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Apr 2016

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Fluorophore-Based Mitochondrial Ca2+ Uptake Assay
基于荧光团的线粒体Ca2+摄取测定实验   

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Abstract

The physiological importance of mitochondrial calcium uptake, observed in processes such as ATP production, intracellular calcium signaling, and apoptosis, makes desirable a simple, straightforward way of investigating this event with unambiguous results. The following protocol uses a calcium-sensitive, membrane-impermeable fluorophore to monitor extra-mitochondrial calcium levels in the presence of permeabilized mammalian cells harboring activated mitochondria.

Keywords: Mitochondrial (线粒体), Calcium (钙), Flux (通量), MCU (MCU), Uniporter (单向转运体), HEK (HEK)

Background

Mitochondrial Calcium Uniporter (MCU)-mediated calcium flux is the primary way in which calcium enters the mitochondria. Mitochondrial calcium is important for several reasons, three of which are cited often in the literature. One, calcium in the mitochondria activates key dehydrogenases in the Krebs cycle which leads to increased ATP production. A more recent study indicates that mitochondrial calcium has a direct effect on both F1FO-ATPase and cytochrome chain activity (Glancy and Balaban, 2012), further enhancing its role as pivotal in cellular energy production. Two, because of the low, micromolar affinity of MCU and the large amount of cytosolic calcium mitochondria can sequester, MCU-mediated calcium uptake also plays a critical role in clearing transient increases in cytoplasmic calcium and in turn, shapes cellular signaling pathways that use calcium as a secondary messenger (Wheeler et al., 2012). Three, modulations of mitochondrial calcium play an important role in the regulation of apoptosis (Zoratti and Szabo, 1995). Steep increases in mitochondrial calcium levels initiate cell death by inducing the opening of the mitochondrial permeability transition pore in the inner membrane, an event that dissipates the inner mitochondrial membrane potential and releases cytochrome C, Diablo/Smac, and Caspase enzymes from the intermembrane space (Zoratti and Szabo, 1995; Pacher and Hajnoczky, 2001). While these phenomena have been well-characterized, the genetic identity of MCU has only recently been observed, and with that discovery has come the demand for a speedy and reliable way of observing mitochondrial calcium flux.

Our fluorophore-based mitochondrial calcium uptake assay is easy to set up and provides several advantages over other popular methods. HEK-293 cells with activated mitochondria are suspended in a recording buffer along with a membrane-impermeable calcium-sensitive fluorophore. The plasma membrane is permeated with a detergent while leaving the mitochondrial inner membrane in-tact, bringing the mitochondria in direct contact with the buffer. Following this, calcium is added to the cell-suspension and MCU-mediated calcium flux can be followed by observing the changing fluorescence of the fluorophore, which cannot follow calcium into the mitochondria. The highly specific MCU inhibitor, Ru360, is finally added to the cell-suspension to show that the observed change in fluorescence (i.e., calcium flux) is mediated by MCU. One of the most attractive features of the protocol is the speed of set-up and acquisition of flux data. Once cells are ready to harvest, calcium-flux data can be obtained in less than ten minutes. Another important quality of the protocol lies in its simplicity, specifically, in the straight-forward way in which the assay reports calcium flux and identifies MCU as the pathway. Implicating MCU as the sole calcium uptake pathway in this protocol is the observation that no calcium uptake of any kind is observed in cells lacking MCU. One drawback to the protocol is the inability of it to carefully quantify calcium flux, and for this, a calcium-45 uptake protocol is much preferable. In fact, limited quantification of mitochondrial calcium flux using this protocol is possible if one reports only the relative calcium flux, for example, by comparing two fluxes as a ratio of one over the other.

One of the opaquer yet more popular methods of observing MCU activity aims to follow the changes in mitochondrial calcium levels in intact cells whose mitochondria have been pre-loaded with a membrane-permeable calcium sensitive fluorophore. Because the plasma membranes of these cells are intact, intracellular calcium modulation depends on the release of calcium from the other major intracellular calcium sink, the endoplasmic reticulum (ER), which can be triggered by the addition of histamine to the extracellular buffer. Histamine achieves this by activating the phospholipase C/IP3 pathway, which results in the production of IP3 and concomitant activation of the IP3-receptor in the ER membrane, thereby releasing calcium stores from the ER into to cytoplasm. Because of the spatial proximity of the ER to the mitochondria, activation of the IP3 receptor transiently bathes mitochondria with a high dose of free calcium, which is in turn sequestered by the mitochondrial matrix. In this system, intra-mitochondrial calcium levels are monitored by observing changes in fluorescence of the pre-loaded, membrane-permeable, calcium sensitive probes which have presumably migrated to the mitochondrial matrix. Because MCU is the primary way through which calcium traverses the inner-mitochondrial membrane, it is taken for granted that the observed changes in fluorescence are due to the activation of MCU by these local increases in free calcium. It has even been suggested that the degree to which the fluorescence signal changes upon histamine stimulation is directly proportionate to the degree of MCU functionality. The complexity of this experimental design naturally raises doubts about what’s being inferred, namely, that the fluorescence changes upon histamine stimulation are a function of MCU functionality alone and not of, for example, the successful localization of the probe to mitochondria, or of the potential changes to any part of the phospholipase C/IP3 pathway, or of changes in proximity of the ER to mitochondria, or of the amount of calcium stored and/or released by the ER in various cell types under various experimental conditions, any of which may explain the observed fluoresce differences between the conditions tested and which may actually have little to do with MCU functionality. The experimental design described in detail below aims to reduce these sorts of ambiguities and to clearly report mitochondrial calcium flux mediated by MCU.

Materials and Reagents

  1. Pipette tips
  2. Cell culture dishes (Corning, catalog number: 430293 )
  3. Pasteur pipette
  4. 15 ml tube
  5. HEK 293 cells (Incubation: 37 °C, 5.0% CO2) (ATCC, catalog number: CRL-1573 )
  6. DMEM (high glucose, no glutamine) (store at 4 °C) (Thermo Fisher Scientific, GibcoTM, catalog number: 11960051 )
  7. Trypsin
  8. Premium fetal bovine serum (store at -20 °C) (Atlanta Biologicals, catalog number: S11150 )
  9. L-glutamine (store at -20 °C) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  10. HEPES (store at RT) (Sigma-Aldrich, catalog number: H3375-1KG )
  11. Potassium chloride (KCl) (store at RT) (Fisher Scientific, catalog number: P217-500 )
  12. Potassium phosphate dibasic (K2HPO4) (store at RT) (Fisher Scientific, catalog number: P290-500 )
  13. Magnesium chloride hexahydrate (MgCl2·6H2O) (store at RT) (Fisher Scientific, catalog number: M33-500 )
  14. Potassium hydroxide (KOH) (store at RT) (for pH to 7.4) (Fisher Scientific, catalog number: P250-500 )
  15. Succinate (store at RT) (Sigma-Aldrich, catalog number: S3674-100G )
  16. Calcium Green 5N - hexapotassium salt (CG-5N) (Thermo Fisher Scientific, InvitrogenTM, catalog number: C3737 )
    Note: Store at -20 °C as powder; 4 °C dissolved in ddH2O. Recommended: dissolve to a concentration of 0.5 μM for stock solution.
  17. Digitonin (Sigma-Aldrich, catalog number: D141 )
    Note: Store at -20 °C as powder. Dissolve in ddH2O immediately before use. Recommended: dissolve to a concentration of 30 mM for stock solution.
  18. Ruthedium 360 (Ru360) (Santa Cruz Biotechnology, catalog number: sc-222265 )
    Note: Store at -20 °C as powder; 4 °C dissolved in ddH2O.
  19. Calcium Chloride Dihydrate (Fisher Scientific, catalog number: C79-500 )
    Recommended: Dissolve in water to a concentration of 10 mM for stock solution.
  20. Growth media (see Recipes)
  21. Wash buffer (WB) (see Recipes)
  22. Recording buffer (RB) (see Recipes)

Equipment

  1. Pipettes
  2. Incubator (Fisher Scientific, IsotempTM, catalog number: 13-255-26 )
  3. Centrifuge (Thermo Fisher Scientific, model: HeraeusTM LabofugeTM 400 , catalog number: 75008164)
  4. Quartz cuvette (Fisher Scientific, Fisherbrand, catalog number: 14-958-128 )
  5. Small stir bar (Hach, catalog number: 2095349 )
  6. Thermo Cell Holder with Stirrer (Hitatchi, model: 251-0148 )
  7. F-2500 Fluorescence spectrophotometer (Hitachi, model: 251-0090 )

Software

  1. IGOR Pro
  2. Microsoft Excel

Procedure

  1. Cell (HEK-293) preparation
    1. Carefully remove growth media from a confluent 10-cm dish (i.e., with a Pasteur pipette. See Note 3 for more on cell density). Add 10 ml WB (Wash Buffer) warmed to 37 °C to the plate with 10 ml pipet, pipetting up and down to gently lift cells from the bottom of the plate, and transfer WB-cell slurry to a 15 ml tube. These preparations are to be carried out on the bench at room temperature. If cells are strongly adherent and not easily dislodged by gentle up-and-down pipetting, add 0.7 ml of Trypsin (or just enough to evenly cover cells) warmed to 37 °C after washing cells once with 10 ml WB and let stand in a 37 °C incubator for 2-3 min. Remove cells from the incubator. Cells should now be easily displaced by gentle up-and-down pipetting with 10 ml WB. Transfer WB-cell slurry to a 15 ml tube.
    2. Gently pellet cells by centrifugation at room temperature at 1,000 x g for 5 min. Discard supernatant.
    3. Wash pellet by re-suspending cells in 10 ml WB, spin for 5 min at 1,000 x g, and discard supernatant.
    4. Resuspend cells in 2.5 ml recording buffer (RB), also warmed to 37 °C. Transfer 2.0 ml of this slurry to the quartz cuvette with a small stir bar. (The remaining 0.5 ml can be used separately to analyze protein content via Western Blot analysis. See Note 5 for more on Western Blots)
      Note: Gently flick cuvette to remove bubbles before the start of the experiment, as these can create unwanted noise in the data.

  2. Flux experiment
    1. Place cuvette with RB-cell slurry into the cell-holder/stirrer affixed to the spectrophotometer. Turn on a stirrer and stir cells slowly (see Note 1).
    2. Set spectrophotometer to these parameters:
      1. Excitation wavelength: 506 nm
      2. Emission wavelength: 531 nm
      3. Em/Ex slit width: 2.5/2.5 nm
      4. Time scan
        1. 600 sec (or longer)
        2. 0 sec delay
      5. PMT Voltage: 700 Volts
      6. Response: 0.04 sec
      7. Data Mode: Fluorescence
    3. Start recording time scan. See Figure 1 below.


      Figure 1. Starting the Recording. Left: cartoon of quartz cuvette with 2 ml recording buffer suspending wild-type (WT) HEK cells, loaded into the stirrer on the fluorescence spectrophotometer (small stir bar not shown). Right: spectrophotometric trace about 45 sec after starting the recording.

    4. Add CG-5N to a final concentration of 0.25 nM (i.e., 1 μl of 0.5 μM stock). CG-5N is a membrane impermeable calcium-sensitive fluorophore (Kd = 14 μM). The fluorescence signal will go up with the addition of the fluorophore as trace amounts of calcium are present in the RB. See Figure 2 below.


      Figure 2. Adding CG-5N. Adding CG-5N to a concentration of 0.25 nM (left) will increase the fluorescence signal in the trace profile (right).

    5. Add digitonin to a final concentration of 30 μM (i.e., 2 μl of 30 mM stock). Digitonin permeabilizes the plasma membrane by extracting cholesterol, leaving those intracellular membranes lacking cholesterol (e.g., the mitochondrial inner membrane) intact. This brings mitochondria in direct contact with the buffer solution and susceptible to influence by experimental reagents (the outer mitochondrial membrane is already in a steady-state with the cytoplasm with regards to small molecules like the reagents used in this protocol). CG-5N (membrane-impermeable) is afterward reporting ‘extra-mitochondrial’ calcium levels. A typical dip in fluorescence is almost always observed upon the addition of digitonin, as can be seen in Figure 3 below.


      Figure 3. Adding digitonin. Adding digitonin to a concentration of 30 μM (left) will cause the spectrophotometric trace profile to drop (right).

    6. Add CaCl2 to a final concentration of 10 μM (i.e., 2 μl of 10mM stock). The fluorescence signal will go up as calcium binds to CG-5N. If the cells are harboring activated mitochondria containing functional MCU (i.e., WT-HEK cells), a precipitous declination in fluorescence will immediately follow, signifying MCU-mediated calcium uptake (see Figure 4).
      Note: If cells lack MCU, the fluorescence signal in the trace profile will increase but then will immediately flatten out, showing that the observed declination in the trace profile of WT cells after the addition of calcium is due to MCU-mediated calcium uptake AND NOT by other intra-cellular calcium uptake pathways such as SERCA (Sarco/Endoplasmic Calcium-ATPase) (Note 6).


      Figure 4. Adding calcium. Adding calcium to a final concentration of 10 μM (left) will produce a sharp increase in the trace profile (right). The following drop in fluorescence signal is indicative of MCU-mediated calcium flux.

    7. After some time has passed, typically between 30 sec to 1 min, add Ru360 to a final concentration of 0.5 μM. Ru360 (i.e., 0.5 μl of 2 mM stock) is a potent inhibitor of MCU (Kd = 340 pM) and will cause the spectrophotometric trace to flatten as the steady-state exchange between the CG-5N-calcium-bound and CG-5N-calcium unbound species returns due to the lack of calcium mobility away from CG-5N. This step testifies to the dependence of MCU on the removal of calcium from the buffer solution observed in the previous step. See Figure 5 below; here, Ru360 is added to the cuvette after a second shot of 10 μM calcium. 


      Figure 5. Ru360. Adding Ru360 to a final concentration of 0.5 μM (left) will cause the spectrophotometric trace profile to flatten (right).

Data analysis

  1. For reference data, please refer to Figure 4–figure supplement 2C, and Figure 4–figure supplement 3A in our paper ‘Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex’ by following the link provided below:
    http://cdn.elifesciences.org/elife-articles/15545/figures-pdf/elife15545-figures.pdf?_ga=1.67863441.1487892113.1470405893
  2. Raw data from the spectrophotometric traces were uploaded into IGOR Pro and the slope of the a.u./time trace, starting immediately following the addition of calcium and for the following 10 sec, was calculated from each graph. The average of 3 slopes from cells expressing WT-hMCU and WT-hEMRE was calculated in Microsoft Excel along with the concomitant standard error. These values were used to normalize all following experimental data sets against.

Notes

  1. Stir bar speed
    The speed of the stir bar can be an important but overlooked factor in determining the quality of the fluorescence trace profile. Spinning too fast can result in cell damage (cells will clump together in the cuvette), while spinning too slowly may limit the speed of reagent mixing within the cuvette, which can be seen in the spectrophotometric data primarily as enhanced noise and hyperbolic-like transitions from one steady-state to another upon the addition of a new reagent to the cuvette. In general, we’ve found that starting the stir bar in its slowest setting first and then slowly ramping up speed is the best way to find the optimal speed leading to intact cells and quick rates of reagent mixing. In our hands, this ideal speed is, qualitatively, on the slow end within the range of possible stir-bar speeds.
  2. Digitonin
    We have found that digitonin from Sigma-Aldrich works particularly well for this experiment. Also, make digitonin stock fresh before each experiment, as this reagent tends to crash out of solution between experiments (within an hour).
  3. Cell density
    While the number of HEK cells used in an experiment is completely up to the experimenter, we have found the ideal number to be 2.0 x 107, or 1 fully confluent 10 cm dish.
  4. Cell types
    We have performed this experiment exclusively with HEK 293 cells, and cannot say how it might work using other cell types.
  5. Western Blot Analysis
    Recording Buffer (RB) does not interfere Western Blot analysis. Simply spin the remaining 0.5 ml of cell slurry down, discard sup (RB), and lyse cells with ice cold lysis buffer (we found RIPA buffer works well for this). Lysing cells with about 50 μl RIPA buffer yields protein concentrations in a range appropriate for the comfortable loading of between 10 μg and 50 μg of protein in a 15 μl or 50 μl per well gel. A high-speed spin step at Step A4 is also required after lysis to pellet cell derbies and to keep the lysate from becoming ‘goopy’ and unmanageable during loading; keep the supernatant. Follow instructions for your lysis buffer of choice (add protease inhibitors, work on ice, etc.) and carefully quantify protein concentration after the high-speed spin with your method of choice. We always run a loading control gel on which we detect Actin (we load 10 μg protein for this blot). Other gels should be run on which to look for the protein of interest (typically MCU or one of its regulatory partners, but this obviously depends on the experiment and what’s desirable to detect). In general, expect to spend time optimizing western blots to see high-quality (high signal to noise) data.
  6. A clear difference between the trace profiles of wild-type cells and MCU-knock out cells (cells in which MCU has been deleted from the genome) is that in the latter, the slope, after the addition of calcium, is nearly zero, while in the former, the slope is clearly negative. The profile’s negative slope after calcium addition is therefore indicative of MCU-mediated calcium uptake.


    Figure 6. Spectrophotometric Profile of MCU-knockout Cells. Trace profile is flat after the addition of Ca2+ when MCU has been deleted from the genome.

Recipes

  1. Growth media
    90% (v/v) DMEM
    4.5 g/L D-glucose, L-glutamate, sodium pyruvate
    10% (v/v) premium fetal bovine serum
    2 mM L-glutamine
    Store at 4 °C
    Notes:
    1. Prepare media under sterile conditions, i.e., in a laminar flow hood.
    2. Filter media through a 0.22 μm filter after preparation.
  2. Wash buffer (WB)
    20 mM HEPES
    125 mM KCl
    2 mM K2HPO4
    1 mM MgCl2
    Adjust pH to 7.4 with KOH
    Store at 4 °C
  3. Recording buffer (RB)
    WB (see above)
    5 mM succinate
    Store at 4 °C

Acknowledgments

This work was carried out in Dr. Christopher Miller’s lab (HHMI; Brandeis University) and was supervised by Dr. Ming-Feng Tsai.
The above protocol is a classic protocol which has been used for decades in the field of mitochondrial calcium handling. Our thanks to those original thinkers who gave it life and to those who have refined it over the years. The author declares that there are no conflicts of interest or competing interests.

References

  1. Glancy, B. and Balaban, R. S. (2012). Role of mitochondrial Ca2+ in the regulation of cellular energetics. Biochemistry 51(14): 2959-2973.
  2. Pacher, P. and Hajnoczky, G. (2001). Propagation of the apoptotic signal by mitochondrial waves. EMBO J 20(15): 4107-4121.
  3. Wheeler, D., Groth, R., Ma, H., Barrett, C., Owen, S., Safa, P., and Tsien, R. (2012). CaV1 and CaV2 channels engage distinct modes of Ca2+ signaling to control CREB-dependent gene expression. Cell 149(5): 1112-1124.
  4. Zoratti, M. and Szabo, I. (1995). The mitochondrial permeability transition. Biochim Biophys Acta 1241(2): 139-176.

简介

在诸如ATP产生,细胞内钙信号传导和细胞凋亡的过程中观察到的线粒体钙摄取的生理学重要性使得以明确的结果研究该事件的简单,直接的方式成为可取的。 以下方案使用钙敏感的膜不可渗透的荧光团来监测在具有活化的线粒体的透化的哺乳动物细胞存在下的线粒体外钙水平。

【背景】线粒体钙单向转运蛋白(MCU)介导的钙通量是钙进入线粒体的主要方式。线粒体钙由于几个原因是重要的,其中三个原因经常在文献中引用。其一,线粒体中的钙激活Krebs循环中的关键脱氢酶,导致ATP产生增加。最近的一项研究表明,线粒体钙对F1FO-ATP酶和细胞色素链活性有直接影响(Glancy和Balaban,2012),进一步增强其在细胞能量产生中的关键作用。二,由于MCU的低微摩尔亲和力和大量细胞溶质钙线粒体可以隔离,MCU介导的钙摄取也在清除细胞质钙的瞬时增加中起关键作用,反过来,形成使用钙的细胞信号通路作为第二信使(Wheeler et al。,2012)。三,线粒体钙的调节在细胞凋亡的调节中起重要作用(Zoratti和Szabo,1995)。线粒体钙水平的急剧增加通过诱导内膜中线粒体通透性转换孔的开放而引发细胞死亡,这一事件消除了线粒体内部膜电位并从膜间隙释放细胞色素C,暗黑/ Smac和半胱氨酸蛋白酶( Zoratti和Szabo,1995; Pacher和Hajnoczky,2001)。虽然已经对这些现象进行了充分表征,但最近才观察到MCU的遗传特性,并且随着这一发现的出现,需要一种快速可靠的观察线粒体钙通量的方法。

我们的基于荧光团的线粒体钙摄取测定易于设置,并提供了优于其他常用方法的几个优点。将具有活化的线粒体的HEK-293细胞与膜不可渗透的钙敏感性荧光团一起悬浮在记录缓冲液中。质膜渗透有去污剂,同时保留线粒体内膜,使线粒体与缓冲液直接接触。在此之后,将钙添加到细胞悬浮液中,并且可以通过观察荧光团的荧光变化来观察荧光团的荧光变化,荧光团不能跟随钙进入线粒体。最终将高度特异性的MCU抑制剂Ru360添加到细胞悬液中,以显示观察到的荧光变化(即,钙通量)是由MCU介导的。该协议最吸引人的特征之一是设置和获取通量数据的速度。一旦细胞准备好收获,就可以在不到十分钟的时间内获得钙通量数据。该方案的另一个重要质量在于其简单性,特别是在测定报告钙通量并将MCU识别为途径的直接方式中。在该方案中暗示MCU作为唯一的钙摄取途径是观察到在缺乏MCU的细胞中没有观察到任何类型的钙摄取。该方案的一个缺点是它不能仔细量化钙通量,为此,更优选钙-45摄取方案。事实上,如果仅报告相对钙通量,则可以使用该方案对线粒体钙通量进行有限的定量,例如,通过比较两个通量作为一个相对于另一个的比率。

观察MCU活动的一种较常见但更受欢迎的方法旨在跟踪完整细胞中线粒体钙水平的变化,所述细胞的线粒体已经预装有膜可渗透的钙敏感荧光团。因为这些细胞的质膜是完整的,细胞内钙调节取决于钙从其他主要细胞内钙汇,即内质网(ER)的释放,其可以通过向细胞外缓冲液中添加组胺来触发。组胺通过激活磷脂酶C / IP3途径实现这一目的,其导致IP3的产生和ER膜中IP3-受体的伴随激活,从而将ER储存从ER释放到细胞质中。由于ER与线粒体的空间接近,IP3受体的活化瞬间用高剂量的游离钙洗涤线粒体,而游离钙又被线粒体基质隔离。在该系统中,通过观察可能迁移至线粒体基质的预加载的,膜可渗透的钙敏感探针的荧光变化来监测线粒体内钙水平。因为MCU是钙穿过线粒体内膜的主要途径,所以理所当然地认为观察到的荧光变化是由于局部游离钙的增加而激活了MCU。甚至有人提出,荧光信号在组胺刺激时的变化程度与MCU功能的程度成正比。这种实验设计的复杂性自然引起了人们对推断内容的怀疑,即组胺刺激后的荧光变化仅仅是MCU功能的一个功能,而不是例如探针成功定位到线粒体或潜在的潜力。磷脂酶C / IP3途径的任何部分的变化,或ER与线粒体接近的变化,或在各种实验条件下ER在各种细胞类型中储存和/或释放的钙量的变化,其中任何一种都可能解释所测试条件之间观察到的荧光差异,这实际上可能与MCU功能无关。下面详细描述的实验设计旨在减少这些模糊性并清楚地报告由MCU介导的线粒体钙通量。

关键字:线粒体, 钙, 通量, MCU, 单向转运体, HEK

材料和试剂

  1. 移液器吸头
  2. 细胞培养皿(康宁,目录号:430293)
  3. 巴斯德吸管
  4. 15毫升管
  5. HEK 293细胞(孵育:37°C,5.0%CO 2 )(ATCC,目录号:CRL-1573)
  6. DMEM(高葡萄糖,无谷氨酰胺)(储存于4°C)(Thermo Fisher Scientific,Gibco TM ,目录号:11960051)
  7. 胰蛋白酶
  8. 优质胎牛血清(-20°C储存)(Atlanta Biologicals,目录号:S11150)
  9. L-谷氨酰胺(储存在-20°C)(Thermo Fisher Scientific,Gibco TM ,目录号:25030081)
  10. HEPES(在RT存储)(Sigma-Aldrich,目录号:H3375-1KG)
  11. 氯化钾(KCl)(在室温下储存)(Fisher Scientific,目录号:P217-500)
  12. 磷酸氢二钾(K 2 HPO 4 )(在室内储存)(Fisher Scientific,目录号:P290-500)
  13. 氯化镁六水合物(MgCl 2 ·6H 2 O)(在室温下储存)(Fisher Scientific,目录号:M33-500)
  14. 氢氧化钾(KOH)(在室温下储存)(pH值为7.4)(Fisher Scientific,目录号:P250-500)
  15. 琥珀酸盐(在RT存储)(Sigma-Aldrich,目录号:S3674-100G)
  16. 钙绿5N - 六钾盐(CG-5N)(Thermo Fisher Scientific,Invitrogen TM ,目录号:C3737)
    注意:在-20°C下保存为粉末; 4℃溶于ddH 2 O.推荐:溶解至浓度为0.5μM的原液。
  17. Digitonin(Sigma-Aldrich,目录号:D141)
    注意:在-20°C下保存为粉末。使用前立即溶于ddH 2 O.推荐:溶解至浓度为30 mM的储备溶液。
  18. Ruthedium 360(Ru360)(Santa Cruz Biotechnology,目录号:sc-222265)
    注意:在-20°C下保存为粉末; 4℃溶于ddH 2 O。
  19. 二水合氯化钙(Fisher Scientific,目录号:C79-500)
    推荐:在水中溶解至浓度为10 mM的原液。
  20. 增长媒体(见食谱)
  21. 洗涤缓冲液(WB)(见食谱)
  22. 录音缓冲区(RB)(见食谱)

设备

  1. 移液器
  2. 孵化器(Fisher Scientific,Isotemp TM ,目录号:13-255-26)
  3. 离心机(Thermo Fisher Scientific,型号:Heraeus TM Labofuge TM 400,目录号:75008164)
  4. 石英比色皿(Fisher Scientific,Fisherbrand,目录号:14-958-128)
  5. 小搅拌棒(哈希,目录号:2095349)
  6. 带搅拌器的热电池座(Hitatchi,型号:251-0148)
  7. F-2500荧光分光光度计(日立,型号:251-0090)

软件

  1. IGOR Pro
  2. Microsoft Excel

程序

  1. 细胞(HEK-293)制备
    1. 小心地从融合的10厘米培养皿中取出生长培养基(即,用巴斯德吸管。有关细胞密度的更多信息,请参见注释3)。将10ml WB(洗涤缓冲液)加热至37℃,用10ml移液管吸移,上下移液以从板底部轻轻提起细胞,并将WB细胞浆液转移至15ml管中。这些制剂应在室温下在工作台上进行。如果细胞粘附强烈且不易通过温和的上下移液移出,则在用10 ml WB洗涤细胞一次后,加入0.7 ml胰蛋白酶(或刚好足以均匀覆盖细胞)温热至37°C,然后静置37°C培养箱2-3分钟。从培养箱中取出细胞。现在,通过使用10 ml WB轻柔上下移液,可轻松取代细胞。将WB细胞浆液转移到15 ml管中。
    2. 通过在室温下以1,000 x g 离心5分钟轻轻沉淀细胞。丢弃上清液。
    3. 通过将细胞重悬于10ml WB中洗涤沉淀,在1,000 x g 下旋转5分钟,并弃去上清液。
    4. 将细胞重悬于2.5ml记录缓冲液(RB)中,也温热至37℃。用小搅拌棒将2.0ml该浆液转移到石英比色皿中。 (剩余的0.5 ml可单独用于通过蛋白质印迹分析分析蛋白质含量。有关Western Blots的更多信息,请参见注释5)
      注意:在实验开始前轻轻地轻推比色杯以去除气泡,因为这会在数据中产生不必要的噪音。
  2. 助焊剂实验
    1. 将具有RB细胞浆液的比色皿放入固定在分光光度计上的细胞固定器/搅拌器中。打开搅拌器,慢慢搅拌细胞(见注1)。
    2. 将分光光度计设置为以下参数:
      1. 激发波长:506 nm
      2. 发射波长:531 nm
      3. Em / Ex狭缝宽度:2.5 / 2.5 nm
      4. 时间扫描
        1. 600秒(或更长)
        2. 0秒延迟
      5. PMT电压:700伏
      6. 回复:0.04秒
      7. 数据模式:荧光
    3. 开始录制时间扫描。见下面的图1.


      图1.开始记录。左:用2ml记录缓冲液悬浮野生型(WT)HEK细胞的石英比色皿卡通,在荧光分光光度计上加载到搅拌器中(小搅拌棒未显示) 。右:开始录制后约45秒的分光光度跟踪。

    4. 加入CG-5N至终浓度为0.25nM(,即,1μl的0.5μM原液)。 CG-5N是膜不可渗透的钙敏感荧光团(Kd =14μM)。随着荧光团的添加,荧光信号将上升,因为RB中存在痕量的钙。见下面的图2。


      图2.添加CG-5N。将浓度为0.25 nM的CG-5N(左)添加会增加痕量曲线中的荧光信号(右图)。

    5. 加入洋地黄皂苷至终浓度为30μM(即,2μl30mM原液)。 Digitonin通过提取胆固醇使质膜透化,使那些缺乏胆固醇的细胞内膜(例如,线粒体内膜)保持完整。这使得线粒体与缓冲溶液直接接触并且易受实验试剂的影响(外部线粒体膜已经与细胞质处于稳定状态,关于小分子,如本方案中使用的试剂)。 CG-5N(膜不可渗透)随后报告“线粒体外”钙水平。在加入洋地黄皂苷后,几乎总能观察到典型的荧光下降,如下图3所示。


      图3.添加洋地黄皂苷。将浓度为30μM(左)的洋地黄皂苷添加将导致分光光度曲线分布下降(右)。

    6. 加入CaCl 2 至终浓度10μM(,即,2μl10mM原液)。当钙与CG-5N结合时,荧光信号将上升。如果细胞含有活化的线粒体含有功能性MCU(即,WT-HEK细胞),则会立即出现荧光的急剧下降,表明MCU介导的钙摄取(参见图4)。
      注意:如果细胞缺乏MCU,则痕量分布中的荧光信号会增加但随后会立即变平,表明加入钙后WT细胞的痕量分布中观察到的下降是由于MCU介导的钙摄取而不是其他细胞内钙摄取途径,如SERCA(Sarco / Endoplasmic Calcium-ATPase)(注6)。


      图4.添加钙。添加最终浓度为10μM的钙(左图)会使痕量曲线急剧增加(右图)。荧光信号的下降表明MCU介导的钙通量。

    7. 经过一段时间后,通常在30秒至1分钟之间,加入Ru360至终浓度为0.5μM。 Ru360(即,0.5μl的2 mM原液)是MCU的有效抑制剂(Kd = 340 pM),并且随着CG-5N-之间的稳态交换,将导致分光光度迹线变平。由于缺乏远离CG-5N的钙迁移,钙结合和CG-5N-钙未结合物质返回。该步骤证明了MCU对从前一步骤中观察到的缓冲溶液中钙的去除的依赖性。见下面的图5;在这里,在第二次注射10μM钙后,将Ru360添加到比色杯中。 


      图5. Ru360。将Ru360添加到最终浓度为0.5μM(左)将导致分光光度曲线分布变平(右)。

数据分析

  1. 有关参考数据,请参阅下图提供的图4-图补充2C和图4-图补充文件3A在我们的论文中“线粒体钙单向转运复合体中小调节亚基的双重功能”,如下所示:
    http://cdn.elifesciences .org / elife-articles / 15545 / figures-pdf / elife15545-figures.pdf?_ga = 1.67863441.1487892113.1470405893
  2. 将来自分光光度迹线的原始数据上载到IGOR Pro中,并且从每个图表计算在加入钙之后立即开始的a.u./time迹线的斜率和随后的10秒。在Microsoft Excel中计算表达WT-hMCU和WT-hEMRE的细胞的3个斜率的平均值以及伴随的标准误差。这些值用于标准化所有后续实验数据集。

笔记

  1. 搅拌棒速度
    在确定荧光迹线轮廓的质量时,搅拌棒的速度可能是一个重要但却被忽视的因素。旋转太快会导致细胞损伤(细胞会在比色皿中聚集在一起),而旋转太慢可能会限制试剂在比色杯内混合的速度,这可以在分光光度数据中看到,主要是增强的噪声和类双曲线过渡在向比色皿中添加新试剂时,从一种稳态到另一种稳态。一般来说,我们发现首先以最慢的速度启动搅拌棒然后慢慢加快速度是找到导致完整细胞的最佳速度和快速混合试剂的最佳方法。在我们的手中,这种理想的速度定性地在可能的搅拌棒速度范围内的慢速端。
  2. Digitonin
    我们发现来自Sigma-Aldrich的洋地黄皂苷在该实验中特别有效。此外,在每次实验之前使新鲜的洋地黄皂苷库存新鲜,因为这种试剂在实验之间(一小时内)趋向于从溶液中崩溃。
  3. 细胞密度
    虽然实验中使用的HEK细胞数量完全取决于实验者,但我们发现理想数量为2.0 x 10 7 ,或1个完全融合的10 cm培养皿。
  4. 细胞类型
    我们专门用HEK 293细胞进行了这个实验,并且不能说它如何使用其他细胞类型。
  5. 蛋白质印迹分析
    记录缓冲液(RB)不会干扰蛋白质印迹分析。简单地旋转剩余的0.5ml细胞浆液,丢弃sup(RB),并用冰冷的裂解缓冲液裂解细胞(我们发现RIPA缓冲液对此有效)。用约50μlRIPA缓冲液裂解细胞产生的蛋白质浓度适合于在15μl或50μl/孔凝胶中舒适加载10μg至50μg蛋白质。在裂解以沉淀细胞德比并且保持裂解物在加载期间变得“粘性”并且难以控制时,还需要步骤A4的高速旋转步骤;保持上清液。按照您选择的裂解缓冲液的说明(添加蛋白酶抑制剂,在冰上工作,等),并使用您选择的方法仔细量化高速旋转后的蛋白质浓度。我们总是运行一个加载对照凝胶,我们在其上检测肌动蛋白(我们为此印迹加载10μg蛋白质)。应该运行其他凝胶来寻找感兴趣的蛋白质(通常是MCU或其监管伙伴之一,但这显然取决于实验以及需要检测的内容)。一般而言,期望花时间优化Western印迹以查看高质量(高信噪比)数据。
  6. 野生型细胞和MCU敲除细胞(MCU已从基因组中删除的细胞)的痕迹谱之间的明显差异在于,在后者中,加入钙后的斜率几乎为零,而在前者中,斜率显然是负的。因此,加入钙后曲线的负斜率表明了MCU介导的钙摄取。


    图6. MCU敲除细胞的分光光度分布。当从基因组中删除MCU时,添加Ca 2 + 后,痕量分布是平坦的。

食谱

  1. 成长媒体
    90%(v / v)DMEM
    4.5g / L D-葡萄糖,L-谷氨酸,丙酮酸钠
    10%(v / v)优质胎牛血清
    2 mM L-谷氨酰胺
    储存在4°C
    注意:
    1. 在无菌条件下,即在层流罩中准备培养基。
    2. 制备后通过0.22μm过滤器过滤介质。
  2. 洗涤缓冲液(WB)
    20 mM HEPES
    125 mM KCl
    2 mM K 2 HPO 4
    1 mM MgCl 2
    用KOH将pH调节至7.4 储存在4°C
  3. 录音缓冲区(RB)
    WB(见上文)
    5 mM琥珀酸盐
    储存在4°C

致谢

这项工作是在Christopher Miller博士的实验室(HHMI; Brandeis大学)进行的,并由蔡明峰博士监督。
上述方案是一种经典方案,已在线粒体钙处理领域中使用了数十年。我们感谢那些赋予它生命的原始思想家以及多年来对其进行改进的人。作者声明没有利益冲突或竞争利益。

参考

  1. Glancy,B。和Balaban,R。S.(2012)。 线粒体Ca 2 + 在调节细胞能量中的作用。 生物化学 51(14):2959-2973。
  2. Pacher,P。和Hajnoczky,G。(2001)。 通过线粒体波传播凋亡信号。 EMBO J 20(15):4107-4121。
  3. Wheeler,D.,Groth,R.,Ma,H.,Barrett,C.,Owen,S.,Safa,P。和Tsien,R。(2012)。 Ca V 1和Ca V < / sub> 2个通道参与不同的Ca 2 + 信号模式以控制CREB依赖性基因表达。 Cell ,149(5),1112-1124。
  4. Zoratti,M。和Szabo,I。(1995)。 线粒体通透性转换。 Biochim Biophys Acta 1241( 2):139-176。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Phillips. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Phillips, C. (2018). Fluorophore-Based Mitochondrial Ca2+ Uptake Assay. Bio-protocol 8(14): e2934. DOI: 10.21769/BioProtoc.2934.
  2. Tsai, M. F., Phillips, C. B., Ranaghan, M., Tsai, C. W., Wu, Y., Willliams, C. and Miller, C. (2016). Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex. Elife 5: e15545.
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