The authors of this paper would like to thank Prof. Jiayan Xie for technical assistance and equipment usage. This work was financially supported by the National Natural Science Foundation of China (41406137).

1.细胞的制备<ol><li> 细胞恢复 <ol><li>转移冷冻保存的人胚胎肾293（HEK293）细胞从液氮罐到37℃水浴中，并用于1连续摇 - 2分钟。 </li><li>加入10 mL的平衡的生长培养基（Dulbecco改良的Eagle培养基（DMEM），10％胎牛血清（FBS）和1％青霉素 - 链霉素溶液），以10厘米的细胞培养皿中。轻轻将解冻的细胞添加到平衡生长培养基。 </li><li>孵育细胞在37℃的水夹套CO 2培养箱用含95％空气和5％CO 2 2的潮湿气氛中- 4小时。 </li><li> 2个后 - 4小时，检查该细胞附着。替换另外24平衡的生长培养基和培养 - 48小时。 注意:因为它消除二甲亚砜（DMSO），并防止细胞损伤改变平衡生长介质是有用的。 </li></ol></li><li> &lt;强&gt;细胞通道<ol><li> 24个后 - 48小时，当细胞已经生长到接近汇合，弃去平衡生长培养基并用2ml磷酸盐缓冲盐水（PBS）洗涤细胞。轻轻摇动菜。 </li><li>弃去PBS和加入1毫升胰蛋白酶-EDTA（盐水，0.25％胰蛋白酶和0.02％EDTA）。以完全消化细胞，小心摇动培养皿中并吸脱胰蛋白酶-EDTA溶液。放于37℃的水夹套CO 2培养箱培养皿3分钟。 注意:在此期间，准备了三个新的10cm细胞培养皿，并添加10毫升平衡生长培养基到每个。 </li><li>使用光学显微镜，确定是否在细胞已经从培养皿的底部升起。加入2毫升平衡生长培养基的培养皿中。通过移液混合，并立即吸管0.6毫升细胞悬浮液的每一个的新菜肴。轻轻用移液管混合并允许细胞在37℃和5％CO 2孵育。 </li></ol></l我&gt; </ol> 2.细胞接种<ol><li> 24个后 - 48小时，当传代的细胞已经生长到接近汇合，弃去平衡生长培养基，并用2毫升的PBS洗涤细胞。轻轻摇动菜。 </li><li>弃去PBS和加入1毫升胰蛋白酶-EDTA。以完全消化细胞，小心摇动培养皿中并吸脱胰蛋白酶-EDTA。放于37℃的水夹套CO 2培养箱培养皿3分钟。 注意:在此期间，加入2毫升平衡生长培养基到每个孔中制备一个6孔板中。 </li><li>加入1毫升平衡生长培养基的菜，如果，在显微镜观察下，将细胞已经从培养皿底部升起。通过移液混合，并立即吸管0.1毫升细胞悬浮液（体积根据细胞生长密度调节），以所制备的6孔板（用2mL平衡生长培养基的细胞较少6孔板）的每个孔中。轻轻吹打混合，并允许细胞在37℃下温育和5％CO 2。 </li></ol> 3.细胞转染<ol><li> 转染方法1，用脂质体制剂 注:请参阅材料的表为转染试剂1。 <ol><li>次日，确保细胞应该是85 - 95％汇合。 </li><li>添加质粒DNA（500毫微克/微升）和100μL减少血清培养基中的3微克到无菌微量离心管中，并轻轻混合。孵育在室温下5分钟。 </li><li>制备9μL试剂1和在另一微量离心管中加入100μL低血清培养基中，并轻轻混合。孵育在室温下5分钟。 </li><li>结合用稀释的试剂1（总体积= 215微升）稀释的质粒混合物。轻轻混匀并孵育在室温下20分钟。 </li><li>吸出培养基断细胞培养板，并加入1.5毫升新鲜不含FBS的DMEM。 </li><li>添加DNA-TRANSF的215微升挠度试剂混合物到每个孔中的溶液，并通过旋动板轻轻混匀。 </li><li>孵育在37℃和5％CO 2的4 - 8小时。用新鲜的含10％FBS更换。 </li><li>孵育细胞在37℃和5％CO 2的另一个24 -运行的任何实验之前48小时。 </li></ol></li><li> 转染方法2，用脂质体制剂 注:请参阅材料的表为转染试剂2。 <ol><li>添加质粒DNA的2微克（500毫微克/微升），试剂2 4μL，和100μL减少血清培养基到无菌微量离心管中。轻轻混匀并孵育在室温下15分钟。 </li><li>将转染混合物加入到细胞中以逐滴方式。 </li><li>转染后，孵育细胞24 -在37℃下48小时和5％CO 2，在运行任何实验之前。 </li></ol></li></ol> 4.在盖玻片上的细胞培养<ol><li>回覆移动从培养6孔板的生长培养基中。 </li><li>洗用1mL PBS的细胞单层没有钙和镁。 </li><li>添加0.5毫升胰蛋白酶-EDTA。小心摇动培养皿中并吸弃胰蛋白酶-EDTA。孵育培养板在37℃下3分钟和5％CO 2。实际的培养时间与所使用的细胞系不同而不同。 注意:在此期间，准备一个新的12孔板，并添加直径15mm无菌盖玻片到每个孔中。涂有赖氨酸，层粘连蛋白或胶原盖玻片可以改善HEK293细胞容易脱离细胞附着。 </li><li>当细胞被分离，添加预热的1毫升完全生长培养基到每个孔中。通过移液混合，并立即将所需数量的细胞与盖玻片转移至12孔板。新鲜的完全的生长培养基添加到每个孔中以调节总体积至1mL </li><li>孵育细胞16 -在37℃下24小时和5％CO 2。 注:30后 -60分钟后，大部分细胞将已经粘附到无菌盖玻片上。 </li></ol> 5.共聚焦显微镜检测<ol><li> 细胞定位 <ol><li> 16个后 - 24小时，当细胞已经生长到接近汇合于在12孔板中的无菌盖玻片，添加膜探针的DiI，向每个孔中，得到的5μM的终浓度。使细胞温育在37℃下30分钟和5％CO 2。 </li><li> 30分钟后，取出平衡生长培养基并在冷PBS中洗涤细胞两次（2 - 8℃）。 </li><li>通过加入1毫升4％的多聚甲醛溶液（在PBS中）到每个孔中固定细胞。轻轻搅动15分钟。 注意:多聚甲醛是通过皮肤接触或吸入有中等毒性，并指定为可能的人类致癌物。化学品通风柜，排出平衡外壳或其它保护性措施应的多聚甲醛的称重和处理过程中被使用。&lt;/ LI&gt; <li>洗固定细胞两次在PBS中。添加200-300 DAPI（4&#39; ，6-二脒基-2-苯基吲哚）溶液（0.5微克/毫升）的μL到每个孔中，并孵育在室温下10分钟。 </li><li>用PBS洗细胞两次。小心地从板上取下盖玻片和灯芯掉过量的PBS。倒置到装载有安装平台（50％v / v甘油/水）一滴显微镜载玻片上。从载玻片移除过量的安装平台。 </li></ol></li><li> 细胞内 <ol><li> 16个后 - 24小时，当细胞已经生长到接近汇合于在12孔板中的无菌盖玻片，取出平衡生长培养基并加入新鲜的，温热的DMEM培养基（37℃）。孵育所述细胞在37℃下1个小时和5％CO 2。 </li><li>处理细胞用不同浓度（0，10 -12，10 -9，和10 -6 M）的配体的30分钟，并用10 -6 M的配体不同的时间（0对待它们，10，30和60分钟）。 </li><li>清洗细胞两次在冷PBS（2 - 8℃）。通过加入1毫升4％的多聚甲醛溶液（在PBS中）到每个孔中固定细胞。轻轻搅动15分钟。 </li><li>清洗细胞两次在PBS中。小心地从板上取下盖玻片和灯芯掉过量的PBS。倒置到装载有安装平台（50％v / v甘油/水）一滴显微镜载玻片上。从载玻片移除过量的安装平台。 </li></ol></li><li> 共聚焦显微镜 <ol><li>打开共焦显微镜的硬件，包括汞灯功率，PC显微镜，扫描仪和激光。登录到操作系统帐户，启动软件，检查配置和选择激光。按照说明书的顺序打开激光共聚焦显微镜系统的不同单元。 </li><li>放置制备玻片上的显微镜载物台上。在使用阶段控制器物镜样本位置。加上雪松油一滴被选择的油浸镜头时盖玻片。将盖玻片上面对镜头的幻灯片。 </li><li>找到感兴趣的细胞中的荧光显微镜下。 </li><li>切换到扫描模式。选择激光功率和发射荧光团的光谱范围。捕获由调谐激光强度和其它参数的高质量的共聚焦图像。 </li></ol></li></ol>

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The authors declare no conflicts of interest.

这里介绍的协议提供了一种有效和容易解释的方法来检测在HEK293细胞中的亚细胞定位和表达GPCR融合蛋白的内在化。该技术可以容易地适用于许多不同基因和细胞类型，诸如用于从家蚕在HEK293和家蚕细胞16 corazonin受体的细胞定位和内化。 这个功能强大的试验的成功应用依赖于几个关键因素。最重要的是细胞的身体健康，这是转染效率和数据质量至关重要。将转染的培养时间，转染和其它实验中，和转染的细胞的通过成像之前盖玻片之间的间隔，是所有的步骤，其中所述培养的细胞的损伤是可能的。因此，保持细胞的健康是在这个实验中的所有步骤的重要前提。此外，避免了在密集的培养物中紧密压缩细胞是为了获取具有共焦显微镜的高品质的图像的关键。为了尽量减少对GPCR的本地化的荧光标签的影响，我们产生用EGFP受体融合于细胞内的C末端。该基因的成功和高效转染到细胞也很重要。转染效率可以在不同细胞系变化很大。因此，在协议某些步骤的优化可能是必要的，这取决于所使用的特定细胞类型。例如，在转染方法1，在DMEM转染的细胞的不含FBS的培养持续时间可以根据小区条件进行了优化。除了DNA质粒的剂量，脂质体试剂的还原血清培养基的剂量，并应根据特定的细胞系进行调整。否则，electrotransfection技术可同时进行，而不是脂质体试剂介导的转˚F或在特定的细胞系上可接受的转染效率。然而，额外的硬件是必需的，并且在细胞悬浮液，并在小体积难以通过electrotransfection转染。当前测定的真实强度，但是，在于急性处理，如Sj的 GnRH受体的响应于Sj的促性腺激素释放激素配体的内在化后相同的培养条件下在相同的细胞的变化之间的比较。 内化是主要的机制控制GPCR信号，以确保适当的细胞应答的刺激11中的一个。我们可以很容易地通过激光共聚焦显微成像观察内在的变化。 Sj的 GnRH受体的以剂量和时间依赖的方式内化在图4中清楚地显现出来。 我们已经描述了该协议可以很容易地适应其它真核PR的亚细胞追踪oteins，如核雌激素受体17。然而，在该协议某些步骤的优化将是必要的，这取决于所使用的特定细胞类型，这将需要进一步的探索和实践。不过，考虑到蛋白的亚细胞定位对基因，蛋白质修饰的识别有很大的影响，和信号通路，这个协议的基础上，发表的研究，可以提供可靠的数据和信息。

细胞具有贩卖货物机械运送细胞外材料-如配体，微生物，营养素，和跨膜蛋白，成信息，能源和其他目的1，2中的单元格。它是细胞内环境稳定，组织的功能和整体细胞存活的关键。荧光融合蛋白的基于细胞的表达是一个功能强大的方法来研究的定位和跨膜受体，如G蛋白偶联受体（GPCR），内化在信号通路3。 从水母Aequorea victoria的标记有绿色荧光蛋白（GFP）的融合蛋白的表达，与直接荧光检测技术相结合，在已获得广泛的接受蛋白靶向研究4，5，6。基于GFP-detecti上具有允许活细胞，同时避免固定的工件7的实时成像的优点。一系列通用的克隆载体的已被构造成便于在各种细胞中8蛋白质融合到GFP的表达，9。的质粒pEGFP-N1载体是编码增强型绿色荧光蛋白（EGFP），它已被从野生型GFP优化用于在哺乳动物细胞更亮的荧光和更高的表达一种广泛使用的并商业化的质粒载体。观察EGFP的荧光信号在哺乳动物细胞中表达时，荧光显微术，应在488nm处激发和在507 nm处的发射，最好用共聚焦显微镜下进行。 监测的亚细胞定位和受体配体-介导的内是一种常用的技术来研究的GPCR 10的信号转导和功能</s向上&gt;，11。在大多数情况下，内化导致的GPCR脱敏（尽管激动剂的存在下的刺激反应的衰减）12，13。内化受体可以结合到溶酶体和降解，或它们可以被再循环回到细胞表面，这取决于受体和在实验中使用的细胞系的性质。 促性腺激素释放激素受体（GnRHRs）属于GPCR家族，并具有典型的GPCR蛋白结构，具有细胞外氨基末端，一个细胞内-COOH终端，和七个跨膜（TM）结构域14。重组质粒Sj的 GnRH受体-EGFP的转染（从曼氏粳稻的促性腺激素释放激素受体基因）和EGFP标记的GnRH受体Sj的 （在HEK293细胞中表达）的共焦显微镜检测报道上10sly和GFP荧光确立为跨膜蛋白定位试验15个记者。现在，SJ GnRH受体的内在化，通过S.粳稻促性腺激素释放激素（GnRH的Sj的 ）激活，表现在通过共聚焦显微镜时间和剂量依赖性。

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			<td height="21" style="height:21px;width:300px;">HEK293 cell line&#160;</td>
			<td style="width:236px;"></td>
			<td style="width:119px;"></td>
			<td style="width:203px;"></td>
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			<td height="21" style="height:21px;width:300px;">DMEM/High glucose</td>
			<td style="width:236px;">Hyclone</td>
			<td style="width:119px;">SH30022.01</td>
			<td>High glucose 4.0 mM L-glutamine</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Foetal Bovine Serum (FBS)</td>
			<td style="width:236px;">PuFei</td>
			<td style="width:119px;">1101-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:236px;">Genom</td>
			<td style="width:119px;">GNM10010</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Penicillin-Streptomycin</td>
			<td style="width:236px;">Genom</td>
			<td style="width:119px;">GNM15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">0.25% Trypsin &#38; 0.02% EDTA</td>
			<td style="width:236px;">Genom</td>
			<td style="width:119px;">GNM25200</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Opti-MEM (1X)</td>
			<td style="width:236px;">GIBCO, by Life Technologies</td>
			<td style="width:119px;">31985-062</td>
			<td>reduced serum media</td>
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		<tr height="24">
			<td height="24" style="height:25px;width:300px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:236px;">Invitrogen</td>
			<td style="width:119px;">11668027</td>
			<td>Reagent 1</td>
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		<tr height="24">
			<td height="24" style="height:25px;width:300px;">X-tremeGENE HP DNA Transfection Reagent</td>
			<td style="width:236px;">Roche</td>
			<td style="width:119px;">6366236001</td>
			<td>Reagent 2</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:300px;">DAPI Staining Solution</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1006</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">DiI</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Antifade Mounting Medium</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">P0126</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:300px;">Paraformaldehyde</td>
			<td style="width:236px;">Sinopharm Chemical Reagent Co.(SCRC)</td>
			<td style="width:119px;">80096618</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">100 mm cell culture</td>
			<td style="width:236px;">CORNING</td>
			<td style="width:119px;">430167</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">6-well plate&#160;</td>
			<td style="width:236px;">ExCell Bio</td>
			<td style="width:119px;">CS016-0092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">12-well plate&#160;</td>
			<td style="width:236px;">ExCell Bio</td>
			<td style="width:119px;">CS016-0093</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Cover Glass</td>
			<td style="width:236px;">NEST</td>
			<td style="width:119px;">801007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Microscope Slides</td>
			<td style="width:236px;">Citoglas</td>
			<td style="width:119px;">10127105P-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Thermo Scientific Forma CO2 Incubator</td>
			<td style="width:236px;">Thermo</td>
			<td style="width:119px;">3111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">TCS SP5II laser scanning confocal microscope</td>
			<td style="width:236px;">Leica&#160;</td>
			<td style="width:119px;">5100001311</td>
			<td></td>
		</tr>
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detection of ligand-activated g protein-coupled receptor internalization by confocal microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

图1显示了共焦显微镜系统的一个例子。 图2呈现的pEGFP-N1的在HEK293细胞中的表达。在细胞质和细胞核中检测到GFP信号。 图3显示了促性腺激素释放激素受体的从国槐在HEK293细胞中的亚细胞定位，与我们先前公布的结果一致15。该融合蛋白与GnRH受体Sj的 （Sj的 GnRH受体-EGFP）的C末端的EGFP标签在这个实验中出现绿色CLSM下时。用DAPI将细胞核揭示了蓝色。的DiI染色以红色突出显示的细胞膜。合并后的图像中的细胞表面上的黄色信号所表示的红色和绿色的重合，表明Sj的 GnRH受体的质膜定位。 图4和5显示在HEK293细胞中的Sj的促性腺激素释放激素受体的内化测定法的结果。为了显现Sj的 GnRH受体的内在化，将细胞与不同浓度的促性腺激素释放激素（Sj的促性腺激素释放激素）15处理30分钟（ 图4），或它们与单一浓度进行处理-为不同的持续时间（10 -6 M）（ 图5）。在没有Sj的促性腺激素释放激素的，控制（CTL）受体融合有质膜定位。 如图4中所示，在不同的配体浓度下，Sj的促性腺激素释放激素受体的内在化以剂量依赖的方式进行，并且该受体被完全从与10的细胞表面内化- -6 M的配体。 如图5所示，用10受体内在化的时间过程分析的结果- -6 M配体表明内化Sj的 GnRH受体可检测到的激动剂刺激后10分钟，通过发生60分钟极端内化。观察这两个数字共聚焦显微镜显示，荧光Sj的 GnRH受体-EGFP融合蛋白在细胞膜主要定位并且是显着和迅速内化至细胞中响应于在HEK293细胞中的Sj的 GnRH的肽。 <img alt="图1" src="/files/ftp_upload/55514/55514fig1.jpg" /> 图1.一个共聚焦显微镜系统的一个例子。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55514/55514fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55514/55514fig2.jpg" /> 图2.在控制GFP蛋白的共聚焦显微镜的pEGFP-N1转染的HEK293细胞<html> 。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55514/55514fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55514/55514fig3.jpg" /> 在HEK293细胞中通过共聚焦显微镜Sj的 GnRH受体-EGFP融合蛋白的图3位置。用DAPI染色细胞核显示为蓝色。的DiI染色，以红色显示，突出了细胞膜。 SJ的GnRHR-EGFP融合蛋白显示为绿色。在合并后的图像，黄色表示红色和绿色信号的巧合。所有图像代表至少三个独立的实验。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55514/55514fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ig4.jpg" /&gt; 图4.内化在时程由共聚焦显微镜HEK293细胞中的Sj的 GnRH受体-EGFP融合蛋白的。与GnRH受体Sj的 -EGFP质粒转染的HEK293细胞通过活化处理用不同浓度的GnRH的30分钟。该控制的情况下是没有GnRH刺激（CTL）。所有图像代表至少三个独立的实验。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55514/55514fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55514/55514fig5.jpg" /> 图5.内在Sj的 GnRH受体-EGFP融合蛋白在HEK293细胞中通过共聚焦显微镜以剂量依赖方式的。与GnRH受体Sj的 -EGFP质粒转染的HEK293细胞通过活化处理，用10 - -6 M的配体不同的Durations。该控制的情况下是没有GnRH刺激（CTL）。所有图像代表至少三个独立的实验。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55514/55514fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy at JoVE.com

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

这个协议描述了在哺乳动物细胞中的G蛋白偶联受体（GPCR）的内化共聚焦显微镜检测。它包括基本细胞培养，转染，和共聚焦显微镜的程序和提供了一个高效和容易解释的方法来检测融合表达GPCR的亚细胞定位和内化。

配体激活的G蛋白偶联受体内在化的检测通过共聚焦显微镜

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

detection-ligand-activated-g-protein-coupled-receptor-internalization

Nanyang Technological University

Research

JoVE Journal

Biology

10.6K 次观看.  Zhejiang Ocean University. 这个协议描述了在哺乳动物细胞中的G蛋白偶联受体（GPCR）的内化共聚焦显微镜检测。它包括基本细胞培养，转染，和共聚焦显微镜的程序和提供了一个高效和容易解释的方法来检测融合表达GPCR的亚细胞定位和内化。 

视频: Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

科研

生物学

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

量化在G蛋白偶联受体激动剂活性

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...