Name: monitoring er/sr calcium release with the targeted ca2+ sensor catcher+
Uploaded: 2017-05-19T15:00:00
Description: Watch this Scientific Journal Video about Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+ at JoVE.com

这项工作由美国国立卫生研究院GM62999，NIH EB007268，NIH AG15820，B＆B种子授予和NIH补充赠款给FR，BB奖学金，CM，CDT奖学金资助给RG。

幻灯片准备<ol><li>将22 mm x 40 mm玻璃显微镜放置在6 cm细胞培养皿中，每道1张。 </li><li>将每个载玻片的每一面以及6厘米的盘暴露于紫外（UV）光15-20分钟，以在无菌罩中消毒。 </li><li>用碟形玻璃盖上碟子，并放置在4°C直到准备使用。 </li></ol> 2.制备介质，缓冲液，溶液和试剂<ol><li>在去离子水中制备1升Hank&#39;s平衡盐溶液（HBSS），并加入10mM HEPES，5mM NaHCO 3和1mM EGTA。用NaO...

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荧光探针如CatchER +的活单细胞成像是分析每个细胞响应于受体激动剂或拮抗剂的复杂ER / SR Ca 2+信号传导过程的有效技术。这种技术对于同时使用多个波长的成像也是有用的，例如Fura-2所需要的或将Catcher +和Rhod-2一起图像分别监测ER和胞质钙变化。该协议有几个关键步骤;细胞转染可以对成像的可行性有很大的影响。低转录率可能导致GECI表达不足以监测钙的反应。另一方面?...

细胞内钙（Ca 2+ ）瞬态的时空属性激活各种生物学功能1 。这些Ca 2+信号事件通过不同的刺激细胞外触发，并由主要的Ca 2+储存细胞器和许多Ca 2+泵，通道和Ca 2+结合蛋白在细胞内控制。由于信号调制的缺陷，Ca 2+瞬变可能会显着改变，导致不同的疾病2 。由于Ca 2+信号传导系统的速度和复杂性，与内质网（ER）和肌质网（SR）在中心，需要基因编码的Ca 2+探针已被优化用于具有快速动力学的哺乳动物表达观察不同细胞的全球和局部Ca 2+变化3 。 ER和SR，其对应的肌肉细胞，是主要的细胞内Ca 2+储存细胞器，并充当Ca 2+水槽，有助于放大Ca 2+信号4 。 ER / SR是Ca 2+信号的组成部分，具有作为信号5的发射器和接收器的双重作用。兰诺定受体（RyR）和肌醇1,4,5-三磷酸受体（IP 3 R）是位于由Ca 2+ 6调节的ER / SR膜上的Ca 2+释放受体。其他药物直接或间接刺激这些受体的功能。 4-氯间甲酚（4-cmc）是RyR的有效激动剂，其具有比用于诱导SR Ca 2+释放的咖啡因高10倍的灵敏度，其中两者均经常用于研究RyR介导的Ca 2+释放健康和患病细胞7 。 ATP增加IP 3介导的Ca 2+通过IP 3 R 8租赁。 ATP结合嘌呤能受体P2YR，一种G蛋白偶联受体（GPCR），触发产生IP 3 ，结合IP 3 R从ER 9,10释放Ca 2+ 。肌质内质网钙ATP酶（SERCA）泵是一种P型ATP酶泵，也位于ER / SR膜上，可减少细胞溶质Ca 2+ ，并通过主动将离子注入ER / SR内腔来补充ER / SR 11 。 SERCA泵的特异性抑制剂包括来自加拿大的Thapsia garganica的thapigargin和来自曲霉和青霉的环磷酸（CPA）。 CPA对泵具有低亲和力，并可逆地阻断Ca 2+接入点12 。另一方面，毒胡萝卜素在M3螺旋中的残留物F256与纳摩尔不可逆地结合到不含Ca 2+的泵ar亲和力11 。分析和量化涉及Ca 2+刺激事件的变化已经并且仍然是一个挑战。由于ER / SR是Ca 2+信号传播过程中具有中枢功能的主要亚细胞Ca 2+含量隔室，因此大部分工作集中在了解ER / SR Ca 2+信号5 。 合成的Ca 2+染料的创建有助于推动Ca 2+成像的领域和实践。尽管诸如Mag-Fura-2的染料已被广泛用于测量不同细胞中的间隔Ca 2+ ，但它们具有诸如染料负载不均匀，光漂白和不能针对特定细胞器的限制。绿色荧光蛋白（GFP）的发现和荧光蛋白 -基于Ca 2+的探针已经推动了Ca 2+成像领域的发展。一些现有的GECI是涉及黄色荧光蛋白（YFP），青色荧光蛋白（CFP），钙调蛋白和M13结合肽17,18的 Förster共振能量转移（FRET）对。肌钙蛋白C基GECI也可作为FRET对的CFP和柠檬酸和单一荧光团探针19,20,21 。 其他，如GCaMP2和R-GECO是涉及钙调素22,23的单一荧光团传感器。为了克服狭窄调节K d和与其Ca 2+结合结构域24中发现的多个Ca 2+结合位点相关的协同结合的限制，一类新的钙通过在增强型绿色荧光蛋白（EGFP） 25,26的发色团敏感位置设计β桶表面上的Ca 2+结合位点来产生细胞。这种称为CatchER的高度传播的传感器在扩散极限附近具有〜0.18mM的k d ，在700s -1附近ak。 CatchER已被用于监测不同哺乳动物细胞系（如HeLa，HEK293和C2C12 25）中受体介导的ER / SR钙释放。由于其快速动力学，CatchER用于年轻和老朋友病毒B NIH Jackson（FVB）小鼠的屈肌腱肌肉纤维（FDB）肌纤维，以揭示在FDB中经过2秒的去极化后，更多的Ca 2+保留在SR中与老鼠相比，老鼠的纤维27 。克服其在37℃的低荧光，这阻碍了其在哺乳动物的钙成像中的应用细胞，我们开发了一个名为CatchER +的改进版本的CatchER。 CatchER +在37℃下表现出增强的荧光，以更好地应用于哺乳动物细胞。在CatchER中加入了另外的突变以提高37℃ 28,29 ℃的热稳定性和荧光，以产生CatchER + 。 CatchER +与CatchER 30相比，其信噪比（SNR）提高了6倍。 在这里，介绍了使用CatchER +培养和转染HEK293和C2C12细胞的方案及其用于监测ER / SR受体介导的钙瞬变的方案。显示了在用4-cmc，CPA和ATP处理的HEK293细胞中表达的CatchER +的代表性结果。我们还提供了一种用于确定C2C12成肌细胞和qua中CatchER +的 原位 K d的方案基础[Ca 2+ ]的鉴定。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-Chloro-3-methylphenol (4-CmC)</td>
			<td>Sigma-Aldrich</td>
			<td>C55402</td>
		</tr>
		<tr>
			<td>515DCXR dichroic mirror</td>
			<td>Chroma Technology Corp.</td>
			<td>NC338059</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A26209</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>EMD Millipore</td>
			<td>102382</td>
		</tr>
		<tr>
			<td>Corning tissue-culture treated culture dishes (100 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430167</td>
		</tr>
		<tr>
			<td>Corning tissue-culture treated culture dishes (60 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430166</td>
		</tr>
		<tr>
			<td>Cyclopiazonic Acid (CPA)</td>
			<td>EMD Millipore</td>
			<td>239805</td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>ACROS Organics</td>
			<td>41095-0010</td>
		</tr>
		<tr>
			<td>Dow Corning 111 Valve Lubricant &#38; Sealant</td>
			<td>Warner Instruments</td>
			<td>64-0275</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D7777</td>
		</tr>
		<tr>
			<td>Ethylenebis(oxyethylenenitrilo) 
			tetraacetic Acid (EGTA)</td>
			<td>ACROS Organics</td>
			<td>409911000&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher</td>
			<td>26140087</td>
		</tr>
		<tr>
			<td>Fisherbrand Cover Glasses 22x40 mm</td>
			<td>Fisher Scientific</td>
			<td>&#160;12-544B</td>
		</tr>
		<tr>
			<td>Hanks&#8217; Balanced Salts (HBSS)</td>
			<td>Sigma-Aldrich</td>
			<td>H4891</td>
		</tr>
		<tr>
			<td>HEPES, Free Acid, Molecular Biology Grade</td>
			<td>EMD Millipore</td>
			<td>391340</td>
		</tr>
		<tr>
			<td>Immersion Oil without autofluorescence</td>
			<td>Leica</td>
			<td>11513859</td>
		</tr>
		<tr>
			<td>Ionomycin, Free Acid</td>
			<td>Fisher Scientific</td>
			<td>50-230-5804</td>
		</tr>
		<tr>
			<td>Leica DM6100B inverted microscope with a cooled EM-CCD camera</td>
			<td>Hamamatsu</td>
			<td>C9100-13</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>ThermoFisher</td>
			<td>11668019</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>ThermoFisher</td>
			<td>L3000015</td>
		</tr>
		<tr>
			<td>Low Profile Open Diamond Bath Imaging Chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26GLP</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate</td>
			<td>Fisher Scientific</td>
			<td>M33-500</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>ThermoFisher</td>
			<td>51985034</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>EMD Millipore</td>
			<td>PX1405</td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Dibasic</td>
			<td>EMD Millipore</td>
			<td>PX1570</td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Monobasic</td>
			<td>EMD Millipore</td>
			<td>PX1565</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036</td>
		</tr>
		<tr>
			<td>SimplePCI Image Analysis Software</td>
			<td>Hamamatsu</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Scientific</td>
			<td>S233-3</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
		</tr>
		<tr>
			<td>Sterivex-GV 0.22 &#181;m filter</td>
			<td>EMD Millipore</td>
			<td>SVGVB1010</td>
		</tr>
		<tr>
			<td>Till Polychrome V Xenon lamp</td>
			<td>Till Photonics</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Trypsin (2.5%), no phenol red (10X)</td>
			<td>ThermoFisher</td>
			<td>15090046</td>
		</tr>
	</tbody>
</table>

monitoring er/sr calcium release with the targeted ca2+ sensor catcher+

细胞外刺激诱发的细胞内钙（Ca 2+ ）瞬态引发了生物体内的多种生物过程。在细胞内钙释放的中心是细胞内主要的钙储存细胞器，内质网（ER）和肌肉细胞中更专门的肌浆网（SR）。来自这些细胞器的钙的动态释放由兰诺定受体（RyR）和肌醇1,4,5-三磷酸受体（IP 3 R）介导，通过肌肉/内质网钙ATP酶（SERCA）泵进行再灌注。创建了一种称为CatchER的遗传编码钙传感器（GECI），用于监测ER / SR的钙释放速度。在这里，HEK293和C2C12细胞中改良的ER / SR靶向GECI CatchER +的转染和表达的详细方案及其在监测IP 3 R，RyR和SERCA泵介导的钙瞬变中的应用在HEK293细胞中使用荧光显微镜概述。受体激动剂或选择的抑制剂分散在室溶液中，并且强度变化被实时记录。使用这种方法，用4-氯 - 间甲酚（4-cmc）的RyR活化，用三磷酸腺苷（ATP）间接激活IP 3 R和抑制具有环磷酸腺苷酸的SERCA泵，观察到ER钙的降低酸（CPA）。我们还讨论了确定C2C12细胞中原位 K d和定量基础[Ca 2+ ]的方案。总之，与CatchER +结合使用的这些方案可以从ER中引发受体介导的钙释放，并将来应用于ER / SR钙相关病理学研究。

本节将说明使用先前描述的使用优化的ER / SR靶向GECI CatchER +的方法通过不同的受体介导途径监测ER / SR Ca 2+的变化而实现的结果。 图1说明通过用200μM4-cmc刺激的RyR排空ER。 4-cmc是RyR的激动剂。通过CatchER +荧光强度的降低测量，添加药物会诱导ER钙的降低。如标示，本文概述的方案?...

Watch this Scientific Journal Video about Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+ at JoVE.com

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

我们提出了使用我们的靶向基因编码钙指示剂（GECI）CatchER +的方案 ，用于使用实时荧光显微镜监测HEK293和骨骼肌C2C12细胞的内质网/肌浆网（ER / SR）中的快速钙瞬变。还讨论了原位测量和校准的协议。

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 ...

Research

JoVE Journal

Biology

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

Monitoring Actin Disassembly with Time-lapse Microscopy

拆装时间推移显微镜监测肌动蛋白

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 ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

加固，Eyetracking，生理监测的功能成像

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

多巴胺的释放突触前货箱码头在个别的可视化与FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

在人T淋巴细胞的全细胞钙释放激活的钙记录（CRAC）的电流

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

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 ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

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

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

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 ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...