Name: live imaging assay for assessing the roles of ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds
Uploaded: 2013-08-25T14:00:00
Description: Watch this Scientific Journal Video about Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds at JoVE.com

这项工作是由美国国立卫生研究院资助R37 AI34867和R01 GM064625为NWA我们感谢R. Twete​​n博士从俄克拉何马大学的SLO表达质粒的支持。

1。 ASM的转录沉默<ol><li>种子1.5×10 5个HeLa细胞于2ml DMEM生长培养基（DMEM高葡萄糖，10％胎牛血清（FBS），2mM的L-谷氨酰胺，1％青霉素-链霉素）在35 mm的玻璃底培养皿（MatTek）和在5％CO2 培养箱孵育过夜，在37℃。 </li><li>在第二天，吸出物离开的生长培养基，并添加siRNA转染混合物之前，用2ml的DMEM低血清培养基（DMEM，用4％FBS）中，至少1小时更换。 </li><li>准备4管的寡核苷酸的siRNA?...

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J. 231, 205-208 (1985).</li><li>Keefe, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin+triggers+a+plasma+membrane-repair+response+that+facilitates+CTL+induction+of+apoptosis.">Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis.</a> Immunity. 23, 249-262 (2005).</li><li>Idone, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+injured+plasma+membrane+by+rapid+Ca2+-dependent+endocytosis.">Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis.</a> J. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol-dependent+cytolysins,+a+family+of+versatile+pore-forming+toxins.">Cholesterol-dependent cytolysins, a family of versatile pore-forming toxins.</a> Infect. Immun. 73, 6199-6209 (2005).</li><li>Zha, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingomyelinase+treatment+induces+ATP-independent+endocytosis.">Sphingomyelinase treatment induces ATP-independent endocytosis.</a> J. Cell Biol. 140, 39-47 (1998).</li><li>Buckley, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+three+membranes.+Channel+formation+by+aerolysin.">Crossing three membranes. Channel formation by aerolysin.</a> FEBS Lett. 307, 30-33 (1992).</li><li>Parker, M. 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机械损伤和细胞膜修复早期的研究依赖于细胞损伤的机制多种多样，从掉落的玻璃珠，micropipetting，剪切，划伤或者刮伤。所有这些测定的读数是定性而非定量的，并没有给于修复机制的动力学精确的信息。细胞的这些形式的损伤之后重新密封其质膜的能力被加入到在细胞质中，渗透膜不透染料，细胞内的酶，ATP水平，或长期的损失外液示踪物的存在或不存在下测定细胞存活。虽然几个这些测定?...

人们已经知道了几十年了机械性损伤后质膜修复是的Ca 2 +依赖性过程1,2。后来的研究表明，通过伤口的Ca 2 +内流触发细胞内囊泡的胞吐一场轰轰烈烈的过程在损伤部位，这是需要重新密封3,4。加载到细胞的细胞质中的荧光染料的损失率被用来评估修复的速度，并得出结论，被重新密封于&lt;30秒5伤后完成。两款车型最初提出来解释在质膜修为胞吐作用的要求：1）“补丁”的模式，这表明，Ca 2 +的内流，通过病灶触发细胞内囊泡最初同型融合，形成了一个庞大的“补丁”，将然后可以应用到质膜重新密封伤口6和2）的张力减小模型，其中提出，膜ADDEð通过在伤口附近的Ca 2 +依赖性胞吐作用将降低细胞膜张力，促进双层7的重新密封。 钙在细胞膜修复+触发胞吐作用通过研究表明，溶酶体含有Ca 2 +的传感器分子，突触七，这有利于它们的胞外分泌和质膜修复受损细胞，进一步加强8,9,10,11 。 然而，更多的证据表明，单纯胞吐作用不足以促进细胞膜修复。除了 ​​在质膜上的机械眼泪，细胞损伤的常见形式是透由细菌12,13或免疫细胞14,15产生的成孔毒素。不像机械眼泪，成孔蛋白插入本身对质膜形成稳定，蛋白质内衬的孔隙无法通过应用膜“补丁”或由红眼简单地重新密封ING膜的张力。有趣的是，研究发现，哺乳动物细胞具有与透孔形成蛋白后，以修理其质膜一个有效的机制，而这个过程也需要胞外Ca 2 +的12的存在。这一发现提出了从细胞表面跨膜孔隙去除是否也是一个快速的过程的问题，作为具有机械伤口5观察到。出人意料的是，我们最近的研究表明，细胞透化与成孔毒素的再密封具有非常相似的性质，以机械创伤的修复：这个过程需要细胞外Ca 2 +的，并且在约30秒完成。更详细地调查这个过程中，我们最近了解到，除了Ca 2 +的调节溶酶体的胞吐，质膜修复涉及内吞作用的快速形式，它是由溶酶体的释放引发酶的酸性鞘磷脂酶（ASM），是必需的不仅为日ë去除跨膜孔的，而且对机械创伤16的维修。 为了确定通透与成孔蛋白后细胞再密封的动力学，在我们的实验室，我们改编以前已经用于评估激光隔离受伤的肌肉纤维17的重新密封的实时成像方法。该测定依赖于亲脂性染料FM1-43，它稳定地可嵌入到脂质双层中的荧光强度增加的外小叶的属性。当细胞膜双分子层被破坏的细胞外染料得以进入细胞内的膜，提供了一个敏感的检测，以检测细胞膜损伤和修复18,16,19,20。要通透与成孔蛋白后细胞再密封的评估适应这个实验中，我们预孵育细胞在4℃与细菌毒素链球菌溶血素为O（SLO），其中结合细胞膜胆固醇21。同步单元透然后可以通过移动从冰细胞温热介质中加热的显微镜载物台，其激活低聚和变化中的构象，导致跨膜孔形成很容易地实现。这种方法的一个优点，通过使用激光伤人先前公布的测定中，是细胞中的更大的数目可以在一个显微镜视野同时进行分析，提供上述细胞群的较好采样。给定的机械损伤和透化与成孔毒素后见到的细胞重新密封过程之间的机械的相似性，我们在这里描述的测定法提供了解剖参与质膜修复的基本过程的因素非常灵活和强大的方法。作为一个例子，我们表明，它有可能使用该测定，以确定在修复过程中的Ca 2 +依赖性和独立的步骤。

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>HeLa 229 cell line</td>
			<td>ATCC</td>
			<td>CCL2-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose </td>
			<td>Invitrogen</td>
			<td>11965</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose No Calcium</td>
			<td>Invitrogen</td>
			<td>20168</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Optimem Reduced Serum</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Control medium GC content RNAi oligo</td>
			<td>Invitrogen</td>
			<td>12935300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>SMPD1 RNAi oligo</td>
			<td>Invitrogen</td>
			<td>HSS143988</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum Heat-inactivated</td>
			<td>Gemini-Bioproducts</td>
			<td>100-106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sphingomyelinase from Bacillus cereus</td>
			<td>Sigma</td>
			<td>S7651</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 mm-glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-14</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material</td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C9100-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spinning disk confocal microscope</td>
			<td>PerkinElmer</td>
			<td>UltraViewVoX</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software analysis software</td>
			<td>PerkinElmer</td>
			<td>Volocity Suite</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Environmental chamber</td>
			<td>Pathology Devices</td>
			<td>LiveCell System Chamber</td>
		 </tr>
		</tbody>
 </table>

live imaging assay for assessing the roles of ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds

细胞膜损伤是一种常见的事件，伤口必须迅速修复，以确保细胞存活。的Ca 2 +的内流，触发机械创伤的修复上内〜30秒质膜中的关键信号传导事件。最近的研究表明，与透孔形成在一个的Ca 2 +依赖性的过程，涉及溶酶体酶酸性鞘磷脂酶随后孔隙的内吞作用的胞外分泌的毒素后的哺乳动物细胞也重新密封的质膜。在这里，我们描述了用于证明细胞毒素链球菌溶血素Ô透的再密封也是快速和依赖的Ca 2 +内流的方法。该法的设计允许伤害事件的同步和细胞的成像，恢复细胞膜的完整性，并通过量化其liphophilic染料FM1-43达到细胞内膜的程度的能力的精确动力学测量。这活法阿尔斯O允许的外源能力的评估敏感添加可溶性因子如鞘磷脂酶抑制FM1-43大量涌入，反映细胞的修复他们的质膜的能力。该测定法允许我们表明：第一次，鞘磷脂酶的作用的Ca 2 +的依赖性胞吐作用的下游，由于胞外加入酶促进细胞透化在不存在的Ca 2 +的再密封。

低浓度的细菌鞘磷脂（SM）的救援细胞膜修复细胞贫溶酶体酸性鞘磷脂酶。 用FM1-43的成像方法，我们以前表明，细胞缺乏的溶酶体酶ASM和暴露于SLO伤人中的Ca 2 +的存在下具有质膜缺陷是由细胞外加入的重组人酶19的获救。由于人溶酶体ASM具有低的pH最佳值，我们研究是否重新密封也可以恢复增加的SM从蜡状芽孢杆菌 （Sigma公司），它是在中性pH下完全有活性...

Watch this Scientific Journal Video about Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds at JoVE.com

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

细胞暴露于所述亲脂性染料FM1-43的实时成像可以精确测定由该成孔毒素从质膜除去动力学。这是一个敏感的测定法可以用于评估要求的Ca 2 +，鞘磷脂和其它因素对质膜修复。

实时成像检测方法的评估钙的作用<sup&gt; 2 +</sup&gt;和鞘磷脂酶在成孔毒素伤口的修复

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In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

在体内的Ca 2 + -蜜蜂的嗅觉学习期间的蘑菇体神经元的成像

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Techniques for Imaging Ca2+ Signaling in Human Sperm

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

电子自旋共振氧测绘活体物种显微成像

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

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 ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

活细胞成像早期自噬活动：Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...