Red Fluorescent Protein-Aequorin Fusions as Improved Bioluminescent Ca2+ Reporters in Single Cells and Mice

Bioluminescence recording of Ca2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca2+ in various cell compartments. In addition, they would also serve to monitor Ca2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R0) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca2+ oscillations in single HeLa cells expressing tdTA. Ca2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca2+ activity of HeLa cells injected subcutaneously into mice, and Ca2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca2+ sensor reported to date and is, therefore, a promising probe to study Ca2+ dynamics in whole organisms or tissues expressing the transgene.

利用光蛋白水母素（aequorin）记录钙离子信号的生物发光无需辐射能量输入，且可在低背景条件下以优异的时间分辨率进行检测。将水母素的发射波长红移，在维多利亚水母（Aequorea victoria）中可通过生物发光共振能量转移（BRET）至绿色荧光蛋白（GFP）自然实现。该过程已在分子融合体GFP-水母素与单体红色荧光蛋白（mRFP）-水母素中得以重现，但后者的能量转移效率有限。具备强红色发射特性的融合蛋白，将有助于同时成像不同细胞区室中的钙离子信号。此外，由于红光穿过动物组织时散射更少，这类融合体也可用于监测活体生物体内的钙离子动态。本研究中将水母素与橙色及多种红色荧光蛋白进行融合，以筛选红色发射波段下性能最优的能量受体。其中，串联二聚体Tomato-水母素（tdTA）在所有研究的融合体中，展现出最高的生物发光共振能量转移效率（最大能量转移临界距离R0）以及最高的红色波段计数占比。此外，tdTA在细胞内的红色荧光基团成熟速率快于其他融合体。其发光强度足以对表达tdTA的单个HeLa细胞中ATP诱导的钙离子振荡进行成像观测。我们还记录了原代培养小鼠神经元细胞去极化引发的钙离子升高，并对神经元细微突起的变化实现了空间分辨成像。最后，我们还成功实现了对皮下注射入小鼠体内的HeLa细胞钙离子活性的成像，以及对在肌肉或腹腔内注射重组tdTA后产生的钙离子信号的检测。本研究表明，tdTA是目前已报道的亮度最高的红色生物发光钙离子传感器，因此是研究表达该转基因的整体生物体或组织中钙离子动态变化的极具潜力的分子探针。