, Ikura M, Tsien R Y
Department of Pharmacology, University of California, San Diego, La Jolla 92093-0647, USA.
Nature. 1997 Aug 28;388(6645):882-7. doi: 10.1038/42264.
Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.
胞质溶胶和细胞器中重要的Ca2+信号通常极其局限，难以测量。为克服这一问题，我们构建了新型Ca2+荧光指示剂，它们由基因编码，无需辅因子，且可靶向特定细胞内位置。我们将这些荧光指示剂称为“钙黄绿蛋白”。它们由绿色荧光蛋白（GFP）的蓝色或青色发射突变体、钙调蛋白、钙调蛋白结合肽M13以及增强型绿色或黄色发射GFP的串联融合体组成。Ca2+的结合使钙调蛋白环绕M13结构域，增加侧翼GFP之间的荧光共振能量转移（FRET）。钙调蛋白突变可调节Ca2+亲和力，以测量10(-8)至10(-2) M范围内的游离Ca2+浓度。我们已经可视化了用编码带有适当定位信号的嵌合体的互补DNA转染的单个HeLa细胞的胞质溶胶、细胞核和内质网中的游离Ca2+动态变化。单个细胞内质网中的Ca2+浓度在静息时为60至400 microM，Ca2+动员后为1至50 microM。FRET也是青色GFP标记的钙调蛋白与黄色GFP标记的M13可逆分子间缔合的指示剂。因此，GFP突变体之间的FRET可监测单个活细胞中的局部Ca2+信号和蛋白质异二聚化。
