The authors are deeply indebted to all the members of the scientific community working on pH and ion transport, who have originated and improved the measurements described here. They particularly thank Dr. Jacques Pouyss&#233;gur who originated the H+-killing selection technique used here. They acknowledge the University of Nice-Sophia Antipolis, the CNRS, the ANR (JCJC SVSE1 NHEint) and the ICST Labex for support.

1. H +杀戮的选择<ol><li>细胞系<ol><li>稳定地转染NHE缺陷型细胞（ 例如 ，CCL39衍生PS120细胞系16），使用哺乳动物表达载体和转染方法的任意组合，将产生有效的转染率和选择在一个成纤维细胞系。 注:多年来磷酸钙沉淀19与良好的收益率转用。这已被替换最近被商业试剂如Lipofectamine2000，正在执行按照制造商的说明转染。 </li></ol></li><li>解决方案<ol><li>制备描述如下3无菌溶液。通过过滤（0.22微米）灭菌这些溶液。 <ol><li>制备NH + 4加载溶液（pH7.4）中含有50mM的氯化铵 ，70毫氯化胆碱，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM的GLucose和15mM MOPS pH为7.4。 </li><li>制备由120毫氯化胆碱，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES，在pH 7.0漂洗溶液（pH 7.0）中。 </li><li>制备包含120mM氯化钠，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES，在pH 7.4的回收溶液（pH7.4）中。 </li></ol></li></ol></li><li>在开始选择之前，获得细胞培养孵化器，可用于在37℃下，没有额外的CO 2，从外部罐（称为CO 2 - "自由"孵化），5％的CO 2将导致缓冲可能损害酸化。扩增表达感兴趣向上NHE至约2×10 8个细胞（通常为约20汇合100毫米平板）的细胞系。 </li><li> H +查杀方法: <ol><li>孵育表达目的细胞内NHE的细胞1小时，在加载溶液中，在37°C，在无二氧化碳排放的孵化器。 </li><li>吸出装载溶液，然后漂洗两次它们在上述漂洗溶液。有效地执行这一步，以消除残余细胞外氯化铵 ，将损害创造一个陡峭的NH 4 +梯度，将产生的酸化。 </li><li>消除通过抽吸漂洗介质，并在回收溶液中的CO 2培养箱自由再次孵育细胞一小时，在37℃。 </li><li>替换常规的培养基（通常的DMEM以7.5％FCS）中的恢复中，生长中的细胞的标准培养条件下（37℃，在5％的CO 2和95％空气的潮湿气氛中）。 </li></ol></li><li>每周重复选择两次这样的循环，直到平稳克隆出现。 注:要特别注意有关细胞培养和选择条件。工作几个月可以通过任何的污染是比较容易Ø毁经过文化和重复单元操作长时间CCur函数。 <ol><li>在这里，选择是否放大克隆并分别在第2-3为进一步说明他们的特点，或集中在一起，生成特征之前，细胞群体。 </li></ol></li><li>适用偶尔选择（每两周一次左右）通过反选择那些可能已经恢复到亲酸敏感的表型（ 图1B）保留酸化抗性细胞。 </li></ol> 2.细胞内pH测量 2.1）荧光pH值成像<ol><li>使用比例PH敏感荧光染料BCECF / AM，这是pH敏感当在490nm激发，并具有一个445纳米的等吸光点，按照制造商的协议。 <ol><li>可替代地，可以使用其他pH敏感的探针，按照制造商的协议。 </li></ol></li><li>使用的成像设置CONsisting倒置显微镜耦合到一个高灵敏度的摄像机的。装备这套450 nm和搭配激发适当的石英中性密度滤光片490 nm的窄带干涉滤光片。 <ol><li>如果可能的话，可以使用适当的一组过滤器和照明条件设置可比荧光值λ1和λ2，使得比接近1。此外，还要避免被设置或者饱和或过低，都为λ1和/或λ2基础荧光值。这可能会产生重要的文物和当时一样的比例不得被检测到，虽然细胞内pH值发生变化。 注意:此系统必须在适当的发射波长（535纳米的BCECF）使快速灌注，延时激励在两个上述的波长和采集。装备该系统用软件实现自动数据采集和存储。 </li></ol></li></ol> NHE转发活动2.2）测量（挤压从胞浆质子） <ol><li>酸化的细胞用1小时温育中的NH + 4加载溶液（参见步骤1.4），在不存在的CO 2。 </li><li>添加BCECF-AM（5μM的终浓度），以将溶液的最后五分钟，然后用NH冲洗4 +加载溶液，以消除外探头。 </li><li>安装在显微镜的细胞，并采取若干图像来记录一个稳定的基线。灌注冲洗液（见上文）。这导致了降荧光对应于酸化。 </li><li> pH值稳定后，灌注任何下面给出的解决方案，以测量由锂+ / H +，的Na + / H +或K + / H +交换介导的pH值回收率。每个溶液含有潜在耦合阳离子为H +交换进行测试。如果这些中的一个被传送，这将导致荧光增加，在495nm处，这将correspOND在细胞内pH加薪: 120毫氯化锂，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES pH 7.4的 120mM氯化钠，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES pH 7.4的 125毫米氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES pH 7.4的</li></ol>对NHE反向激活2.3）测量注:此处的原理是反转跨膜离子梯度。 <ol><li>在测量之前，加载细胞与感兴趣的细胞内阳离子（见下文）。 </li><li>它们孵育5分钟，BCECF / AM（见步骤2.2.2）;冲洗一下，将它们安装在视频显微镜。 </li><li>灌注的加载溶液，并采取若干图像来记录一个稳定的基线。 </li><li>基线稳定，图像当细胞灌注细胞外酸性介质中含有120米pH值变化后，M氯化胆碱，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM MES pH为6.5。 </li><li>为氯化锂装载，在一个在pH7.4组成的120毫氯化锂，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES溶液孵育细胞2小时。用原子吸收光谱测定胞内锂产生的约60mM的值。 </li><li>对Na +的负荷，在1mM的哇巴因的存在下孵育细胞两个小时在120 mM氯化钠，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES组成的溶液在pH7.4挡住的Na / K ATP酶。在这些条件下，细胞内的Na +浓度为40毫摩尔的范围内。 注:K +负荷是不必要的，因为细胞内K +浓度为140mM的范围内和向外定向的K +梯度，因此容易实现。 </li></ol> 2.4）数据处理和校准: 注意:此步骤既适用于正向和反向测量。 <ol><li>在每个实验结束时，灌注细胞的140mM的氯化钾，20mM的HEPES和5μM的尼日利亚菌素溶液6.5和7.4之间调节至pH值。 </li><li>收集（代表亮度级的灰度级）的数据作为从图像中荧光测量和出口下的文本格式和下方使用电子表格程序当作说明。 </li><li>计算每个单独的细胞或使用以下等式关心区域内pH值: pH值= pKa值+（日志（R-Rmin的）/（R 最大 -R））X F 分钟（λ2）/ F MAX（λ2） </li><li>使用F 分钟（λ2）/ F 毫安×（λ2）的比例，如果有探针漂白或渗漏，从而导致在整个实验中的450纳米的荧光的减少。然而，这是非常罕见的，在沙漠中使用的条件下观察cribed实验。 </li><li>作为校准数据存在于每个pH测量结束时，检查相应的计算值是否与用于校准的pH值不同。如果是这种情况，则通过以下步骤调整所有实验确定的pH值下与校准: <ol><li>计算以下商数（Q，校准值之间的测量值/差分之间的差）。乘以整个数据集由Q设为通过加法或减法的最终调整，以使计算出的值将等于相应的校准值。 </li></ol></li></ol> 3.测量初始价格NHE7通过快速锂离子吸收<ol><li>多孔板种子细胞（6至24个孔），并使用任一所述的NH 4上方或交替的尼日利亚菌素/牛血清白蛋白（BSA）在20中所述酸化描述+加载技术酸化它们。 </li><lI&gt;冲洗板两次快速使用上述冲洗溶液，然后孵育细胞在含1-10毫锂（略低于Km值）的吸收溶液和阳离子的所需浓度和/或感兴趣的抑制剂所需量的时间。 </li><li>保持短和一致的吸收的持续时间（一般为一分钟或以下），以确保运输工作在初始速率条件下。另外，还要确保所有用于摄取的解决方案是等渗。 </li><li>在摄取时间结束时，小心地消除吸收介质并用冰冷的磷酸盐缓冲盐水（PBS）洗涤细胞4次。作为快速执行这些洗涤越好（低于10秒，其中的四个是理想的），以防止锂流出。 </li><li>裂解的细胞在25％的硝酸。应用250微升每25％的硝酸井并让它静置至少1小时，然后废每个孔与吸头的端部和在一个新的英里传送整个井悬浮crocentrifuge管。 </li><li>转移溶解物在1.5ml微量离心管中，离心它们在15000×g离心（室温）5分钟以除去细胞碎片。 </li><li>测量上清液用原子吸收光谱21的锂含量。 注意:不同的公司提供的原子吸收光谱仪，其中必须配备有一个锂空心阴极灯。按照制造商的说明，因为这些机器非常精确，但也相当细腻。 </li></ol>

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The authors have nothing to disclose.

这个协议描述如何选择细胞表达细胞内Na + / H +交换器在质膜而不通过定点诱变改变其一级序列。这些换热器现在可以表征。 该方法是基于细胞内的质子的细胞毒性来选择细胞系，将表达水疱的Na + / H +交换器在质膜。它是可能的，在原则上，以使用其它阳离子可怀疑被运输，例如Li +，K +或Cs +。然而，这种不确定性增加了一个?...

大部分胞内隔室显示酸性腔的pH值，这对于成熟，贩卖，回收蛋白质或激素和神经递质加载的一个关键参数。它已被证明是受液泡H + ATP酶1产生胞质溶胶和水泡性内容之间的pH梯度，耦合到水疱的ClC氯化物转运2。无论是在敲除（KO）小鼠和人患者，这些转运的重要性已经被强调在他们的基因3-6突变引起的重表型。 钠氢交换SLC9A家族的成员，也称为NHEs其Na + / H +交换剂，已被证明是关键效应物在细胞内的pH和调节细胞体积，以及在酸-碱当量的通过上皮的向量转运。除了 ​​质膜NHEs，三高度保守的Na + / H +交换剂，NHE 6,7和图9表示在反式高尔基网络和在早期内涵体7。在他们的基因突变已与安琪儿状或玮综合征8-9，以家庭为基础的自闭症10和多动症11-12。这些交换机还参与了神经退行性问题，如阿尔茨海默病的易感性13和X连锁智力低下基因连续证候14。两者合计，这些研究突出在脑发育和/或功能的这些细胞内NHEs的重要性。 这些交换机的细胞内定位防止其离子选择性精确的测量，传输方向，动力学参数，和监管。作为用于表达的细胞内区室的所有转运的情况下，这是非常困难的，以评估其生化活性，因此，以充分理解其生理作用和mechan主义的基本病理意义。基于高细胞内K +浓度时，最普遍接受的假设是，他们的工作为K +加上质子外流转运。这样的质子泄漏的存在已经被推测的，因为它可能会以维持稳定状态的pH水疱抗衡质子泵通过V-ATP酶。这种视觉本文的目的是（i）至展示出一种方法，其允许基因筛选表达这样水疱转运在它们的质膜，和（ii），以显示两个独立的方法来测量这些转运体的功能的细胞系。 三十年前，Pouysségur和弗兰基已经开创了一个，使该NHE家族15成员的分子克隆和鉴定遗传方式。这是基于对细胞内的质子作为筛选方法的毒性。第一步是获得的细胞系缺乏在任何的Na + / H +交换表示在质膜，使用这种转运的可逆性。成纤维细胞（CCL39细胞系）预装的Na +或Li +，然后置于酸性胞外培养基（pH6.5）中2小时。这导致了细胞的表达的功能性的Na + / H +交换的死亡，并逆向转运缺陷细胞（PS120细胞系）16的选择。当在碳酸氢盐的培养基培养，这些细胞是急性细胞酸化非常敏感。因此，在质膜的任何官能质子外排机制的表达将阳性选择（见17），如果这样的细胞被提交到急性细胞内acidifications。这样酸化技术可用于分离细胞系贩卖缺陷有利的WT细胞内NHEs的强制表达在细胞膜。 作为真核生物的Na + / H+交换是电中性的，他们是无法衡量由已经使用了巨大的成功来衡量通道电生理的方法。因此本手稿演示了如何通过细胞内pH值测量和锂的吸收快速动力学测量此热交换器的活性。作为基本概念是相同的，但有趣的是注意到，许多供选择部分开发的进程，也可直接用于功能测量。 有趣的是，我们已观察到，贩卖缺陷存在于使用本手稿描述的方法选择的细胞系将导致其它水泡性蛋白质在质膜的更大的表达，如水泡性钾通道TWIK1 18。这指出了朝向的一般保留缺陷机制水泡性跨膜蛋白的选择。因此，本次评选程序，它生成5月细胞构成一个有前途的工具，科学界上工作的细胞内区室的膜蛋白。以及这里介绍的测量技术可能适用于研究其他非电的转运。

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard cell culture equipement</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td>Used for H+ killing selection composed of many devices with different catalog numbers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator with CO2</td>
			<td style="width:119px;">Sanyo</td>
			<td style="width:119px;">MCO15A</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Incubator without CO2</td>
			<td style="width:119px;">Heraeus instrument</td>
			<td style="width:119px;">BB6220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laminar flow hood PSM1200NF</td>
			<td style="width:119px;">Fisher&#160;</td>
			<td style="width:119px;">52010120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM medium</td>
			<td style="width:119px;">sigma</td>
			<td style="width:119px;">D5796</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS gold</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:119px;">A15-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicilin/streptomycin</td>
			<td style="width:119px;">PAA</td>
			<td>P11-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin 10X</td>
			<td style="width:119px;">PAA</td>
			<td style="width:119px;">L11-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Atomic Absorption spectrometer with Zeeman furnace system</td>
			<td style="width:119px;">Thermo Scientific</td>
			<td style="width:119px;">ICE 3500 GFZ</td>
			<td></td>
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			<td style="width:119px;">L4408</td>
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			<td style="width:119px;">sigma</td>
			<td align="right" style="width:119px;">438073</td>
			<td></td>
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			<td></td>
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			<td style="width:119px;">Leica</td>
			<td style="width:119px;"></td>
			<td>Composed of many devices with different catalog numbers</td>
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			<td style="width:119px;">DMI6000B</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LED transmission light&#160;</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">8097321</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;"></td>
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			<td height="21" style="height:21px;width:216px;">motorized plate</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11522068</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">camera output</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888373</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888259</td>
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		<tr height="21">
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			<td style="width:119px;"></td>
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			<td></td>
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		<tr height="21">
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">images acquisition/analysis software</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">optics</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11506507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11506243</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11506203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fluorescence Xenon lamp</td>
			<td style="width:119px;">leica</td>
			<td style="width:119px;">DMI6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">camera</td>
			<td style="width:119px;">hamamatsu</td>
			<td align="right" style="width:119px;">8100601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">metafluor/Mmfluor software</td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11640905</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pH sensitive probe, BCECF-AM</td>
			<td style="width:119px;">life technologies</td>
			<td style="width:119px;">B1170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nigericin</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">N7143</td>
			<td></td>
		</tr>
	</tbody>
</table>

functional characterization of na+/h+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

内含体酸化是对广泛的过程，如蛋白质的回收和降解，受体脱敏，并装载神经递质在突触小泡的关键。此酸化被描述为通过质子ATP酶，其耦合到的ClC氯化物转运介导。高度保守的电中性的质子转运，所述的Na + / H +交换器（NHE）6,7和9还表示在这些隔室中。在他们的基因中的突变已与人类认知和神经变性疾病。奇怪的是，他们的角色仍然是难以捉摸的，因为他们的细胞内定位阻止详细的功能特性。这个手稿表示的方法来解决这个问题。这包括突变体的细胞系，能够通过在质膜保持细胞内NHEs幸存急性胞质酸化的选择。然后，它描绘了两个互补的协议来测量离子选择性和活性这些交换器:（i）一种基于使用荧光视频显微镜的细胞内pH值的测量，以及（ii）一种基于锂摄取的快速动力学。这样的协议可以外推到测量其他非电的转运。此外，这里介绍的选择过程生成的细胞与细胞内保留有缺陷的表型。因此，这些细胞也将表达其它水泡性膜蛋白在细胞膜。因此此处所描述的试验策略可能构成潜在有力工具，研究其它细胞内蛋白质，这将在质膜被再表达连同水疱的Na + / H +用于选择热交换器。

选择: 在H +杀选择是基于所述铵弱碱的扩散，如在图1A中所描绘。弱碱和酸对细胞内pH值扩散的影响已率先由Walter硼和合作者22。优雅的主意，使用这种现象产生一种致命的酸化积极基因筛选，然后由雅克Pouysségur17开发的。在这样的协议中，细胞内pH值降低至约5.5的漂洗步骤之后。细胞不表达功能性NHE在质膜不能恢复中性pH值，并显示具有平...

Watch this Scientific Journal Video about Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane at JoVE.c

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

娜功能分析<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt;细胞内的隔间使用质子打死选择器来表达它们的质膜

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

functional-characterization-na-h-exchangers-intracellular

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

9.2K 次观看.  Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics. The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers. 

视频: Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

科研

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

娜功能分析

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