Name: direct imaging of er calcium with targeted-esterase induced dye loading (ted)
Uploaded: 2013-05-07T13:00:00
Description: Watch this Scientific Journal Video about Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED at JoVE.com

这项工作已经得到了德意志研究联合会（DFG）BL567/3-1和弗里德里希·鲍尔基金会的资助。我们要感谢钱永健，在圣地亚哥加州大学的霍华德·休斯医学研究所的实验室为我们提供了标签RFP-T2。值得庆幸的是，我们承认，帕萨迪纳加州理工学院的戴维·巴尔的摩，和为我们提供的慢病毒质粒FUGW，psPAX2/pMD2.G，日内瓦，日内瓦大学Didier TRONO的。

该协议介绍TED应用细胞株，海马神经元及大脑皮质神经胶质细胞。 TED性能最好的时候，TED向量稳定表达，例如慢病毒载体。 TED的方法在图4中所示的示意性概观。 1。溶液的制备开始之前，应准备以下的解决方案，并可以被存储为指示。我们通常使用的玻璃和塑料材料和无菌水消毒。 <ol><li>准备HEPES林格（毫米）：125氯化钠，氯化钾，25 HEPES...

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TED的方法的优点和缺点已进行了广泛的讨论，在最近的出版物34,35。 TED情况下，与上述的其他方法相比，细胞破坏和灌注的问题。此外，需要强调两个方面。 TED ER钙成像的原则要求（1）目标TED的载体构建的帮助下，内质网腔，和（2）的高效率和低亲和力的合成AM酯优先释放活性羧酸酯酶表达内质网腔的钙染料。 1。 TED载体构建 图3总?...

为了解决ER钙的生理反应，我们开发了一个新的策略，以提高合成钙敏感染料进入ER诱捕。该方法能够直接的，非破坏性的实时监控自由ER钙在细胞外钙离子的存在。 ER钙的功能和信令钙信号被发现的不同类型的细胞， 如肌肉细胞，神经元和神经胶质细胞，其功能范围从调解肌肉收缩，参与突触传递的学习和记忆1,2。游离钙离子浓度的变化有很高的科学价值，因为钙参与调节基因转录，细胞增殖，神经细胞的兴奋性，细胞死亡和其他细胞信号事件1-7。所有这些细胞钙信号连接功能，并有助于细胞内钙存储动态8-10。 所有的钙信号中的一个共同特点是钙外空间之间的流动，细胞质和细胞器，主要是ER和线粒体。这会导致这些细胞器内的钙离子浓度的动态变化，由不同的信令组件感测。一般情况下，内质网中的钙浓度的范围在100至800微米之间，在细胞质中的钙离子浓度接近100纳米，和在细胞外空间中的浓度大约是1-2毫米。因此，存在很高的化学驱动力细胞质中的钙离子流向2,9,10。 最常研究的ER-源性钙信号依赖于G-蛋白偶联受体（GPCR），然后激活磷脂酶C（PLC）的刺激。反过来，PLC在生产肌醇1,4,5 -三磷酸肌醇（IP 3）1。 IP 3结合后其REC在ER膜的eptor（IP 3， 图1（记录）），钙离子从内质网腔中释放。在历史上，第一IP-3介导从内质网释放钙-间接即使-测量胰腺腺泡细胞由Streb 等的。于1983年11。此出版物建议在第一次涉及乙酰胆碱，磷脂酶C和IP 3的信号级联。这种方式的钙离子的释放一般被称为IP 3引起的钙离子的释放（IiCR）（ 图1）。依赖激酶激活磷脂酶Cγ受体酪氨酸激酶生长因子和神经营养因子的作用ER钙信号后IP海拔3 12。钙海拔在除了IiCR，可介导的离子型钙条目，例如兰尼再通过电压门控钙通道（CA V），以及随后的钙诱导钙释放（CICR）与其受体（RyR的）。 IiCR和CICR生理链接库操纵的钙进入（SOCE）。 SOCE包括STIM（基质相互作用的分子），这是一个传感器，用于ER钙离子释放的动作。激励信号已被证明，通过瞬时受体电位通道色氨酸（Trp），13，14 Orai钙离子通道和电压门控钙离子通道15（ 图1）刺激细胞外钙离子条目。 ER钙丢失是动态救出由的肉瘤内质网钙ATP酶（SERCA），积极的泵钙背到急诊室的作用。阻断药物，如毒胡萝卜素SERCA ER钙胞质室推出了一个连续亏损。该ER钙“泄漏”Sec61蛋白复合物16,17（ 图1）配合物，如ER膜内孔隙所造成的。 贝里奇1998年，出版了一本模型中，“神经元内的神经元模型”，这ħ建议的原则生理作用的ER结合神经元钙5。该模型认为，存在一个连续的的ER膜系统形成一个细胞内的“形象”的神经细胞质膜5。这个二进制真核生物膜系统是自称是快与慢的神经元钙信号的时间与空间整合的基本前提。钙信号发生随之而来，随后在不同的相同的神经元树突棘或赋予细胞的胞体或细胞核通过急诊室，在那里他们总结了5,18。然后，其总和可能会影响神经元的兴奋性，调节基因转录或集成的信号级联。因此，ER支持钙信号的整合。这一概念的一个先决条件是所声称的一些研究，并已被证明至少为躯体-D在单个细胞中的雌激素受体的连续性endritic地区和短距离轴突预测19-21。是否有内长轴突预测ER连续性是一个有争议的问题。 来测量其流量超过ER膜游离钙的战略钙信号的最频繁监测在细胞质中22,23。因此，它可以很容易地区别是否的Ca 2 +流进入细胞质从细胞外或细胞内储存6,24。为了克服这个限制，方法策略直接ER钙成像已经制定出来。总之，使用下列策略：（1）ER-针对性的基因工程蛋白质指标25-27。基于蛋白质的低亲和力的Ca 2 +的指标，使用的生物发光蛋白水母发光蛋白结合钙传感蛋白或GFP。这些基因工程的Ca 2 +的指标（GECIS）定位到内质网的信号肽的帮助下，积极地保持在ER中使用的保留和检索动机。常见的ER的Ca 2 +指标上卡梅里安原则基础卡默莱昂YC4.3 26,28; 29分裂YC7.3ER卡梅里安，卡梅里安D1 30。 （2） 直接酯酶基染料装载AM酯低亲和力的Ca 2 + 指标 31,32 AM衍生工具的指示剂染料（MAG-Fura2-AM，MAG-Fluo4-AM-AM Fluo5N）通过生物膜的亲脂性，钙不敏感的状态。然后，在细胞质中，以及在ER中，内源性酯酶对AM-酯基裂开，释放的Ca 2 +指示剂，留下一定量的活性染料在细胞质中，在ER。因此，这种方法在ER中的钙离子浓度高的条件下是有用的，只要细胞内游离钙离子的浓度保持低于去低亲和性的指标的TECTION限制，特别是在特征钙信号（ 例如，纳米低μM）。 （3）AM酯组合质膜通透性32加载，任何剩余的胞浆Ca 2 +指示剂除去质膜通透性与少量的“温和”的清洁剂（ 例如皂甙）在人工细胞内的缓冲。因此，细胞内的膜的可能的刺激， 例如 ，与在细胞内的缓冲液中的IP 3，可直接通过质膜中的“孔”。 （4） 胞液中透析，根据全细胞配置和同时测量的Ca 2 +在ER腔体和的胞质溶胶32,33。细胞被第一次装载有低亲和力的Ca 2 +的指示器（ 例如，幅度- Fura2上午，比例，UV光）。之后，与帮助的补丁吸管，任何其它细胞色素osolic低亲和性的Ca 2 +指示剂透析的胞液中的缓冲液含有高亲和力的Ca 2 +的指示器（ 例如萤光-3，可见光）。这一战略使细胞质和ER派生的信号同步记录。 （5） 目标酯酶诱导染料加载 8,34。甲羧酸酯酶（CES）上被定位到内质网的内腔中，并提供了一个高的酯酶活性对AM-酯的形式存在的低亲和力的Ca 2 +的指标的有效捕获。 有针对性的酯酶诱导染料吸附量（TED） 为了提高针对低亲和力的Ca 2 +的指标内质网腔，TED的开发。 TED需要一个ER靶向小鼠羧酸酯酶（CES2）（ 图2），这是实现通过表达构建的过表达。对AM-酯的形式存在的钙孵育细胞表达重组CES构造的dicator染料（Fluo5N-AM， 图2）。然后，在ER中，染料被转换到的Ca 2 +敏感的，不透膜的Ca 2 +指示剂络合物（Fluo5N/Ca 2 +）的高的酯酶活性，因此，捕获的内质网腔8中以高浓度的染料，34。该方法是特别有用的调查通过IiCR途径例如雌激素受体 ​​的钙释放，通过代谢，嘌呤或谷氨酸受体8,34和可视化ER钙耗竭后，在SERCA 17的封锁，通过“泄漏通道”，例如直接，34。据我们的经验，低亲和力的Ca 2 +指标Fluo5N-AM是目前最好的可用指标，使用TED的染料加载策略。 Fluo5N-AM具有低亲和力的Ca 2 +（解离常数K D〜90微米， 图2），其AM-形式几乎无荧光，但提供高荧光发射后calciu米8,34约束力。 〜490 nm处，对应于标准染料如FITC标记的Alexa 488，或绿色荧光蛋白与一个标准的光源，可兴奋的Fluo5N/Ca 2 +配合物。细胞内钙信号几乎达到在低浓度μM范围内，因此，几乎没有检测到在细胞质中Fluo5N 35。 为了提高的TED性能，开发一些重组载体构建（ 图3）。本来，式载体的基础上的编码序列（RefSeq的登记号为NM_145603，CES2c CES2）和最佳的TED性能是观察到与CES的结构稳定表达。新TED载体表达红色荧光蛋白TagRFP-T2 36 CES2融合的核心要素。这些载体的优点是，它们可以被用来识别转导的细胞，使用的红色荧光变化的归一化在Fluo5N/Ca 2作为内部控制+荧光。红色荧光还提供了可能性，以可视化的刺激条件下的ER和ER动态变化的结构分布。

<table border="1">
	 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>(S)-3,5-DHPG (Dihydroxyphenylglycine)</td>
 <td>Tocris</td>
 <td>0805</td>
 <td></td>
 </tr>
 <tr>
 <td>5';-ATP-Na2, ATP disodium salt hydrate</td>
 <td>Sigma</td>
 <td>A26209-1G</td>
 <td></td>
 </tr>
 <tr>
 <td>B-27 supplement,50x</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td></td>
 </tr>
 <tr>
 <td>Carbamoylcholine chloride (Charbachol)</td>
 <td>Sigma</td>
 <td>C4382-1g</td>
 <td></td>
 </tr>
 <tr>
 <td>Cyclopiazonic acid (CPA)</td>
 <td>Ascent scientific</td>
 <td>Asc-300</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethylsulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM with Glutamax</td>
 <td>Invitrogen-Gibco</td>
 <td>31966-047</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's PBS without Ca/Mg 1x</td>
 <td>PAA</td>
 <td>H15-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal calf serum</td>
 <td>Invitrogen-Gibco</td>
 <td>10270-106</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-5N-AM, 10 x 50 &mu;g</td>
 <td>Invitrogen</td>
 <td>F14204</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamate</td>
 <td>Ascent scientific</td>
 <td>Asc-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamax</td>
 <td>Invitrogen</td>
 <td>35050-038</td>
 <td></td>
 </tr>
 <tr>
 <td>Ham's F12 nutrients mixture</td>
 <td>Invitrogen-Gibco</td>
 <td>21765-029</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's BSS(1x) without Ca,without Mg, with Phenol Red, HBSS</td>
 <td>PAA Laboratories</td>
 <td>H15-010</td>
 <td></td>
 </tr>
 <tr>
 <td>Horse serum</td>
 <td>SHD3250YK</td>
 <td>Linearis</td>
 <td></td>
 </tr>
 <tr>
 <td>Ionomycin Ca2+ salt</td>
 <td>Ascent scientific</td>
 <td>Asc-116</td>
 <td></td>
 </tr>
 <tr>
 <td>murine EGF </td>
 <td>PreproTech</td>
 <td>315-09</td>
 <td></td>
 </tr>
 <tr>
 <td>N2 supplement 100x</td>
 <td>Invitrogen</td>
 <td>17502-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Neurobasal medium</td>
 <td>Invitrogen-Gibco</td>
 <td>21103-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127, low UV absorbance,2g</td>
 <td>Invitrogen</td>
 <td>P6867</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-DL-ornithine hydrobromide PORN</td>
 <td>Sigma</td>
 <td>P8638</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-D-lysine hydrobromide</td>
 <td>Sigma</td>
 <td>P7280</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-L-Lysin</td>
 <td>Sigma</td>
 <td>P2636</td>
 <td></td>
 </tr>
 <tr>
 <td>Thapsigargin</td>
 <td>Calbiochem</td>
 <td>586005-1mg</td>
 <td></td>
 </tr>
 <tr>
 <td>TryLE Express</td>
 <td>Invitrogen</td>
 <td>12605-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Worthington</td>
 <td>LS-003707</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsininhibitor from Glycine MAX (soybean)</td>
 <td>Sigma</td>
 <td>T6522</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Materials</td>
 </tr>
 <tr>
 <td>Confocal system: inverted laser scanning microscope: FV1000 with IX81</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: laser combiner FV10 and diode lasers (405, 473, 559, 635)</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: scan head FV10-SPD with spectraldetector</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Heater controller TC-344B</td>
 <td>Warner Instruments</td>
 <td>64-0101</td>
 <td>to control in line solution heater</td>
 </tr>
 <tr>
 <td>NIH ImageJ software</td>
 <td>WS Rasband, ImageJ, US National Institutes of Health, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>, 1997--2006</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UAPON20XW340 NA 0,7 WD 0,35</td>
 <td>Olympus</td>
 <td>N2709100</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLFLN40XO</td>
 <td>Olympus</td>
 <td>N1478700</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLSAPO60XO/1.35, WD=0.15</td>
 <td>Olympus</td>
 <td>N1480700</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, inverse</td>
 <td>custom-made</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, RC-49FS</td>
 <td>Warner Instruments</td>
 <td>W4 64-1709 </td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion tube: PT-49</td>
 <td>Warner Instruments</td>
 <td>64-1730</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Peristaltic pump MiniPlus 3 (4 channels)</td>
 <td>Gilson</td>
 <td>n.a. </td>
 <td>perfusion pump</td>
 </tr>
 <tr>
 <td>Single inline solution heater SH-27B</td>
 <td>Warner Instruments</td>
 <td>64-0102</td>
 <td>controlled by heater controller</td>
 </tr>
 <tr>
 <td>Suction tubes: ST3R</td>
 <td>Warner Instruments</td>
 <td>64-1407</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Heating insert: Tempocontrol 37-2 digital 2-channel</td>
 <td>Pecon</td>
 <td>n.a. </td>
 <td></td>
 </tr>
	 </tbody>
 </table>

direct imaging of er calcium with targeted-esterase induced dye loading (ted)

重要的是要了解钙的作用在细胞生理钙动态的可视化。要检查钙动力学，合成的Ca 2 +荧光指示：已成为流行。在这里，我们展示了TED（=有针对性的酯酶诱导染料吸附量），一种方法来提高释放的Ca 2 +指示剂染料在不同类型的细胞内质网腔。迄今为止，TED在细胞系，神经胶质细胞和神经元在体外使用。 TED快递羧酸酯酶（CES）的基础上高效，重组目标高羧酸酯酶活性使用载体构建到内质网腔。最新的TED载体包含CES2红色荧光蛋白融合的核心要素，从而使两色同时成像。在ER中的游离钙离子的动态成像的一种颜色，而相应的ER​​结构以红色显示。的程序开始时，用慢病毒转导细胞。随后，受感染的细胞重新接种于盖玻片终于使活细胞成像的。然后，活细胞培养与乙酰甲酯（AM -酯）的形式，低亲和力的Ca 2 +指标，例如，MAG-Fluo4-AM Fluo5N-AM或MAG Fura2的调幅。在ER内酶切关闭从AM的Ca 2 +指示剂和亲水性荧光染料/ Ca 2 +的复合物形式的疏水性侧链的酯酶活性的内质网腔中形成并聚集。染料吸附量后，将细胞在一个倒置的共聚焦激光扫描显微镜分析。细胞连续灌流林格类似的解决方案和ER钙动力学直接可视化的时间推移成像。从内质网的钙离子的释放是确定的感兴趣区域的荧光强度下降，ER钙库的再填充而产生的荧光强度的增加。最后，荧光强度随时间的变化ΔF/ F 0通过计算确定。

该协议提供了一种非破坏性的方法，免费的ER钙的直接成像。低亲和性的合成的Ca 2 +释放指标被困于内质网腔的帮助下，一个对象的ER，重组酯酶的酶的活性。改进ER钙库动态加载的Ca 2 +指示剂染料的内质网腔，实现了直接和快速成像。 TED的细胞类型该方法的可行性已被证明在细胞系BHK21，HEK293 34，HeLa细胞17，SH-SY5Y，星形胶?...

Watch this Scientific Journal Video about Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED at JoVE.com

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

有针对性的酯酶诱导染料吸附量（TED）支持通过荧光成像分析细胞内钙库动态。该方法的基础上针对重组羧酸酯酶的内质网（ER），在那里它提高了合成的低亲和性钙本地揭露<sup&gt; +</sup&gt;指示剂染料在ER腔。

直接成像诱导ER钙有针对性的酯酶染料装载（TED）

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

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Biology

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Ex vivo Mechanical Loading of Tendon

体外机械装载肌腱

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

科研

生物学

Loading Drosophila Nerve Terminals with Calcium Indicators

载入果蝇与神经末梢钙指标

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Calcium Imaging of Cortical Neurons using Fura-2 AM

使用FURA - 2上午皮层神经元的钙成像

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Expression and Purification of Virus-like Particles for Vaccination

表达和疫苗病毒样颗粒的纯化

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

监测ER / SR钙释放与目标Ca<sup&gt; 2+</sup&gt;传感器捕捉器<sup&gt; +</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

2维直接随机光学重建显微成像中间丝和微管

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

黄荧光蛋白标记 p62 在攻击样诱导结构中光漂白后的荧光恢复

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...