Name: visualization of g3bp stress granules dynamics in live primary cells
Uploaded: 2014-05-21T13:00:00
Description: Watch this Scientific Journal Video about Visualization of G3BP Stress Granules Dynamics in Live Primary Cells at JoVE.com

作者要感谢在那里进行了收购的蒙彼利埃力成像（MRI）平台。他们感谢伊莎贝尔克里斯蒂娜·洛佩兹·梅希亚，亚历山德拉梅斯，伊琳娜Lassot，索朗Desagher，法比安斯基Loust​​alot和维尔Georget他们在协议中的不同部分的帮助。这项工作是支持的基金会倒拉RECHERCHEMédicale（FRM）（队报FRM 2011-N°DEQ20111223745）。

在这个协议的所有动物的程序都在严格遵守与1986年11月24日（86-609/EEC）的欧共体理事会指令的指引。房子的小鼠组，从而允许食物和水随意 。他们保持在受控环境中（22±1℃，55±5％相对湿度）与12小时：12小时光照：黑暗周期（灯亮在7:00上午）。 1小鼠原代细胞培养：小鼠胚胎成纤维细胞（MEF中） <ol><li>高压灭菌镊子薄，以及弯曲镊子和解剖剪刀。存放在无菌容器中...

<ol>
	<li>Thomas, M. G., Loschi, M., Desbats, M. A., Boccaccio, G. 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Binds G3BP2 and Regulates Stress Granule Formation Following Cellular Stress.</a> PLOS ONE. 7 (4), (2012).</li><li>Tourri&#232;re, H., 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=RasGAP-associated+endoribonuclease+G3Bp:+selective+RNA+degradation+and+phosphorylation-dependent+localization.">RasGAP-associated endoribonuclease G3Bp: selective RNA degradation and phosphorylation-dependent localization.</a> Molecular and Cellular Biology. 21 (22), 7747-7760 (2001).</li><li>Costa, M., Ochem, A., Staub, A., Falaschi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+DNA+helicase+VIII:+a+DNA+and+RNA+helicase+corresponding+to+the+G3BP+protein,+an+element+of+the+ras+transduction+pathway.">Human DNA helicase VIII: a DNA and RNA helicase corresponding to the G3BP protein, an element of the ras transduction pathway.</a> Nucleic acids research. 27 (3), 817-821 (1999).</li><li>Solomon, S., 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=Distinct+structural+features+of+caprin-1+mediate+its+interaction+with+G3BP-1+and+its+induction+of+phosphorylation+of+eukaryotic+translation+initiation+factor+2alpha,+entry+to+cytoplasmic+stress+granules,+and+selective+interaction+with+a+subset+of+mRNAs.">Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mRNAs.</a> Molecular and Cellular Biology. 27 (6), 2324-2342 (2007).</li><li>Soncini, C., Berdo, I., Draetta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ras-GAP+SH3+domain+binding+protein+(G3BP)+is+a+modulator+of+USP10,+a+novel+human+ubiquitin+specific+protease.">Ras-GAP SH3 domain binding protein (G3BP) is a modulator of USP10, a novel human ubiquitin specific protease.</a> Oncogene. 20 (29), 3869-3879 (2001).</li><li>Zekri, L., 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=Control+of+fetal+growth+and+neonatal+survival+by+the+RasGAP-associated+endoribonuclease+G3BP.">Control of fetal growth and neonatal survival by the RasGAP-associated endoribonuclease G3BP.</a> Molecular and Cellular Biology. 25 (19), 8703-8716 (2005).</li><li>Martin, S., 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=Deficiency+of+G3BP1,+the+stress+granules+assembly+factor,+results+in+abnormal+synaptic+plasticity+and+calcium+homeostasis+in+neurons.">Deficiency of G3BP1, the stress granules assembly factor, results in abnormal synaptic plasticity and calcium homeostasis in neurons.</a> Journal of neurochemistry. , (2013).</li><li>Mazroui, R., Huot, M. -. E., Tremblay, S., Filion, C., Labelle, Y., Khandjian, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+of+messenger+RNA+by+Fragile+X+Mental+Retardation+protein+into+cytoplasmic+granules+induces+translation+repression.">Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression.</a> Human molecular genetics. 11 (24), 3007-3017 (2002).</li><li>Colombrita, C., 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=TDP-43+is+recruited+to+stress+granules+in+conditions+of+oxidative+insult.">TDP-43 is recruited to stress granules in conditions of oxidative insult.</a> Journal of neurochemistry. 111 (4), 1051-1061 (2009).</li><li>Thomas, M. G., Tosar, L. J. M., Desbats, M. A., Leishman, C. C., Boccaccio, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+Staufen+1+is+recruited+to+stress+granules+and+impairs+their+assembly.">Mammalian Staufen 1 is recruited to stress granules and impairs their assembly.</a> Journal of Cell Science. 122 (4), 563-573 (2009).</li><li>Chen, Y., Stevens, B., Chang, J., Milbrandt, J., Barres, B. A., Hell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NS21:+re-defined+and+modified+supplement+B27+for+neuronal+cultures.">NS21: re-defined and modified supplement B27 for neuronal cultures.</a> Journal of neuroscience. 171 (2), 239-247 (2008).</li><li>Xia, Z., Dudek, H., Miranti, C. K., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Influx+via+the+NMDA+Receptor+Induces+Immediate+Early+Gene+Transcription+by+a+MAP+Kinase/ERK-Dependent+Mechanism.">Calcium Influx via the NMDA Receptor Induces Immediate Early Gene Transcription by a MAP Kinase/ERK-Dependent Mechanism.</a> The Journal of Neuroscience. 16 (17), 5425-5436 (1996).</li><li>Dudek, H., Ghosh, A., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+phosphate+transfection+of+DNA+into+neurons+in+primary+culture.">Calcium phosphate transfection of DNA into neurons in primary culture.</a> Current protocols in neuroscience. 3, (2001).</li><li>Reineke, L. C., Dougherty, J. D., Pierre, P., Lloyd, R. 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在协议中关注的转染和更具体的时间间隔采集，它必须以降低下来的细胞毒性进行仔细的监测中最关键的步骤。 原代细胞培养并不是一件困难的部分，只要在无菌条件得以维持，谨慎采取以防止在解剖和细胞分离步骤的损害。 MEFs细胞可以冷冻保存在早期传代。神经元的转染与磷酸钙已被证明工作良好21和从夏适于此协议等人 20使转染的良好的水平，但...

应力颗粒剂（SGS）是形成为对环境胁迫，如升高的温度下，氧化应激，缺氧，渗透压休克，紫外线照射，葡萄糖剥夺，或病毒感染1的蜂窝式保护性反应的非膜质细胞质灶。它们可以化学诱导治疗与像亚砷酸钠，触发氧化应激的化合物。超导重力积累停滞翻译被捕信使核糖核蛋白（mRNPs）配合物2中，从机械翻译的mRNA螯合剂，和它们的组件可以通过eIF2α蛋白的磷酸化（真核起始因子2α）来触发。超导重力仪是它与核糖体和其他颗粒，如P小体交换组件的动态结构。它们构成了一个“分流中心”，其中的mRNA进行分类和处理要么翻译，再引发，降低或包装成稳定的非多核糖体mRNPs 3。超导重力仪的组装速度快bUT它是一个渐进的过程，最初众多的小聚集体，其凝聚成较大的颗粒。使用该中断或稳定微管的化学抑制剂表明，微管网络是必需的法国兴业动态，包括组装，聚结和拆卸过程。 超导重力的动态组件也促进特定RNA结合蛋白（RNA-BP），如TIA-1（T细胞内抗原-1）和聚集TIAR（TIA-1相关蛋白），其能够二聚化促进多聚核糖体拆卸和mRNA的路由到的SG 4。 G3BP（RasGAP SH3结构域结合蛋白）是这样的RNA-BP的本地化的SGS当细胞被强调用砷或高的温度，并去磷酸化G3BP的过表达可诱导的SG组件5。 G3BP是一个进化上保守的RNA-BP，最初表征通过其与一个RAS-GTP酶激活蛋白相互作用（RasGAP p120 6）;但此相互作用是最近重新7。该G3BP家族包括哺乳动物中两个成员，G3BP1（简称G3BP）和G3BP2 8。这两个蛋白共定位于SGS，当细胞受到应力9。典型的RNA识别基序（RRM）与保守RNP1和RNP2基序：G3BPs包括一个N-末端结构域NTF2建议以影响其定位和低聚，接着富含脯氨酸（PxxP基）基序，则C-末端基序与RNA结合相关，其次是精氨酸 - 甘氨酸丰富（RGG）框。有趣的是，通过构建融合到EGFP的不同结构域的蛋白质的不同部分的分析表明，NTF2样结构域和RNA结合结构域是最有效地募集到的SG，表明二聚化的性质的重要性和RNA的结合超导重力仪的组装。多样化的车型已经在体外 10-13透露G3BP蛋白的不同功能。G3BP在小鼠干扰已经表明这种蛋白在发育的生长和存活14，以及用于G3BP在中枢神经系统（CNS），在空间工作记忆15特征在于，共济失调和缺陷中起重要作用的重要性。 G3BP缺乏导致改变神经元的可塑性和钙稳态，建立SG的形成和神经退行性疾病15之间的直接联系。能够学习的SG的动力学在初级细胞如神经元因此，这是很重要的。 该协议提供了一种简单的方法来观察下砷治疗的原代细胞含G3BP1-SGS的组装。它可以被用来研究的SG组件在不同条件下，例如不同类型的应力。它也可以适用于其它颗粒的SG的或其他成分。事实上，这个协议的重点G3BP1，但也有其他应力的颗粒标记物像TIA-1 / R，TTP（tristetraprolin）2，FMRP（脆性X智力低下综合征蛋白）16，TDP-43（有中介的响应DNA结合蛋白43），17或18诗道芬。特别是，像TIA-1 / R蛋白是一样G3BP，成核RNA结合蛋白，可诱导的SG组件过度表达时，即使不同形成的SG可以在功能，调节和相关的转录物不同。的任意这些关键部件或成核剂的SG的荧光团标记的版本转染可以执行图像特定的SG组件和动态。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Name of Material/ Equipment</td>
			<td style="width:153px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:312px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">DMEM/F12</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">21331</td>
			<td>Pre-warm at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sodium Pyruvate</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">11360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Non essential amino acids</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">11140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">L-Glutamine</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Trypsin</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">15096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glass bottom B-35</td>
			<td style="width:153px;">Greiner</td>
			<td style="width:119px;">627860/627861</td>
			<td>Treated or not (treated: increases attachment of adherent cells)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Poly-L-lysine</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">DMEM</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">31966</td>
			<td>Pre-warm at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">HeBS</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">51558</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Neurobasal</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">21103</td>
			<td>Pre-warm at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">2-mercaptoethanol</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">31350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Forceps</td>
			<td style="width:153px;">Biotek</td>
			<td style="width:119px;">DU-110-A</td>
			<td>Very thin, tips 0,1 mm (useful to remove meninges)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Curved forceps</td>
			<td style="width:153px;">Biotek</td>
			<td style="width:119px;">P-110-BUF</td>
			<td>Very thin, tips 0,1 mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Small scissors</td>
			<td style="width:153px;">Biotek</td>
			<td style="width:119px;">CM-85-BS</td>
			<td>Can be useful to remove hippocampi</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyplus transfection JetPEI reagent&#160;</td>
			<td style="width:153px;">Ozyme</td>
			<td style="width:119px;">101-10</td>
			<td>MEFs transfection, follow the forward protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted laser scanning confocal microscope</td>
			<td style="width:153px;">Leica&#160;</td>
			<td style="width:119px;">SP5</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of g3bp stress granules dynamics in live primary cells

SGS能按特定的蛋白质成分或聚腺苷酸+ mRNA的免疫染色进行可视化的细胞。超导重力仪是高度动态的，其装配和命运的研究是重要的，了解应激的细胞反应。在超导重力仪的关键因素，如G3BP（RasGAP SH3结构域结合蛋白）的缺乏导致小鼠和中枢神经系统的改变发育缺陷。若要从一个有机体，人们可以培养的原代细胞研究超导重力仪的动态细胞和遵循的SG的转染标记组件的国产化。我们描述时间推移实验，观察含G3BP1-SGS在小鼠胚胎成纤维细胞（MEF中）。这种技术也可以用来研究含G3BP-SGS在活神经元，这是至关重要的，因为它是最近表明，这些超导重力形成在神经变性疾病如阿尔茨海默病的发作。这种方法可以适用于任何其他的蜂窝体和颗粒的蛋白质成分，以及执行以转基因动物，从而允许例如颗粒动力学在没有这些颗粒的具体因子的活研究。

应力颗粒的形成是重要的细胞对应激的反应，允许一个细胞适应与停滞翻译，直到压力已被清除，相关的预防凋亡。此法允许以研究在原代细胞的SGS，通过以下关键蛋白质的SG部件国产化（ 图1）。 G3BP，SGS组件的一个重要因素，是存在而漫射在MEF中培养的或神经元（ 图2A中a和 c）的细胞质中，并且显然存在于离散的颗粒下在MEF中砷治疗以及神经元（ 图2A b和D）。</st...

Watch this Scientific Journal Video about Visualization of G3BP Stress Granules Dynamics in Live Primary Cells at JoVE.com

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

中G3BP应力颗粒动力学在Live原代细胞可视化

应力颗粒剂（SGS）是含有受阻核糖核蛋白颗粒（的RNP），并且在对各种逆境的细胞应答重要的细胞质RNA的颗粒。超导重力仪的动态特性可以遵循的活细胞通过可视化的SG在应激后转染的原代细胞标记一个组件的国产化。

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Research

JoVE Journal

Biology

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

一个现场的固定化酵母细胞可视化的快速技术

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

科研

生物学

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

在原代培养海马神经元的密集核心囊泡的实时成像

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Isolation and Primary Culture of Rat Hepatic Cells

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

将细胞质Foci分类为哺乳动物应激颗粒的方法

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

高速超分辨速度显微镜在活原纤毛中的应用

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...