Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.
Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.
钙离子是人体的每一个细胞中大量存在一个非常重要的离子。它有许多不同的细胞功能,包括生长,增殖,迁移,凋亡和1-4显著的作用。这是公认的Ca 2+可以通过直接和间接的行为影响这些过程,因此,改变该离子的正常细胞内浓度可以很容易地得到用于受影响的细胞中的负面结果。的SR被认为是细胞5内的最大细胞内Ca 2+存储。同时在SR和胞质钙离子稳态水平,通过不断变化进入和退出这个细胞器的钙 -transporting渠道的正常维护。由于任何形式的伤害或疾病在细胞施加的压力条件通常与SR钙离子的去到对长期影响他显著变化相关细胞ALTH。在最严重的情况下,一个强调细胞无法收回,并保持稳定状态SR钙离子水平甚至可能死亡的细胞凋亡6-8高潮。
目前的研究到细胞钙动力学的事实,一些研究试验细胞器的 Ca 2+存储内容直接9-11限制。最常见的做法,而不是涉及SR 钙含量12-14胞质钙离子水平的变化间接测量的测量。在这些实验中,钙离子通常诱导从SR通过使用引起细胞器的耗尽药剂( 如毒胡萝卜素)的释放。结论然后就基于在细胞质的 Ca 2+浓度的波动变化到SR 的 Ca 2+绘制。尽管剩余研究者以一种迂回的马得出这样的结论的能力nner,SR 钙这种测量方法显然是来收集这些信息,提供关于收集的数据的解释很多局限性一种间接的方式。为了绕过此明确的限制,有必要测量的 Ca 2+的SR管腔网络内直接发现的量。
至关重要的是能够直接记录腔内的SR 的 Ca 2+水平的最终结果是细胞培养工具和所用的钙离子指 示剂。在当前的手稿中引用的数据,必须指出,使用血管平滑肌细胞从冷冻的细胞系来是很重要的。细胞在Dulbecco改进的Eagle培养基(DMEM)+ 10%新生小牛血清(NCS)以上的概述实验的通道22-26,在一个恒定的37℃,5%CO 2的供应孵育培养。使用当前的方法,例如,细胞已经非常成功地生长在模拟胞外蛋白质混合物许多不同类型的组织15的环境。重要的是沿着SR Ca的静脉成功2+研究是用于蛋白质混合物的类型;在这种情况下,一个低生长因子各种必要避免从影响常规的Ca 2+信号和运动不断测试的细胞内发生的该工具的组件。以下的试验细胞的成功的增长,所述钙离子指 示剂也必须有效地引入到这些培养细胞。这已经通过使用携带SR-驻留的 Ca 2+指示剂D1SR的腺病毒载体成为可能。实现了高转染效率,细胞必须用病毒载体孵育成像前至少36-48小时。该制剂提供了测量的Ca以高精度和再现性的SR腔内2+瞬变的可靠工具。
在这个协议中使用D1SR指标是D1ER钙的改良变种2+我最初由钱永健博士的实验室在加州大学圣地亚哥分校,美国9,16创建ndicator。原始D1ER属于第二代的遗传编码的 Ca 2+指标称为cameleons,超过一个更广泛的范围(0.5-160微米)显示的 Ca 2+敏感性与先前的 Ca 2+指标9,16。该新变种D1SR,韦恩Chen博士(加拿大阿尔伯塔大学)是一种恩赐,但是,携带突变集钙蛋白序列(而不是钙网蛋白序列原D1ER)。突变体肌集钙蛋白具有结合降低的 Ca 2+,消除在对SR管腔11内结合的 Ca 2+内源性肌集钙蛋白竞争的问题。该D1SR指标进行截断的增强型青色荧光蛋白(CFP)和黄色荧光蛋白(YFP),其结合含改性钙调素(CAM)和一个M13连接蛋白(T他26个残基结合CAM)序列的肌球蛋白轻链激酶的肽。凸轮M13序列已被修饰以防止M13的结合内源性钙调蛋白。同时,为了确保SR滞留,一个集钙蛋白序列已经添加CFP 11的5'端。当结合的 Ca 2+,凸轮-M13域经过导致的增加的侧翼CFP和YFP,其被记录为一个增加的FRET信号强度之间的能量传递的构象变化。另一方面,当SR管腔的 Ca 2+的浓度下降,凸轮-M13域经过反向构象变化,从而导致在侧翼CFP和YFP,并在FRET信号强度的显著降之间的能量传递减少。
这个协议描述平滑肌的转染与D1SR腺病毒到SR的内腔中研究的 Ca 2+的浓度。此方法允许的 Ca 2+的此细胞器中的直接测量,以及它的运动进/出SR的不同钙移动剂的应用的结果。该方法对朝更改SR和胞质钙离子水平如何影响整体的健康细胞,以及这些变化是相互关联的一个全面的了解调查工作的许多优点。细胞内Ca 2+的移动的传统观测涉及改变仅细胞质<su…
The authors have nothing to disclose.
这项工作是由健康研究加拿大学院资助[MOP-1112666至CVB&ME]支持。
Dulbecco's Modified Eagle's Medium (high glucose, pyruvate) | Life technologies | 11995-065 | Warm in 37°C water bath before use |
Matrigel Basement Membrane Matrix Growth Factor Reduced, 5mL | Corning | 356230 | Keep at -20°C until right before use |
Newborn Calf Serum | Sigma-Aldrich | Keep at -20°C until right before use | |
35mm glass-bottomed plates | MatTek Corporation | P35G-1.5-14-C | |
250 mL 0.2 μM vented blue plug seal cap flask | BD Falcon | 353136 | |
Trypsin/EDTA Solution | Life technologies | 25200056 | Keep at -20°C until right before use |
50 mL Polypropylene Conical Tube | BD Falcon | 352070 | |
D1SR | N/A | N/A | Gift |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D8779 | |
Pluronic F-127 | Sigma-Aldrich | P2443 | |
Fluo 4-AM | Life technologies | F-23917 | Keep at -20°C until right before use |
1.5 mL Black Microcentrifuge Tubes | Argos Technologies | T7100BK | |
Leica TCS SP5 confocal microscope | Leica Microsystems | ||
Leica Application Suite 2.6.3 (with FRET SE Wizard application) | Leica Microsystems | ||
GraphPad Prism 5.0 | GraphPad Software, Inc. |