构建钙荧光探针细胞用于探究胞浆和线粒体钙对ATP刺激的响应
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摘要
目的:构建表达基因编辑钙探针(GECIs)的细胞系HeLa-GECIs,探究细胞应答外界ATP刺激中钙离子在细胞内的响应和变化。方法:分别用能够直接通过荧光强度反映细胞胞浆内和线粒体内钙离子相对浓度的2种钙探针cyto-GCaMP6和4mt-GCaMP6感染HeLa细胞,获得2种表达钙离子探针的HeLa细胞系;在感染了2种腺病毒探针24 h后,用共聚焦荧光显微镜检测荧光探针在HeLa细胞内的表达情况;在表达2种钙探针的细胞的培养基中加入外源ATP,用Time-lapse成像动态观测技术观察HeLa细胞内钙离子对外环境中ATP的响应。结果:共聚焦荧光显微镜观察,确定95%以上的细胞表达了对应的钙离子指示荧光探针;Time-lapse成像动态观测技术观察发现,在细胞培养基中加入ATP后,细胞胞浆钙探针荧光强度瞬时(3～6 s)升至10倍,200 s后逐渐降低到基础水平;线粒体钙到达峰值(4倍)的时间稍滞后(5～8 s),并且回落更慢,300 s时至1.5倍。在ATP受体P2X7抑制剂A438079预处理的实验组,上述胞浆钙和线粒体钙浓度上升不明显。结论:构建了能在活体细胞内通过荧光探针实时监测钙离子响应胞外ATP刺激的细胞实验体系,为进一步深入探究ATP等危险信号导致细胞的炎性损伤机制奠定了基础。
Objective: To investigate the intracellular calcium response to extracelluar ATP stimulation by generating human cervical cancer cell lines(HeLa) expressing genetically encoded Ca~(2+) indicators(GECIs). Methods:We obtained HeLa cell lines expressing the two calcium probes cyto-GCaMP6 or 4 mt-GCaMP6,whose fluorescence intensity reflects the free Ca~(2+) level in cytosol or mitochondria respectively by infecting HeLa cells with adenovirus containing the calcium probes. 24 hours after virus infection, the expression of the two fluorescent probes in HeLa cells was checked by confocal fluorescence microscope. We observed the intracellular calcium response of cancer cells under ATP stimulation by Time-lapse analysis. We also observed the ATP stimulated intracellular calcium response in cells pretreated with A438079, the inhibitor of ATP receptor channel P2X7. Results: 24 hours after infecting viruses, we found that 95% of the cells expressing corresponding probes by Confocal microscope imaging. Through time-lapse imaging analysis, we found that the fluorescence intensity dynamic trace of cytoGaMP6 exhibited an about 10 times elevation in 3～6 seconds after ATP stimulation and returned to the basal level after 200 seconds. By contrast, it took a little more time(about 5-8 seconds after stimulation) for mitochondrial calcium trace to reach the highest fluorescence intensity. The decay time of the probe in mitochondria was slow as the fluorescence intensity stayed 1.5 times the basal level even after 300 seconds. When pretreating cells with the ATP receptor P2X7 inhibitor, A438079, the amplitude of both cytosolic and mitochondrial calcium transient was reduced. Conclusion: In this study, we successfully generated fluorescent Ca~(2+) indicators within living cells to monitor dynamic responses of intercellular calcium upon ATP stimulation.
Objective: To investigate the intracellular calcium response to extracelluar ATP stimulation by generating human cervical cancer cell lines(HeLa) expressing genetically encoded Ca~(2+) indicators(GECIs). Methods:We obtained HeLa cell lines expressing the two calcium probes cyto-GCaMP6 or 4 mt-GCaMP6,whose fluorescence intensity reflects the free Ca~(2+) level in cytosol or mitochondria respectively by infecting HeLa cells with adenovirus containing the calcium probes. 24 hours after virus infection, the expression of the two fluorescent probes in HeLa cells was checked by confocal fluorescence microscope. We observed the intracellular calcium response of cancer cells under ATP stimulation by Time-lapse analysis. We also observed the ATP stimulated intracellular calcium response in cells pretreated with A438079, the inhibitor of ATP receptor channel P2X7. Results: 24 hours after infecting viruses, we found that 95% of the cells expressing corresponding probes by Confocal microscope imaging. Through time-lapse imaging analysis, we found that the fluorescence intensity dynamic trace of cytoGaMP6 exhibited an about 10 times elevation in 3～6 seconds after ATP stimulation and returned to the basal level after 200 seconds. By contrast, it took a little more time(about 5-8 seconds after stimulation) for mitochondrial calcium trace to reach the highest fluorescence intensity. The decay time of the probe in mitochondria was slow as the fluorescence intensity stayed 1.5 times the basal level even after 300 seconds. When pretreating cells with the ATP receptor P2X7 inhibitor, A438079, the amplitude of both cytosolic and mitochondrial calcium transient was reduced. Conclusion: In this study, we successfully generated fluorescent Ca~(2+) indicators within living cells to monitor dynamic responses of intercellular calcium upon ATP stimulation.
引文
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[10] Llinas R,Blinks J R,Nicholson C. Calcium transient in presynaptic terminal of auid giant synapse:detection with aequorin[J]. Science,1972,176(4039):1127-1129.
[11] Stefani D D,Rizzuto R, Pozzan T. Enjoy the trip:calcium in mitochondria back and forth[J]. Annu Rev Biochem, 2016,85(1):161-192.
[12] De Stefani D, Raffaello A, Teardo E, et al. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter[J]. Nature, 2011,476(7360):336-340.
[13] Kasuya G,Yamaura T,Ma X B,et al. Structural insights into the competitive inhibition of the ATP-gated P2X receptor channel[J]. Nat Common, 2017,8:876.
[14] Munoz F M, Gao R, Tian Y, et al. Neuronal P2X7 receptor-induced reactive oxygen species production contributes to nociceptive behavior in mice[J]. Sci Rep,2017,7(1):3539.
[15] Zimmermann H. Extracellular ATP and other nucleotides-ubiquitous triggers of intercellular messenger release[J]. Purinergic Signal, 2016,12(1):25-57.
[2] Kurashima Y,Amiya T,Nochi T, et al. Extracellular ATP mediates mast cell-dependent intestinal inflammation through P2X7 purinoceptors[J]. Nat Common,2012,3:1034.
[3] Melani A, Turchi D, Vannucchi M G, et al. ATP extracellular concentrations are increased in the rat striatum during in vivo ischemia[J]. Neurochem Int, 2005,47(6):442-448.
[4] Wang Y,Martins I, Ma Y, et al. Autophagy-dependent ATP release from dying cells via lysosomal exocytosis[J]. Autophagy, 2013,9(10):2.
[5] Idzko M, Ferrari D, Eltzschig H K. Nucleotide signalling during inflammation.[J]. Nature, 2014,509(7500):310-317.
[6] Baron L, Gombault A, Fanny M, et al. The NLRP3 inflammasome is activated by nanoparticles throughATP, ADP and adenosine[J]. Cell Death Dis, 2015,6(2):e1629.
[7] Carafoli E, Krebs J. Why calcium? How calcium became the best communicator[J]. J Biol Chem, 2016,291(40):20849-20857.
[8] Horikawa K. Recent progress in the development of genetically encoded Ca2+indicators[J]. J Med Invest,2015,62(1-2):24.
[9] Chen T W, Wardill T J, Sun Y, et al. Ultrasensitive fluorescent proteins for imaging neuronal activity[J].Nature, 2013,499(7458):295-300.
[10] Llinas R,Blinks J R,Nicholson C. Calcium transient in presynaptic terminal of auid giant synapse:detection with aequorin[J]. Science,1972,176(4039):1127-1129.
[11] Stefani D D,Rizzuto R, Pozzan T. Enjoy the trip:calcium in mitochondria back and forth[J]. Annu Rev Biochem, 2016,85(1):161-192.
[12] De Stefani D, Raffaello A, Teardo E, et al. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter[J]. Nature, 2011,476(7360):336-340.
[13] Kasuya G,Yamaura T,Ma X B,et al. Structural insights into the competitive inhibition of the ATP-gated P2X receptor channel[J]. Nat Common, 2017,8:876.
[14] Munoz F M, Gao R, Tian Y, et al. Neuronal P2X7 receptor-induced reactive oxygen species production contributes to nociceptive behavior in mice[J]. Sci Rep,2017,7(1):3539.
[15] Zimmermann H. Extracellular ATP and other nucleotides-ubiquitous triggers of intercellular messenger release[J]. Purinergic Signal, 2016,12(1):25-57.
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