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Ca2+ Measurements in Mammalian Cells with Aequorin-based Probes
使用基于水母发光蛋白的探针测定哺乳动物细胞中的Ca2+   

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参见作者原研究论文

本实验方案简略版
EMBO Molecular Medicine
May 2016

Abstract

Aequorin is a Ca2+ sensitive photoprotein suitable to measure intracellular Ca2+ transients in mammalian cells. Thanks to recombinant cDNAs expression, aequorin can be specifically targeted to various subcellular compartments, thus allowing an accurate measurement of Ca2+ uptake and release of different intracellular organelles. Here, we describe how to use this probe to measure cytosolic Ca2+ levels and mitochondrial Ca2+ uptake in mammalian cells.

Keywords: Ca2+ (Ca2+), Aequorin (水母发光蛋白), Probes (探针), Luminescence (发光)

Background

Aequorin is a 21 kDa photoprotein isolated from jellyfish Aequorea victoria that emits blue light in the presence of Ca2+. In its active form the photoprotein includes an apoprotein and a covalently bound prosthetic group, called coelenterazine. The apoprotein contains four helix-loop-helix ‘EF-hand’ domains, three of which are Ca2+-binding sites. These domains confer to the protein a particular globular structure forming the hydrophobic core cavity that accommodates the coelenterazine. When Ca2+ ions bind to the three high affinity EF-hand sites, coelenterazine is irreversibly oxidized to coelenteramide, with a concomitant release of CO2 and emission of light (Head et al., 2000).

Aequorin began to be widely used when the cDNA encoding the photoprotein was cloned, thus opening the way to recombinant expression. In particular, recombinant aequorin can be expressed not only in the cytoplasm, but also in single intracellular compartments by including specific targeting sequences in the engineered cDNAs (Hartl et al., 1989). To expand the range of Ca2+ sensitivity that can be monitored, point mutations in the EF-hand motives that lower the affinity for Ca2+ have been introduced (Granatiero et al., 2014a and 2014b). Reconstitution of an active recombinant aequorin in living cells is obtained by simple addition of coelenterazine into the medium. Coelenterazine is highly hydrophobic and permeates cell membranes of various cell types. Different coelenterazine analogues have been synthetized and are now commercially available.

Materials and Reagents

  1. Round glass coverslips 12 mm (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 1014355112NR15 ) - sterile upon autoclave cycle
  2. 24-well plate (Corning, Costar®, catalog number: 3524 )
  3. MDA-MB-231 cell line
  4. MDA-MB-468 cell line
  5. BT-549 cell line
  6. Aequorin-expressing plasmids (Brini, 2008)
  7. Gelatin
  8. Collagen
  9. Coelenterazine 0.5 mM stock solution, in methanol (IS Chemical Technology, catalog number: I14-2266 ) - aliquot in 50 μl aliquots. Store at -80 °C. Save it from light
  10. Agonist (e.g., ATP, Histamine, Bradykinin, Caffeine, Carbachol, Glutamate)
    Note: In this protocol example 100 µM ATP is used.
  11. Milli-Q water
  12. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  13. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
  14. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
  15. Potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich, catalog number: NIST200B )
  16. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M2773 )
  17. HEPES (Sigma-Aldrich, catalog number: H3375 )
  18. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 71687 )
  19. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 449709 )
  20. Glucose (Sigma-Aldrich, catalog number: G8270 )
  21. Digitonin (Sigma-Aldrich, catalog number: D5628 or D141 )
  22. Krebs-Ringer modified buffer (KRB) (see Recipes)
  23. Digitonin lysis solution (see Recipes)

Equipment

  1. Windows-based computer
  2. Perfusion chamber (Elettrofor)
  3. Low noise photomultiplier (Hamamatsu Photonics K. K., model: H7360-01)
  4. Peristaltic pump (Gilson’s MINIPULS® 3)
  5. Water bath (temperature-controlled)
  6. Photon-counting unit (Hamamatsu Photonics K. K., model: C8855-01 )

Note: Equipment is assembled as depicted in Figure 1.


Figure 1. Equipment. A. Fully equipped aequorinometer is composed of: (1) computer, (2) perfusion chamber, (3) photomultiplier, (4) peristaltic pump, (5) water bath, (6) photon-counting unit. B. During the experiment, the perfusion chamber is placed in close proximity to the photomultiplier, protected from light.

Procedure

  1. Day 1
    Plate cells on 12 mm round glass coverslips, in a 24-well plate at 30-50% confluence (about 70,000 cells/well) and let them grow in their specific medium (Figure 2). Pre-treatment of coverslips with gelatin, collagen or poly-lysine to increase cell adherence is not mandatory, but could be recommended for specific cell types.


    Figure 2. Different mammalian cell lines. MDA-MB-231 (A), MDA-MB-468 (B) and BT-549 (C) breast cancer cell lines at around 40% confluence.

  2. Day 2
    Transfect cells with the proper aequorin-expressing plasmids, according to the expected free [Ca2+] present in the subcellular compartment (Brini et al., 1995; Brini, 2008). Choose the best transfection/infection protocol depending on the targeted cell type. In this example protocol, Aequorin-wt and mitochondrial mutated aequorin (Asp119Ala) are used.

  3. Day 3
    1. 24 h after transfection remove medium from the cell plate.
    2. Wash cells with KRB solution once.
    3. Incubate cells at 37 °C for 90 min with 5 µM coelenterazine in 200 µl KRB solution.
    4. Transfer a coverslip containing the transfected cells to the perfusion chamber. Fix the perfusion chamber in close proximity to the photomultiplier (2-3 mm distance) (Figure 3).
    5. Continuously perfuse cells with KRB, at 37 °C in a water bath. Agonists and other drugs should be added to the same solution.
    6. Switch on the photomultiplier and start recording the light emission, ideally with a time span of 1 sec (the output of the amplifier-discriminator is captured by the photon-counting unit connected to a Windows-based computer).
    7. As soon as the background values are stable, stimulate cells with an appropriate agonist concentration, in order to induce Ca2+ release from the endoplasmic reticulum (ER).
    8. Follow light emission until suppression of Ca2+ signals.
    9. Terminate the experiment by lysing cells with the digitonin solution, thus discharging the remaining aequorin pool.
    10. Proceed with data analysis and calibration, as described below.


      Figure 3. Experimental procedure. The coverslip coated with transfected cells is moved from the plate (A) to the perfusion chamber (B). The perfusion chamber is then placed near the photomultiplier (C).

Data analysis

In order to convert the luminescence signal in [Ca2+], an algorithm has been developed. Three variables are required:

  1. The total amount of luminescence that each sample would emit in presence of saturating [Ca2+]. In order to calculate this parameter, at the end of each experiment cells are lysed with a digitonin-containing solution (as reported in the Procedure section), and the luminescence emitted by the residual aequorin is measured. Since the total amount of luminescence is directly dependent on the amount of aequorin, this parameter takes into account differences in transfection efficiency.
  2. Lmax, i.e., the luminescence that would be emitted at a certain time point if all the aequorin had been suddenly discharged. Since aequorin is being constantly consumed, Lmax progressively decreases during the experiment. At each time point, Lmax is calculated by subtracting the luminescence recorded before that point from the total light output of the whole experiment.
  3. L, i.e., the rate of photon emission at any instant during the experiment.
    The [Ca2+] at each time point is a function of L/Lmax (Figure 4). The presence of three Ca2+ binding sites in the aequorin molecule is responsible for the steep relationship between photon emission rate and free [Ca2+]. The rate of luminescence is independent of [Ca2+] at very high (> 10-4 M) and very low [Ca2+] (< 10-7 M). However, it is possible to expand the range of [Ca2+] that can be monitored by choosing the proper recombinant aequorin construct (Figure 5).
    Data should be plotted as average of Ca2+max peaks from replicates of each condition. At least 6 replicates per condition are recommended. In addition, a representative Ca2+ trace for each group of samples should be shown.


    Figure 4. Representative Ca2+ calibration curve. At the end of the experiment, after cell lysis, the total amount of aequorin can be estimated and L/Lmax can be calculated for each data point, thus allowing the conversion of light emitted into free Ca2+ concentration values. N = 6.


    Figure 5. Representative traces of intracellular Ca2+ measurements. A. Mitochondrial Ca2+ uptake upon 100 µM ATP stimulation, using a mutated isoform (Asp119Ala) of aequorin. B. Cytosolic Ca2+ transients upon ATP stimulation, using the wild-type aequorin. C. ER Ca2+ uptake monitored by adding 1 mM CaCl2 to a Ca2+-free KRB, and subsequent Ca2+ release induced by agonist stimulation (ATP).

Notes

  1. Although aequorin luminescence is influenced neither by K+ nor Mg2+ (which are the most abundant cations in the intracellular environment and thus the most likely source of interference in physiological experiments) both ions are competitive inhibitors of Ca2+-activated luminescence. pH also affects aequorin luminescence at values below 7. For these reasons, experiments with aequorin need to be done in well-controlled conditions of pH and ionic concentrations, notably of Mg2+.
  2. To stimulate Ca2+ release from ER, the appropriate agonist should be selected for each cell type (e.g., ATP, Histamine, Bradykinin, Caffeine, Carbachol, Glutamate). The choice depends on cell-type specific expression of the G protein-coupled plasma membrane receptors.

Recipes

  1. Krebs-Ringer modified buffer (KRB)
    135 mM NaCl
    5 mM KCl
    1 mM MgCl2·6H2O
    0.4 mM KH2PO4
    1 mM MgSO4·7H2O
    20 mM HEPES
    Adjust the pH to 7.4 with NaOH
    Store at 4 °C
    Add 1 mM CaCl2 and 5.5 mM glucose, fresh before use
  2. Digitonin lysis solution
    100 μM digitonin
    10 mM CaCl2
    Store at 4 °C for maximum 4 days

Acknowledgments

The research is supported by grants from the European Union (ERC mitoCalcium, No. 294777 to R.R.); Italian Ministries of Education, University and Research (PRIN to R.R., FIRB to R.R., FIRB Futuro in Ricerca RBFR10EGVP_002 to C.M.); NIH (grant 1P01AG025532-01A1 to R.R.) and French Muscular Dystrophy Association AFM-Téléthon (18857 to C.M.).

References

  1. Blinks, J. R. (1989). Use of calcium-regulated photoproteins as intracellular Ca2+ indicators. Methods Enzymol 172(4): 164-203.
  2. Brini, M. (2008). Calcium-sensitive photoproteins. Methods 46(3): 160-166.
  3. Brini, M., Marsault, R., Bastianutto, C., Alvarez, J., Pozzan, T. and Rizzuto, R. (1995). Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation. J Biol Chem 270(17): 9896-9903.
  4. Granatiero, V., Patron, M., Tosatto, A., Merli, G. and Rizzuto, R. (2014a). Using targeted variants of aequorin to measure Ca2+ levels in intracellular organelles. Cold Spring Harb Protoc 2014(1): pdb-prot072843.
  5. Granatiero, V., Patron, M., Tosatto, A., Merli, G. and Rizzuto, R. (2014b). The use of aequorin and its variants for Ca2+ measurements. Cold Spring Harb Protoc 2014(1): pdb-top066118.
  6. Hartl, F. U., Pfanner, N., Nicholson, D. W. and Neupert, W. (1989). Mitochondrial protein import. Biochim Biophys Acta 988(1): 1-45.
  7. Head, J. F., Inouye, S., Teranishi, K. and Shimomura, O. (2000). The crystal structure of the photoprotein aequorin at 2.3 Å resolution. Nature 405(6784): 372-376.

简介

水母发光蛋白是适合于测量哺乳动物细胞内细胞内Ca 2+ 2+瞬态的Ca 2+。由于重组cDNA表达,水母发光蛋白可以特异性靶向各种亚细胞区室,从而可以准确测量不同细胞内细胞器的Ca 2+摄取和释放。在这里,我们描述了如何使用这种探针来测量哺乳动物细胞中的胞质Ca 2+ / +和/或线粒体Ca 2+。

水母发光蛋白是一种21KDa的光稳定蛋白,其分离自在Ca 2+(2+)存在下发出蓝色光的水母维多利亚水母。在其活性形式中,光蛋白包括脱辅基蛋白和共价结合的假体组,称为肠糜嗪。载脂蛋白包含四个螺旋 - 环 - 螺旋“EF手”结构域,其中三个是Ca 2 + - 结合位点。这些结构域赋予蛋白质特定的球状结构,形成适应肠溶纤维素的疏水性核心腔。当Ca 2 + 离子结合到三个高亲和力EF手部位时,coelenterazine不可逆地被氧化成coelenteramide,同时释放CO 2和发光(Head 等人,2000)。
&nbsp;当编码光蛋白的cDNA被克隆时,水母发酵开始被广泛使用,从而打开重组表达的方式。特别地,重组水母发光蛋白不仅可以在细胞质中表达,而且可以在单个细胞内区室中表达,通过在工程化的cDNA中包括特异性靶向序列(Hartl et al。,1989)。为了扩大可以监测的Ca 2 + 敏感度范围,已经引入了降低Ca 2 + 亲和力的EF-手动因子中的点突变(Granatiero < ema等人
,2014a和2014b)。活性细胞中活性重组水母发光蛋白的重建是通过简单地向培养基中加入肠凝血素来获得的。肠培肠蛋白是高度疏水性的并渗透各种细胞类型的细胞膜。已经合成了不同的肠溶素类似物,现在可商购。

关键字:Ca2+, 水母发光蛋白, 探针, 发光

材料和试剂

  1. 圆形玻璃盖玻片12毫米(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:1014355112NR15) - 在高压釜循环下无菌
  2. 24孔板(Corning,Costar ®,目录号:3524)
  3. MDA-MB-231细胞系
  4. MDA-MB-468细胞系
  5. BT-549细胞系
  6. 水母发光蛋白表达质粒(Brini,2008)
  7. 明胶
  8. 胶原蛋白
  9. Coelenterazine 0.5mM储备液,在甲醇(IS Chemical Technology,目录号:I14-2266)中 - 等分试样在50μl等分试样中。储存于-80°C。从光保存
  10. 激动剂(例如,ATP,组胺,缓激肽,咖啡因,卡巴胆碱,谷氨酸)
    注意:在本协议示例中,使用100μMATP。
  11. Milli-Q水
  12. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  13. 氯化钾(KCl)(Sigma-Aldrich,目录号:P5405)
  14. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670)
  15. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:NIST200B)
  16. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:M2773)
  17. HEPES(Sigma-Aldrich,目录号:H3375)
  18. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:71687)
  19. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:449709)
  20. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  21. Digitonin(Sigma-Aldrich,目录号:D5628或D141)
  22. Krebs-Ringer改良缓冲液(KRB)(见食谱)
  23. Digitonin裂解液(参见食谱)

设备

  1. 基于Windows的计算机
  2. 灌注室(Elettrofor)
  3. 低噪声光电倍增管(Hamamatsu Photonics K.K.,型号:H7360-01)
  4. 蠕动泵(Gilson's MINIPULS ® 3)
  5. 水浴(温度控制)
  6. 光子计数单元(Hamamatsu Photonics K.K.,型号:C8855-01)

注意:设备组装如图1所示。


(1)计算机,(2)灌注室,(3)光电倍增管,(4)蠕动泵,(5)水浴,(3) 6)光子计数单元。 B.在实验过程中,灌注室放置在光电倍增管附近,防止光照。

程序

  1. 第1天
    在12mm圆形玻璃盖玻片上的平板细胞,在24孔板中以30-50%汇合(约70,000个细胞/孔)并使其在其特定培养基中生长(图2)。用明胶,胶原蛋白或聚赖氨酸预处理盖玻片以增加细胞粘附性并不是强制性的,但可以推荐用于特定的细胞类型。


    图2.不同的哺乳动物细胞系 MDA-MB-231(A),MDA-MB-468(B)和BT-549(C)乳腺癌细胞系约40%汇合。 br />
  2. 第2天
    根据存在于亚细胞隔室中的预期游离[Ca 2+],使用合适的水母发光蛋白表达质粒转染细胞(Brini等人,1995; Brini,2008) )。根据目标细胞类型选择最佳转染/感染方案。在这个实施例中,使用水母发酵蛋白-wt和线粒体突变的水母发光蛋白(Asp119Ala)。

  3. 第3天
    1. 转染24 h后,从细胞板中取出培养基。
    2. 用KRB溶液洗涤细胞一次。
    3. 在细胞培养37℃下,用5微升肠杆菌素在200微升KRB溶液中孵育90分钟
    4. 将含有转染细胞的盖玻片转移到灌注室。将灌注室固定在光电倍增器(距离2-3mm)附近(图3)。
    5. 在37℃,水浴中连续灌注细胞与KRB。激素和其他药物应加入同一解决方案
    6. 打开光电倍增管并开始记录发光,理想情况下,时间间隔为1秒(放大器鉴频器的输出由连接到基于Windows的计算机的光子计数单元捕获)。
    7. 一旦背景值稳定,就可以以适当的激动剂浓度刺激细胞,从而诱导内质网(ER)的Ca 2+释放。
    8. 遵循发光,直到抑制Ca 2 + 信号。
    9. 通过用洋地黄皂苷溶液裂解细胞来终止实验,从而排出剩余的水母发酵池。
    10. 继续进行数据分析和校准,如下所述。


      图3.实验程序。 将经转染细胞包被的盖玻片从板(A)移至灌注室(B)。然后将灌注腔置于光电倍增管(C)附近。

数据分析

为了转换[Ca 2 + ]中的发光信号,已经开发了一种算法。需要三个变量:

  1. 每个样品在饱和[Ca 2 O 3]存在下发射的总发光量。为了计算该参数,在每个实验结束时,用含有含元甙的溶液裂解细胞(如方法部分所述),并测量残留的水母发光蛋白发出的发光。由于发光总量直接取决于水母发光蛋白的量,因此该参数考虑了转染效率的差异。
  2. 如果所有的水母发光蛋白都突然放电,则在某个时间点发射的发光即是。由于水母发光蛋白正在不断消耗,所以实验过程中L max最大值逐渐降低。在每个时间点,通过从整个实验的总光输出中减去在该点之前记录的发光来计算L max max。
  3. L,即,即实验期间任何时刻的光子发射速率 每个时间点的[Ca 2 + ]是L/L max max的函数(图4)。在水母发光蛋白分子中存在三个Ca 2+ 2 + 结合位点是造成光子发射速率与游离[Ca 2 + ]之间的陡峭关系的原因。在非常高的(> 10℃-4℃)下,发光速率与[Ca 2+> + +]无关,而非常低的[Ca 2+] >](<10 -7 M)。然而,可以通过选择合适的重组水母发光蛋白构建体来扩大可以监测的[Ca 2 + ]的范围(图5)。
    数据应从每个条件的重复绘制为Ca 2 + 最大峰的平均值。建议每个条件至少重复6次。此外,应显示每组样品的代表性Ca 2 + 迹线。


    图4.代表性Ca 2 + 校准曲线在实验结束时,细胞裂解后,可以估计水母发光蛋白的总量和L /可以为每个数据点计算max ,从而允许将发射的光转换成游离Ca 2+浓度值。 N = 6.


    图5.细胞内Ca 代表性痕迹 2 + 测量。 A。使用水母发光蛋白的突变同种型(Asp119Ala)在100μMATP刺激下摄取线粒体Ca 2+。使用野生型水母发光蛋白,ATP刺激后的胞质Ca 2+升高。通过加入1mM CaCl 2的Ca 2+,不含有KRB的Ca 2+,Ca 2+,Ca 2+,Ca 2+, 2 + 由激动剂刺激(ATP)引起的释放

笔记

  1. 虽然水母发光不受K + 和Mg 2 + (其是细胞内环境中最丰富的阳离子,因此也是最可能的生理实验干扰源)两种离子都是Ca 2+激活发光的竞争性抑制剂。 pH值也会影响低于7的水母发光发光。由于这些原因,水母发光蛋白的实验需要在良好控制的pH和离子浓度条件下进行,特别是Mg 2 +
  2. 为了刺激ER的释放,应为每种细胞类型(例如,例如,ATP,组胺,缓激肽,咖啡因,卡巴胆碱,谷氨酸)选择适当的激动剂。选择取决于G蛋白偶联的质膜受体的细胞型特异性表达

食谱

  1. Krebs-Ringer修饰缓冲液(KRB)
    135 mM NaCl
    5 mM KCl
    1mM MgCl 2·6H 2 O
    0.4mM KH 2 PO 4
    1mM MgSO 4·7H 2 O→// 20 mM HEPES
    用NaOH调节pH至7.4 储存于4°C
    加入1mM CaCl 2和5.5mM葡萄糖,使用前新鲜
  2. 降钙素溶解液
    100μM雌二醇素
    10mM CaCl 2
    在4°C储存最多4天

致谢

这项研究得到了欧盟的赠款(ERC mitoCalcium,第294777号给R.R.)的支持。意大利教育部,大学和研究部(PRIN至R.R.,FIRB至R.R.,Ricerca中的FIRB Futuro RBFR10EGVP_002至C.M.); NIH(授予R.R.授予1P01AG025532-01A1)和法国肌营养不良协会AFM-Téléthon(18857至C.M.)。

参考文献

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  2. Brini,M。(2008)。钙敏感光蛋白。 方法 46(3):160-166。
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引用:Tosatto, A., Rizzuto, R. and Mammucari, C. (2017). Ca2+ Measurements in Mammalian Cells with Aequorin-based Probes. Bio-protocol 7(5): e2155. DOI: 10.21769/BioProtoc.2155.
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