我们要感谢为我们提供的比例，pericam MT的表达构建宫胁淳。这项工作是支持的美国国立卫生研究院（DB）的赠款GM081685。

1。 Pericam - MT转染和细胞制备。 <ol><li>无菌玻璃盖玻片放置在一个标准的6孔培养板转板的HeLa细胞的前一天晚上。 </li><li> 2000年DNA /脂质体复合物准备，制造商（Invitrogen）的建议。我们使用比例pericam - MT的表达载体的4微克和10μL0.5毫升OPTI - MEM稀释为每口井的脂质体2000。细胞转染前〜70％融合。 </li><li>在每孔1.5毫升不含抗生素的同一媒体更换HeLa细胞培养基（贝科的修改鹰中等，10％胎牛血清，青霉素/链霉素）。 </li><li>新增，每孔转染的DNA /脂质体复合物。在37℃，5％的CO 2混合轻轻摇晃，4个小时，在细胞培养孵化器的孵化。 </li><li>更换与使用抗生素的培养基抗生素的自由媒体。允许细胞表达成像前1-2天的比例pericam。 </li></ol> 2。显微镜设置和图像采集<ol><li>与HeLa细胞的盖玻片放入成像解决方案的合适的成像室：107毫米氯化钠，氯化钾7.2mM，1.2MM MgCl 2的，1MM氯化钙，11.5mm的葡萄糖，0.1％牛血清白蛋白和7.2 20MM HEPES 。我们的实验室使用Attofluor细胞室自Invitrogen。安装在倒置显微镜下阶段的成像室。 </li><li>查找包含一个或多个细胞表达比例pericam使用一个40X（或更高）油浸物镜的利益区域。我们发现，比例pericam抗性较差，在380 nm漂白，因此，我们使用这个波长物色合适的细胞。在图1中获得的图像，Superfluor尼康40X目标与1.3数值孔径。高倍率的目标也将是适当的（60 - 100X）。激励过滤器使用的色度科技滤波器D380/30x（380 + / - 30纳米）和D495/10（495 + / - 5纳米），分光镜505DCXR（505nm长波），发射滤波器HQ535/50m（535 + / - 25纳米）。 我们的显微镜，用来获取的图像，在图1包括一个尼康TE2000倒置一个罗珀科学Coolsnap总部Ludl MAC6000快速滤光轮和转换器，CCD摄像头，冷却和MetaFluor数据采集和分析软件配备显微镜。适当的过滤器，能够捕获和处理的比例图像的软件，该协议可以适应任何标准的宽视场显微镜。 </li><li>成立了软件使用的495和380 nm的过滤器的双激发。钙结合比例pericam的增加在495 nm处的吸光度，从而比例应设立495 nm/380 nm的。 成比例pericam钙自由激发峰在415 nm处4。在这个协议中，我们建议使用380纳米过滤器，不仅因为它是无处不在大多数钙成像实验室。如果一个410纳米过滤器是可用的，最好是到380纳米过滤。 </li><li>获取图像顺序由另外495和380 nm的激动人心的。收购前至少30小号激动剂的应用程序通过灌注仪器基线钙水平的图像。马克除了为每个激动剂时间。曝光时间和采集时间间隔应进行优化，以防止漂白，同时仍然允许足够的时间resolution.For例如钙激动剂刺激半快速监测动态，被收购，图像每两秒钟，在图1b和C。快得多的数据采集速率也有可能6。长期成像后星形孢菌素管理是一个较长的时间间隔（30岁）（图1 D和E）之间的收购与收购。 </li></ol> 3。图像处理和分析<ol><li>一旦实验完成后，获得的图像可进行离线分析。定义的感兴趣区域（ROI），在您要监视的钙离子水平的变化。使用该领域的空白区域内的投资回报率减去背景，如果有必要选定一个。应该指出，线粒体是相当运动型和动态，并推断长时间，在一个单一的线粒体事件并不总是可能的。因此，考虑到这种细胞器在某些类型的细胞的高活力，投资回报率的选择应仔细监测。此外，在图1中可见一斑，线粒体响应不均匀各种刺激。因此重要的是离线数据分析和监控RO1的上一帧帧的基础上的位置。测量高度能动的线粒体线粒体钙的一个详细描述已在别处 7 。 </li><li>从投资回报率获得比测量可以导入到Excel或类似graphingsoftware。数据可以作为一个代表在一个单细胞或单细胞线粒体的线粒体钙水平的变化进行的跟踪，或平均FL从几个投资回报率的uorescence信号。我们也使用了这种技术来衡量线粒体钙的水平，平均人口接受由Fas配体 8诱导细胞凋亡的淋巴细胞。 </li></ol> 4。代表性的成果： <img src="/files/ftp_upload/2579/2579fig1.jpg" alt="图1" /> 图1。 （一）亚细胞定位比率pericam - MT。这第一个图像显示生活的HeLa细胞表达的比例，pericam - MT。第二个图像显示与线粒体选择性染料MitoTracker红CMXRos荧光染色。在合并后的图像的黄色荧光显示比例pericam - MT和MitoTracker荧光共定位。，（B）系列伪比率（495:380纳米）4 HeLa细胞的图像，表达MT - 10毫米ATP治疗的比例pericam （C）（二）与10毫米ATP治疗（D）系列伪比例（495:380纳米）MT一个HeLa细胞表达细胞的图像量化的感兴趣区域（ROI）的线粒体钙水平的变化表示比例pericam采用0.5微米的星形孢菌素。个别线粒体钙反应的异质性是显而易见的，以及60分钟线粒体显着的碎片。一些线粒体钙（白色箭头头）振荡上升，而其他不显示钙离子水平的显着变化（黄色箭头头）（E）在全球在一个单一的HeLa细胞的线粒体中钙的含量增加后，诱导细胞凋亡的量化0.5微米星形孢菌素。

<ol>
	<li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1, 11-21 (2000).</li><li>Hajnoczky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+calcium+signalling+and+cell+death:+approaches+for+assessing+the+role+of+mitochondrial+Ca2++uptake+in+apoptosis.">Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis.</a> Cell Calcium. 40, 553-560 (2006).</li><li>Nagai, T., Sawano, A., Park, E. S., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circularly+permuted+green+fluorescent+proteins+engineered+to+sense+Ca2.">Circularly permuted green fluorescent proteins engineered to sense Ca2.</a> Proc Natl Acad Sci U S A. 98, 3197-3202 (2001).</li><li>Rizzuto, R., Simpson, A. W., Brini, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+changes+of+mitochondrial+Ca2++revealed+by+specifically+targeted+recombinant+aequorin.">Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin.</a> Nature. 358, 325-327 (1992).</li><li>Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+Ca2++based+on+green+fluorescent+proteins+and+calmodulin.">Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.</a> Nature. 388, 882-887 (1997).</li><li>Shimozono, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+of+subcellular+Ca2++concentrations+using+a+dual-excitation+ratiometric+indicator+based+on+green+fluorescent+protein.">Confocal imaging of subcellular Ca2+ concentrations using a dual-excitation ratiometric indicator based on green fluorescent protein.</a> Sci STKE. , pl4-pl4 (2002).</li><li>Wozniak, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+biphasic+calcium+release+from+the+endoplasmic+reticulum+for+Fas-mediated+apoptosis.">Requirement of biphasic calcium release from the endoplasmic reticulum for Fas-mediated apoptosis.</a> J Cell Biol. 175, 709-714 (2006).</li><li>Boehning, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+c+binds+to+inositol+(1,4,5)+trisphosphate+receptors,+amplifying+calcium-dependent+apoptosis.">Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis.</a> Nat Cell Biol. 5, 1051-1061 (2003).</li><li>Csordas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+interorganelle+contacts+and+local+calcium+dynamics+at+the+ER-mitochondrial+interface.">Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface.</a> Mol Cell. 39, 121-132 (2010).</li><li>Matsuyama, S., Llopis, J., Deveraux, Q. L., Tsien, R. Y., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+intramitochondrial+and+cytosolic+pH:+early+events+that+modulate+caspase+activation+during+apoptosis.">Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis.</a> Nat Cell Biol. 2, 318-325 (2000).</li><li>Jiang, D., Zhao, L., Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+identifies+Letm1+as+a+mitochondrial+Ca2+/H++antiporter.">Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ antiporter.</a> Science. 326, 144-147 (2009).</li></ol>

在这里，我们提出了一个非常简单的方法测量线粒体钙线粒体有针对性的比例pericam。如图1a所示，使用标准广角光学没有去卷积，它可以轻松地查看个人线粒体在HeLa细胞中可以接受的信号噪声比。这是因为HeLa细胞，如文化中的大多数细胞，扁平化，大大壁时无须共聚焦显微镜或其他专用设备的需要。我们坚持多聚赖氨酸涂层的盖玻片的Jurkat细胞中发现了类似的结果。对比方法，使用的染料，它也有可能使用比例pericam（图1E），如基因编码的钙指标的小时线粒体钙的水平，非侵入性的监测。分析线粒体钙在细胞死亡时，这一点尤为重要。此外，它也可以可视化在细胞凋亡过程中线粒体的裂变/碎片。如在图1D和E所示，星形孢菌素治疗增长缓慢导致线粒体该峰的选择亚群在20分钟中的钙。钙水平下降，在治疗2小时后再次前再次与线粒体碎片伴随。这是与星形孢菌素测量治疗使用FURA - 2 9诱导的胞浆钙的慢波相一致。因此，这是不符合聘用静态测量的方法，可以得到重要的动力学信息。虽然我们提出的比率和图1中的钙离子浓度，它是可以就地校准的传感器来计算绝对钙的水平 ， 10。一个重要的需要考虑的是，比例pericam敏感，pH 值4，10 。由于胞质和线粒体pH值可以改变11细胞凋亡过程中显着，这是一个重要的考虑因素。在孤立的线粒体表达pericam中的pH值滴定表明 ，[H +]的增加排放的pericam兴奋时在495 nm的激发在410 nm，已利用的一个属性，同时测量两个线粒体钙的受激发射的影响不大， pH值12。因此，有选择性地监测与410 nm激发的排放提供了一种非ratiometrically显示器线粒体钙与pH值的降低关注。最后，这里介绍的协议，只需要访问一个广泛使用的过滤器的标准啶显微镜，从而使这项技术访问大多数实验室。

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
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<td>Inverted wide-field fluorescent calcium imaging microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>MEA53310</td>
<td>Any wide-field inverted microscope equipped with 380 nm/495 nm excitation filters and appropriate longpass and emission filter can be easily adapted to use this protocol.</td>
</tr>
<tr>
<td>Imaging Software</td>
<td>&nbsp;</td>
<td>Molecular Devices, Inc</td>
<td>Metafluor</td>
<td>&nbsp;</td>
<tr>
<td>Attofluor cell chamber</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>A-7816</td>
<td>Any imaging chamber for an inverted microscope which allows perfusion would suffice.</td>
</tr>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>11668-019</td>
<td>Most transfection reagents/protocols would be appropriate for these studies </td>
</tr>
<tr>
<td>MitoTracker Red CMXRos</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>M7512</td>
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monitoring dynamic changes in mitochondrial calcium levels during apoptosis using a genetically encoded calcium sensor

在各种刺激的反应的细胞内钙离子浓度的动态变化调节许多细胞过程，如增殖，分化和凋亡1。在促进细胞凋亡，线粒体中钙积累从线粒体释放促凋亡因素， 进入细胞质2。因此，利益直接测量原位活细胞线粒体钙在细胞凋亡过程中。高分辨率荧光成像与双激励的比例和非比例合成钙染料加载细胞已被证明是一个可靠和灵活的工具来研究细胞内钙信号的各个方面。测量胞浆钙通量，使用这些技术相对比较简单。然而，测量使用rhod - 2和rhod - FF如合成钙指标的完整细胞intramitochondrial的钙离子水平，是更具挑战性。有针对性的线粒体合成的指标有减弱的反应在线粒体钙重复增加，破坏线粒体形态 3 。此外，综合指标往往超过几个小时，这使得它们不适合用于长期实验泄漏线粒体。因此，根据基于绿色荧光蛋白（GFP）4或有针对性的线粒体aequorin 5的基因编码的钙指标大大促进线粒体钙的动态测量。在这里，我们描述了一个简单的方法线粒体钙通量实时测量在不同的刺激的反应。该方法是基于“比例pericam&#39;，这是选择性地针对线粒体的荧光显微镜。比率pericam是一种钙指标的基础上，融合的循环置换的黄色荧光蛋白和钙调蛋白4。钙的比例pericam结合导致其激发峰从415纳米到494纳米的转变，而发射光谱，其中峰515纳米左右，保持不变。成比例pericam绑定一个单一的钙离子的解离常数在体外〜1.7 微米 4 。比例pericam这些属性允许线粒体钙离子浓度的快速和长期的变化进行量化。此外，我们描述了这种方法的适应，以一个标准的宽视场的钙成像显微镜用普通的过滤器设置。使用两种不同的受体激动剂，嘌呤受体激动剂ATP和凋亡诱导药物星形孢菌素，我们展示了几秒钟到几小时的时间分辨率监测线粒体钙离子浓度的变化，这种方法是适当的。此外，我们还证明，比例pericam也是用于测量/细胞凋亡过程中线粒体裂变碎片。因此，pericam比例是特别适合长期连续的细胞凋亡过程中线粒体钙的动态测量。

Watch this Scientific Journal Video about Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor at JoVE.com

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

本协议描述了线粒体钙通量实时荧光成像测量方法。该方法采用一个基于循环permutated YFP的双激励比例的钙传感器选择性表达在线粒体（比例pericam吨）的优势。

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, ...

monitoring-dynamic-changes-mitochondrial-calcium-levels-during

Research

JoVE Journal

Biology

23.8K 次观看.  University of Texas Medical Branch. 本协议描述了线粒体钙通量实时荧光成像测量方法。该方法采用一个基于循环permutated YFP的双激励比例的钙传感器选择性表达在线粒体（比例pericam吨）的优势。

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

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

双层弹性性能的监测变化的单分子方法

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

科研

生物学

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

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 ...

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

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2 . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+ or Na+ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8.

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

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived 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 ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

测量心肌细胞的快速钙助焊剂

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

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 ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

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

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

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

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

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 ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

实时在体记录了拟南芥钙信号在使用荧光传感器的昆虫饲喂时

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets.

A forward genetic screen based on Ca2+ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

使用转基因钙处理器 Aequorin 前向基因屏幕，以识别钙信号中的新目标

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...