The authors would like to thank Drs. C. Szalinski, H. Teng, and M. Bruchez for help with Imaris, R. Vistein, and D. Shiwarski for technical help and advice, and Dr. M von Zastrow, Dr. T Kirchhausen, Dr. D Drubin, and Dr. W Almers for reagents and helpful discussion. Funding provided by T32 grant NS007433 to SLB and NIH DA024698 and DA036086 to MAP.

在培养细胞中，使用荧光灯的1表达的标记CME组件 HEK293细胞是已被广泛用于研究G蛋白偶联受体的生物学和细胞内吞作用，并因此被用作本协议模型是有用的模型细胞。使用任何转协议提供无过度和低毒性统一的表达。 <ol><li>处理所有的细胞培养在无菌层流罩。填充4个孔的12孔板的1毫升Dulbecco改良的必需培养基（DMEM）中，用10％胎牛血清（FBS）中。从HEK293细胞的融合T25瓶中稀释1:50，1:25，1:16，1:10分别到每个4口井的。 <ol><li>或者，种子细胞的多孔板所需尺寸的，按照生产商的说明进行操作。种子0.4 - 2×10 4个细胞到12孔板。在无菌的37℃，5％CO 2和95％湿度下过夜生长的细胞。 注：考虑到增长Ř茨取决于条件和细胞系中，它可能是优选的种子多口井，以确保理想的汇合对于转染次日或一式两份进行转染的可能性。 </li></ol></li><li>第二天早上，选择一个好就是〜70％融合的转染（ 见图1B）。如果没有也已经达到这种状态，再等一天。 注：70％汇合是理想的HEK293细胞。转染细胞，更稀疏导致细胞死亡和毒性。转染过的细胞融合产生不善表达。 </li><li>加入60微升缓冲液EC（从转染试剂盒），400纳克质粒编码的标记的G蛋白偶联受体和/或网格蛋白机械的部件，和2.4微升增强的入1.5 ml离心管，如制造商的方案进行说明。涡流，持续2秒，以确保充分混合，并旋转在微量在2000×g离心2秒，以收集液体在底部。 </li><li>添加6微升的Effectene如制造商的方案进行说明。涡流，持续2秒，和自旋在2000×g离心2秒的离心以收集液体在底部。等待20分钟，以允许形成转染复合物的胶束。 </li><li>要转染从井抽吸介质。轻轻地用10％胎牛血清补充0.8毫升DMEM中的转染试剂混合物中，并通过吹吸混合和向下慢慢减少破胶束。从侧面媒体和转染混合物加入到孔中非常缓慢。从微量离心管中，加入剩余的转染混合物用微量的好。 注意：任选地，有10％FBS添加一个额外的0.5毫升DMEM中，以转染井得到的细胞产生平缓的转染和低瞬时表达额外的营养。 </li><li>返回信元到37℃培养箱中培养5小时。 </li><li>吸出介质含有转染混合物。替换用1ml的DMEM含10％FBS。允许细胞生长过夜。 </li><li>第二天，通过将细胞以未涂覆的盖玻片上，预先通过高压灭菌消毒。 <ol><li>用1mM EDTA的加入0.5毫升磷酸盐缓冲盐水（PBS）中的每个孔中。另外，使用加入0.5毫升0.05％胰蛋白酶-EDTA。留在培养箱中培养1分钟。 </li><li>将3轮，无菌25毫米盖玻片到6孔板的不同孔。各用2毫升的媒体以及填补。轻轻盖玻片向下推到井的底部，以防止细胞在盖玻片的底部生长。 </li><li>手动搅拌该井抬离细胞从井的底部。加入1 ml的DMEM含10％FBS重悬。从1井的12孔板的均匀分布之间的3盖玻片的解禁细胞。另外，板上3玻璃底菜细胞。 </li></ol></li><li>使细胞生长在盖玻片上的成像之前至少48小时。确保细胞被分散，持平。 </li></ol> 2。中共影像及货物动态使用TIRFM <ol><li>共轭M1抗FLAG抗体与根据生产商的方案使用的Alexa Fluor蛋白标记试剂盒Alexa染料。可选地，预共轭抗体可以买到。 </li><li>在1ml预热的Leibovitz培养基含10％FBS，或的Opti-MEM用50mM HEPES pH 7.4和10％FBS的10分钟，在37℃下千稀释（摄像：预孵育的细胞与抗体在1培养基）中，以标记细胞表面上的FLAG标记的G蛋白偶联受体。跳过这些步骤，如果基因编码的荧光蛋白，如GFP，作为标记（详见讨论）。 </li><li>盖玻片转移到活细胞成像室。另外，对于玻璃底菜，吸出抗体 - 标签介质和加入700μl预热的成像介质。 <ol><li>使用一对镊子和弯曲25号针头的尖端插入一个钩子。 </li><li>持钳子的惯用手。握住弯针，在OTH呃。转动针，直到钩子曲线向下朝玻璃。针，钩端轻轻的拖累，横跨6孔板的底部，直到它钩到盖玻片。小心不要划伤盖玻片的表面上，因为这将分离的细胞。轻轻提起，从井的底部的盖玻片起来。平衡靠墙井的支持盖玻片。 </li><li>使用镊子抓住盖玻片的边缘附近，并移动盖玻片细胞面朝上的成像室，和装配该室。添加预热的成像介质的700微升（或适量）。小心处理盖玻片没有压力，以防止开裂或断裂。 </li></ol></li><li>传送腔室到显微镜和使用阶段加热器或一个封闭的培养箱中保持电池的温度在37℃。 </li><li>使细胞成为焦点采用100X或60X全内反射荧光油浸物镜（NA 1.45或以上）。图像的细胞常规萤光或反对照明（ 即不TIRFM）的焦模式，以确定细胞中表达的合适的结构。失焦细胞是几乎看不见的薄深度TIRF照明使得难以找到焦平面和细胞。 注意：一些系统使用相同的激光器TIRFM图像在常规落射荧光，通过使上述临界角的激光的入射角度。作为一个重要的安全起见，总是知道激光束的角度，并总是指示它远离观察者。 </li><li>聚焦下降到一个表达细胞的底表面至质膜的细胞周围的轮廓消失且细胞的中心出现。这是一个具有细胞膜局部货物蛋白最明显的，例如μ-阿片受体（参见图2A）。 </li><li>切换到全内反射荧光成像。 <ol><li>如果使用相同的激光器进行成像在常规落射荧光，增加的入射角激光直到离焦的荧光在细胞中的内消失，并且在细胞周围质膜没有轮廓被看见。 （比较图2C到图2A和2B） 请注意：在TIRFM，移动焦平面使得图像去完全失焦及/或暗淡的，并且整体荧光水平由于照明的小深度而减小。因此，另一种方法切换到TIRFM照明是第一焦点上的共焦/常规落射荧光的小区的中心，然后，直到失去大部分的荧光的增加入射角，然后集中向下到质膜。 </li><li>一旦在TIRFM，确保激励用激光反射回来通过物镜而不是上面的目标可见。通过检查，如果激光点是可见的确认。 注：在传统的萤光或共聚焦方式，激光照排高于目标可见，如在天花板上。 </li><li>完善重点以获得清晰的图像。内吞机器的主要部件，如网格蛋白，将会在泪点可见。调整精细的焦点，所以泪点的圆形越好。 注：对于膜局部货，调整精细对焦，直到丝状伪足出现截然不同。 </li><li>使用采集程序来保存所有的成像参数，如反射镜和全内反射荧光的角度。做到这一点的所有必要的波长。 </li></ol></li><li>扫描周围盖玻片发现细胞中表达的所有期望的内吞标记：μ-阿片类受体和适配器β-arrestin的在图3A中所示的例子。选择那些表达，但不能过度表达细胞。详情请参阅讨论。 </li><li>一旦细胞被识别，采集图像，每3秒进行10分钟。这足以捕获中共的寿命，因为中共在HEK293细胞在这些条件下成像的平均寿命是AROUND 40秒10-11。这使得约12-14帧，观察中国共产党。例如看电影1。重复所有步骤的图像更多的细胞。 </li></ol>吞动力学的人工验证3。分析虽然中共寿命的人工验证大大由可检测的CCP的数目的限制，它仍然检测他不顾在图像和检测工件全球变化的精确装置。详情请参阅讨论。 <ol><li>打开ImageJ的文件。影像储存为TIFF文件交错渠道单一堆栈。图像转换为hyperstacks。去图像&gt; Hyperstacks&gt;栈Hyperstack。输入获取的信道数目（ 即 ，如果摄像β-休止和μ-阿片受体，输入2），输入1用于Z堆栈（TIRFM是一个单一的平面），和电影的帧的数量（ 即 10分钟电影的一帧，每3秒í前200），在弹出的窗口中。 </li><li>该hyperstack格式产生两个滚动条的窗口。使用顶栏移动通过渠道和底部的移动时间。滚动到其寿命将被检测的蛋白的通道。 </li><li>移动到第10帧或15中的电影的降低夹杂物预先存在的CCP的整个持续时间是不可见的。画一个ROI框周围随机选择一个点。 </li><li>计数帧的数目的点是可见的。 注：请参阅图3B和3C的三个形态类别内吞事件10,11,16-19的例子。 </li></ol>吞动力学的客观认识四分析目标识别可以检测几乎所有的细胞被成像的CCP的，但也可以是容易出错由于杂散检测错误的结构。有关详细信息，权衡的优点和电子商务的利弊讨论，请参阅ACH法。几个方案，包括定制的算法，可以用来客观地检测的CCP。本协议描述的目标识别使用了Imaris，图像分析软件（见材料 ）的关键控制点。 <ol><li>首先，打开图象分析软件的原始图像文件。默认情况下，会出现一个合并的彩色图像。使用“显示调整”窗口调整通道的亮度。点击一个频道的名称更改显示的颜色。取消选中旁边的通道，以防止其显示的框（ 见图4A）。 </li><li>点击橙色斑点的图标，创建“景点”。 如图4B所示 。选择感兴趣区域（ROI）的单元格周围的区域。参见图4C。使用投资回报率，以排除会不正确地检测明亮前缘和斑块区域。分析多个细胞与分别不同的表达水平。 </li><li>测量点的直径PROVI德初步估计算法。切换到“切片”模式，然后点击一个点的极两端测量其直径。参见图4E。做到这一点的4或5圆斑，看起来指示中共在其稳定的高度，形成后断裂之前。使用这些测量结果来计算平均直径降低到微米的最近的十分之一。 </li><li>检测到的景点。返回到“超越”模式。选择适当的渠道进行检测。输入测得的平均直径通常为0.3或0.4微米。点击蓝色箭头向前量化的景点。参见图4E。如果斑点的直径被低估，荧光假点将被认定为斑点。如果直径过大，真正斑点将被忽略。如果不能确定，估计低一点，后来筛选出不正确的检测。 </li><li>通过质量过滤景点。调整过滤器捕捉到尽可能多的景点尽可能不回来地面上。质量过滤器默认选择20。参见图4F。 <ol><li>使用“质量”的曲线为指导，以设置合适的阈值。将过滤器与鼠标左键的底部门槛的曲线这首畅游。这是一个很好的起点，过滤器，因为这个位置通常是提炼检测到唯一可见的斑点。参见图4F。 </li><li>检测点出现在电影的领域或中心像素。使用“颜色”选项卡中，调整斑点的颜色为对比色，使之更容易看到它们。滚动的电影审查每一帧的景点。我们的目标是检测尽可能多的斑点尽可能为他们的一生的全部。 </li><li>消除暗斑或手动将它们连接成连续的磁道以后，因为它们不被检测以及通过他们的一生的过程。 </li><li>与“强度”FIL删除明亮斑块之三，如果需要的话。点击绿色加号添加一个新的过滤器。参见图4F。创建一个过滤器强度或退出一个给定的荧光信号景点进行筛选。使用生成的强度曲线，（类似于质量曲线在4.5中讨论）来设置门槛。用鼠标左键删除，代表最亮的景点曲线的极端左端。 注：大斑块结构通常是在细胞中最亮的元素中，并且它们容易排除与该过滤器。 </li><li>通过电影滚动，以确保关键控制点被检测为斑点，并且不再检测出最明亮的斑块。 图3D示出了细胞，其中检测的CCP（由白球表示）进行了优化，以质量过滤器和斑块被排除与质量过滤器。 </li><li>减少与品质的过滤器明亮小区边缘。明亮的物体可能仍然需要手动删除，在下一步骤。继续与蓝箭头。 </li><li>删除在编辑斑点屏幕异常斑点。切换到“选择”模式。点击当场。它会变黄。单击“删除”。点击蓝色箭头继续建设的算法。参见图4G。 </li></ol></li><li>测量轨道。设置轨道“布朗运动”。中共应该不表现出任何定向运动，穿过质膜只有最小的漂移。这个算法是最适合该运动。参见图4H。 <ol><li>设定的最大距离为较小的值，如0.7微米。参见图4H。 注：设置一个小的距离最小化邻近景点的连接，仍然允许在小区现场，通过中共或细胞运动的持续跟踪。 </li><li>设定最大间隙大小为1。这样的轨道继续，如果只有一帧相继失踪。点击蓝色箭头来计算轨道。参见图4H。 备注：G型超过3的原因不同轨道AP尺寸被错误地连接在一起。 </li><li>通过电影观测质量检测开关轨迹看，以“神龙摆尾”。滚动。如果邻近的景点都被连接，降低了最大距离低于0.7微米。如果景点都被打破了太容易，增加最大间隙尺寸。点击蓝色箭头创建音轨。这导致滤波器的轨道平面。参见图4H。 </li></ol></li><li>滤波道和导出数据。请使用“强度”过滤除去额外的明亮斑块。使用“位移”滤镜移除了进入全内反射荧光领域内体。请小心，因为较长的景点都会有较长的位移为好。点击绿色的双箭头，完成协议。参见图4I。 <ol><li>使用编辑曲目选项卡，用铅笔图标，以消除无法通过过滤器除去轨道误报检测。检查discontinuities或虚假连接。要连接两条轨道，选择它们使用Ctrl-左键单击，然后在编辑曲目中单击“连接”。要断开曲目到不同的地点，选择曲目，然后点击“断开连接”。选择多点用Ctrl +左键单击，然后单击“连接”来创建一个新的轨道。该方法是相同的编辑斑点，在4.5.7中描述。参见图4J。 </li><li>要导出的数据，到数据卡。点击单个软盘图标导出选定的统计信息。单击看上去像一系列软盘导出所有数据的图标。 “轨迹播放时间”统计量包含内吞轨迹的长度。参见图4K。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这里，我们描述了使用TIRFM的可视化网格蛋白介导的内吞作用（CME）以各自的CCP的电平在实时活细胞。 CME是由许多在空间和时间上分开的单个事件的累积效应介导的快速和高度动态的事件。使用表面生物素化或配体结合，流式细胞术或固定的细胞测定法测定内化的蛋白质，或蛋白质的电子显微镜定位的量目前使用最测定法，如内在的生物化学测量，监视在固定的时间点集合的变化在蛋白质定位?...

网格蛋白介导的内吞作用（CME）的过程是依赖于网格蛋白介导的内吞机制的许多组分的良好的定时到来时，收集货物和操纵质膜进入囊泡1-3。 CME是由细胞膜变形，货物适配器蛋白走到了一起，在1内吞作用的新兴网站开始。这些蛋白质招募外壳蛋白网格蛋白，其组装成笼状结构，其形成的笼形蛋白包被的坑（CCP）4。一旦中共完全组装成球形的形状，膜的断裂，主要是通过大量的GTP酶，dynamin上的动作，产生游离网格蛋白包被小泡（校准检验）5,6。此内化触发网格蛋白涂层的快速拆卸，从而可以再次用于多个回合CME的组件。 参与CME蛋白的发现和鉴定已植根于传统biochemical，遗传和显微技术4-6,8。这些实验已经阐明这些元件内吞作用和交互点。虽然用于限定贩卖机的必要成分非常有用，这些测定法是高度限定在捕获CME部件或货物浓度的动态行为。这是一个重要的限制，因为CME是由套蛋白的模块中定义的步骤的编排组件驱动，并且由于在个体内吞事件的动态微小变化可对细胞内吞作用大累积的后果。另外，最近的数据表明，单独的CCP可能不同无论在组成和行为，这表明该过程的生理调节高度空间和时间上的限制9-14。可视个人的内吞的事件，因此，关键是要理解为什么有参与CME的多个冗余的蛋白质，以及这些蛋白可能被控制bŸ生理信号来调节货物的内在。 在这里，我们描述了使用全内反射荧光显微镜（TIRFM）的个体的CCP的活细胞中的动态水平观察CME。 TIRFM依赖于玻璃盖玻片和细胞15,16的流体环境之间折射率的差异。当激发光被导向所述细胞在超过临界角，它是在内部反射，创建维护薄照明的领域延伸的盖玻片上约100nm的渐逝波。这确保了只有在这个狭窄领域的荧光分子被激发。实际上，这允许上或附近的细胞膜荧光分子的激发，并从该单元的内部部件最小化荧光。这提供了一个显著更高的信号 - 噪声比，并沿z轴的分辨率来可视化在质膜，共同事件mpared更常用的模式，诸如传统的表面荧光或共聚焦荧光显微术。我们还描述，在一个介绍和实际操作层面，采用了常用的图像分析平台，分析和定量简单的形态特征和个别货物吞事件的动态。

<table border="0" cellpadding="0" cellspacing="0" width="909">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:207px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">DMEM/High Glucose with L-glutamine and sodium pyruvate</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>SH3024301</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS), no calcium, no magnesium</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td>21600-010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">EDTA Free Acid</td>
			<td style="width:180px;">Amresco</td>
			<td style="width:157px;">0322-500G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Fetal Bovine Serum</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td style="width:157px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Opti-MEM</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">HEPES</td>
			<td style="width:180px;">CellPURE by Fisher Scientific&#160;</td>
			<td>BP2937-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Effectene&#160;</td>
			<td style="width:180px;">Qiagen</td>
			<td style="width:157px;">301425</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">25 millimeter coverglass</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>12-545-86&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Corning cell culture treated flasks, 25cm2</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>10-126-28</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Cell culture 6-well plate</td>
			<td style="width:180px;">Greiner Bio-One, by VWR</td>
			<td>82050-896</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Monoclonal ANTI-FLAG M1</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td>F3040-5MG</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">[D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO)</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td>E7384-5MG</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Alexa Fluor 647 Protein Labeling Kit</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:157px;">A20173</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">ImageJ</td>
			<td style="width:180px;">NIH</td>
			<td style="width:157px;"></td>
			<td>http://rsb.info.nih.gov/ij/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Imaris Image analysis software</td>
			<td style="width:180px;">BitPLane</td>
			<td style="width:157px;"></td>
			<td>http://www.bitplane.com/imaris/imaris, for automated analysis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Nikon Eclipse Ti inverted microscope and required accessories including filter cubes and filters</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Nikon TIRF arm with required adapters for Nikon Eclipse Ti</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:157px;"></td>
			<td>For adjusting angle of incidence</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">iXon+ EMCCD camera and adapters</td>
			<td style="width:180px;">Andor</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

使用活细胞全内​​反射荧光显微镜，我们已经记录了μ-阿片受体（MOR）的内吞动态，G蛋白偶联受体（GPCR）和它的内吞接头蛋白β-arrestin的。的β-抑制蛋白的构建体瞬时转染使用图1中所概述的协议的稳定表达MOR细胞系，并成像的96小时以后。铁道部在稳定细胞系是N端标有pH敏感的绿色荧光蛋白。该荧光蛋白只发荧光的中性细胞外液。另外，MOR仅endocytoses一次通过的激动剂活性，并保?...

Watch this Scientific Journal Video about Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy at JoVE.com

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

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 ...

visualizing-clathrin-mediated-endocytosis-g-protein-coupled-receptors

Research

JoVE Journal

Biology

80.1K 次观看.  Carnegie Mellon University. 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. 

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

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

科研

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

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 ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

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 ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

使用荧光显微镜可视化中的细菌线虫

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

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 ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

在Cytokineticž圈的活细菌超分辨率成像使用快速三维结构照明显微镜（F3D-SIM卡）

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

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 ...