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。使用无菌的D-PBS（Dulbecco氏磷酸盐缓冲盐水）。准备完整的MEF培养基：Dulbecco改良的Eagle培养基（DMEM）/ F12补充10％胎牛血清（FBS），1 mM的L-谷氨酰胺，1mM丙酮酸钠，1％非必需氨基酸，和0.5mM 2 - 巯基乙醇并在37°C温暖</li><li>安乐死怀孕小鼠（13.5天后coïtum（DPC）），通过颈脱位。从小鼠消毒用90％EtOH中取出子宫角。在无菌和清洁油烟机，将喇叭与胚胎在37℃预热的D-PBS。打开喇叭，地点 在100毫米无菌塑料培养皿的胚胎，从脐带材料清除它们，并用温水D-PBS冲洗，以去除血液中多余的。下一个双目，取出胚胎的四肢，内脏和 包含脑头的上部。 </li><li>在一个新的培养皿中，将剁碎 胚胎成非常小的碎片，用无菌手术 刀片 或剪刀（至少为1分钟）。在不同基因型的胚胎的情况下，小心把每个胚胎在个别培养皿中，并保持尾部或头部上进行基因分型。 </li><li>覆盖胚胎用1X胰蛋白酶-EDTA（0.25％胰蛋白酶，1mM EDTA）中。孵育30分钟至1小时在37℃的培养箱中培养。加入10毫升完整的MEF培养基停止胰蛋白酶反应。吸管上下删除所有聚集。让坐着的细胞悬浮液置于50ml试管（填充有完全培养基）中10分钟，并离心上清为5分钟，在300×g离心室温。重悬沉淀完全培养基（6毫升1胚胎）。可行的有核细胞可使用台盼计算 蓝色 （约5×10 6细胞可以从一个胚胎预期）。板3毫升每60毫米培养皿。 </li><li>第二天改变介质和 使细胞生长 直到菜都汇合。许多 细胞类型中可以看出， 但只有成纤维细胞才能生存的亚文化。 </li><li>分裂细胞，让他们在35mm培养皿生长与玻璃底（时间推移实验很重要），直到50-70％融合的转染。 测试支原体和鼠标的病原体。 </li></ol> 2，文化中的一种在神经元的案例daptations <ol><li>在培养的前一天，外套35毫米玻璃底培养皿用聚-L-赖氨酸（200微升的0.1毫克/毫升）在无菌和洁净罩，并留下过夜。 </li><li>第二天早晨，冲洗用无菌纯净水，5分钟两次，一次是45分钟至1小时。更换用2毫升的DMEM加10％FBS培养基中，并保持在37℃培养箱中培养。 </li><li>解剖胚胎在18.5 DPC。在无菌和清洁油烟机，将喇叭与胚胎冷无菌HBSS（Hank平衡盐溶液）以100毫米的培养皿。新生的幼崽，也可用于代替胚胎，以保持大坝的寿命，使其能够产生更多的后代，尤其是在转基因动物可以是难以获得的情况下。在个别培养皿，采取每个胚胎或新生儿和切头剪。握住头部插入弯钳入眼睛，切开皮肤，小心打开从后面的头骨软头部，直到眼睛，在头部的每一侧。切断视神经及脑干，取出大脑，并把它放在含有HBSS一个新的培养皿。下一个立体镜，删除所有使用两根细镊子脑膜。分离海马，皮质或大脑取决于研究该结构的任何其它部分。 </li><li> 4.5毫升冷的HBSS浸入大脑解剖结构在15ml试管预先准备并保持在冰上，直到用胰蛋白酶消化。加入0.5毫升2.5％的胰蛋白酶和孵育在37℃下进行15分钟至20分钟。冲洗3次胰蛋白酶用HBSS，是非常小心，不要丢弃消化大脑部位。 </li><li>重悬在500微升（海马）至1ml（皮层）的DMEM加10％FBS和吸管上下数次1毫升微量装有1毫升尖，然后装有用1ml加200μl的提示，直到有没有明显的聚集。 </li><li>散布100-200微升细胞悬液至35毫米的玻璃博特米菜的DMEM加10％FBS和让神经元坚持在37℃至少3小时。通过预热神经元完全培养基更换（Neurobasal培养基补充有250μML-谷氨酰胺和NS21，如在Chen 等人 19所述制备），并在37℃下离开以允许神经元生长。转染的神经元在5〜14天在体外 （DIV）（转染效率更高经过几个div，不过突触连接的是从7-10格更好地确立）。 </li></ol>的EGFP-G3BP1构造3。转染转染的细胞用含有您感兴趣的蛋白的基因（SGS的任何成分）稠合到荧光标记（GFP，YFP，等）的载体，用3微克纯化的质粒的每35mm培养皿。 <ol><li>使用市售的转染方法的MEFs中，按照制造商的方案（见表原料/试剂的）。 </li><li>转染的神经元与钙磷酸盐的方法改编自Xia 等 20简要： <ol><li>准备解决方案：DMEM洗：含25毫米氯化钾的DMEM;转染溶液：DMEM洗含有1x DMKY（HEPES 5毫米， 氯化镁 10毫米，酚红）;和冲击的解决方案：HEBS 1X，1X DMKY和DMSO 2％（V / V）;并保持在37°C。 </li><li>从神经元，滤液取出介质，并保持在37°C。用DMEM洗洗脸然后更换转染培养基和磷酸钙质粒DNA沉淀物的制备过程中保持在37℃。 </li><li>在1.5 ml离心管中，加入（按照这个顺序）百灵水（终体积50微升），5微升氯化钙 2.5 m和3微克质粒DNA。放弃这一组合到50微升HEBS 2倍的圆底聚丙烯管已经出台。通过转动沿与滴下混合管。在30分钟期间让沉淀物的形式在室温下进行。 </li><li>添加precipitatË滴加到神经元，并在37℃时30〜50分钟离开。 </li><li>更换1分钟冲击溶液，然后用DMEM洗，最后重新引入预处理介质。保持所述细胞在37℃下</li></ol></li></ol> G3BP 4。含可视超导重力大会和动态<ol><li>更换细胞培养基含有酚红酚红的培养基。 </li><li>利用共聚焦显微镜配有荧光的收购。打开显微镜系统：汞灯，计算机，激光器。热身室至37℃至少20分钟前收购的开始。 </li><li> G3BP的过表达诱导型超导重力的自发形成。这些细胞可能因此无应力诱导转染后可观察到的超导重力仪24小时正确装配。另外，压力可诱发进一步诱发超导重力仪的组装。添加0.5 mM的亚砷酸钠和明星加入化合物（颗粒会在1小时内得到很好的形成）后右T的收购。 </li><li>添加到油中浸没物镜（使用40X或63X的目标），并在适于35毫米显微镜支架安装在培养皿在客观的，稳定的阶段保持器。检查盘是平的，否则收购将被改变。根据相关的荧光打开的荧光光以及可视化的转染细胞。关闭荧光曾经感兴趣的细胞是在现场以尽量减少漂白和细胞毒性。 </li><li>在“获取标签”，请根据所标记荧光基团（在我们的协议，使用绿色荧光蛋白标记的G3BP 488纳米）的激发波长的激光器和选择的PMT的适应发射长度（光电倍增管）。调整激光功率（通常不超过10％），以减少噪声和过饱和以及毒性，并设置增益和偏移修改的信号噪声比。扫描速度快（1000赫兹），以尽量减少激光博览会的持续时间（平均线2，帧平均1）。如果需要的话，设置参数的Z堆叠能够重建三维图像（1微米为Z步骤通常是细带细胞）。确定每个采集间隔时间：较小的间隔允许获得更加相干的影片，但是这可能导致光漂白性和毒性（20秒的时间间隔是在所描述的实验中使用）。总采集的持续时间可取决于应力类型而有所不同。许多含G3BP-SGS是根据砷处理形成非常迅速，而且是1小时的治疗后也可见：在这种情况下，选择快速细胞应激后立即拍摄并开始收购，以收购15的总持续时间 - 20分钟很大程度上足以观察几个超导重力仪的组装。 </li><li>保存图片和重建使用ImageJ软件堆栈和电影。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

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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			<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

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。

中G3BP应力颗粒动力学在Live原代细胞可视化

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 ...

visualization-of-g3bp-stress-granules-dynamics-in-live-primary-cells

Research

JoVE Journal

Biology

18.2K 次观看.  Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535. 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。 

视频: Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

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

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

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

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

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

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

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 ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

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 ...

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

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of 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

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

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

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

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 ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

在成细胞分化和融合过程中利用活成像跟踪核

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...