Name: visualizing clathrin-mediated endocytosis of g protein-coupled receptors at single-event resolution via tirf microscopy
Uploaded: 2014-10-20T13:00:00.000+00:00
Duration: 12 min 40 s
Description: 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

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>

<ol>
	<li>McMahon, H. T., Boucrot, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+and+physiological+functions+of+clathrin-mediated+endocytosis.">Molecular mechanism and physiological functions of clathrin-mediated endocytosis.</a> Nature Reviews Molecular Cell Biology. 12 (8), 517-533 (2011).</li><li>Kaksonen, M., Toret, C. P., Drubin, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Modular+Design+for+the+Clathrin-+and+Actin-Mediated+Endocytosis+Machinery.">A Modular Design for the Clathrin- and Actin-Mediated Endocytosis Machinery.</a> Cell. 123 (2), (2005).</li><li>Taylor, M. J., Perrais, D., Merrifield, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+High+Precision+Survey+of+the+Molecular+Dynamics+of+Mammalian+Clathrin-Mediated+Endocytosis.">A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis.</a> PLoS Biology. 9 (3), (2011).</li><li>Ungewickell, E., Branton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+units+of+clathrin+coats.">Assembly units of clathrin coats.</a> Nature. 289 (5796), 420-422 (1981).</li><li>Bliek, A. M., Meyerowrtz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin-like+protein+encoded+by+the+Drosophila+shibire+gene+associated+with+vesicular+traffic.">Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic.</a> Nature. 351 (6325), (1991).</li><li>Hinshaw, J. E., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin+self-assembles+into+rings+suggesting+a+mechanism+for+coated+vesicle+budding.">Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding.</a> Nature. 374 (6518), (1995).</li><li>Merrifield, C. J., Perrais, D., Zenisek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+between+Clathrin-Coated-Pit+Invagination,+Cortactin+Recruitment,+and+Membrane+Scission+Observed+in+Live+Cells.">Coupling between Clathrin-Coated-Pit Invagination, Cortactin Recruitment, and Membrane Scission Observed in Live Cells.</a> Cell. 121 (4), 593-606 (2005).</li><li>Grabs, D., Slepnev, V. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SH3+domain+of+amphiphysin+binds+the+proline-rich+domain+of+dynamin+at+a+single+site+that+defines+a+new+SH3+binding+consensus+sequence.">The SH3 domain of amphiphysin binds the proline-rich domain of dynamin at a single site that defines a new SH3 binding consensus sequence.</a> The Journal of biological chemistry. 272 (20), 13419-13425 (1997).</li><li>Tosoni, D., Puri, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TTP+Specifically+Regulates+the+Internalization+of+the+Transferrin+Receptor.">TTP Specifically Regulates the Internalization of the Transferrin Receptor.</a> Cell. 123 (5), 875-888 (2005).</li><li>Puthenveedu, M. A., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cargo+Regulates+Clathrin-Coated+Pit+Dynamics.">Cargo Regulates Clathrin-Coated Pit Dynamics.</a> Cell. 127 (1), 113-124 (2006).</li><li>Soohoo, A. L., Puthenveedu, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+modes+for+cargo-mediated+control+of+clathrin-coated+pit+dynamics.">Divergent modes for cargo-mediated control of clathrin-coated pit dynamics.</a> Molecular Biology of the Cell. 24 (11), 1725-1734 (2013).</li><li>Posor, Y., Eichhorn-Gruenig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+endocytosis+by+phosphatidylinositol-3,4-bisphosphate.">Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate.</a> Nature. 10, (2013).</li><li>Mettlen, M., Stoeber, M., Loerke, D., Antonescu, C. N., Danuser, G., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+accessory+proteins+are+functionally+distinguished+by+their+differential+effects+on+the+maturation+of+clathrin-coated+pits.">Endocytic accessory proteins are functionally distinguished by their differential effects on the maturation of clathrin-coated pits.</a> Molecular Biology of the Cell. 20 (14), 3251-3260 (2009).</li><li>Loerke, D., Mettlen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cargo+and+dynamin+regulate+clathrin-coated+pit+maturation.">Cargo and dynamin regulate clathrin-coated pit maturation.</a> PLoS Biology. 7 (3), (2009).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-substrate+contacts+illuminated+by+total+internal+reflection+fluorescence.">Cell-substrate contacts illuminated by total internal reflection fluorescence.</a> The Journal of Cell Biology. 89 (1), 141-145 (1981).</li><li>Merrifield, C. J., Feldman, M. E., Wan, L., Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+actin+and+dynamin+recruitment+during+invagination+of+single+clathrin-coated+pits.">Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits.</a> Nature Cell Biology. 4 (9), 691-698 (2002).</li><li>Ehrlich, M., Boll, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+by+random+initiation+and+stabilization+of+clathrin-coated+pits.">Endocytosis by random initiation and stabilization of clathrin-coated pits.</a> Cell. 118 (5), 591-605 (2004).</li><li>Yarar, D., Waterman-Storer, C. M., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+actin+cytoskeleton+functions+at+multiple+stages+of+clathrin-mediated+endocytosis.">A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis.</a> Molecular Biology of the Cell. 16 (2), 964-975 (2005).</li><li>Rappoport, J. Z., Kemal, S., Benmerah, A., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+clathrin+and+adaptor+proteins+during+endocytosis.">Dynamics of clathrin and adaptor proteins during endocytosis.</a> American journal of physiology. Cell physiology. 291 (5), (2006).</li><li>Doyon, J. B., Zeitler, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+clathrin-mediated+endocytosis+revealed+in+genome-edited+mammalian+cells.">Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells.</a> Nature Cell Biology. 13 (3), 331-337 (2011).</li><li>Hopp, T. P., Prickett, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Short+Polypeptide+Marker+Sequence+Useful+for+Recombinant+Protein+Identification+and+Purification.">A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification.</a> Bio/Technology. 6 (10), 1204-1210 (1988).</li><li>Miesenb&#246;ck, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li><li>Saffarian, S., Cocucci, E., Kirchhausen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Dynamics+of+Endocytic+Clathrin-Coated+Pits+and+Coated+Plaques.">Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques.</a> PLoS Biology. 7 (9), (2009).</li><li>Yudowski, G., Puthenveedu, M., Henry, A., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cargo-Mediated+Regulation+of+a+Rapid+Rab4-Dependent+Recycling+Pathway.">Cargo-Mediated Regulation of a Rapid Rab4-Dependent Recycling Pathway.</a> Molecular Biology of the Cell. 1, 1-11 (2009).</li><li>Batchelder, E. M., Yarar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Requirements+for+Clathrin-dependent+Endocytosis+at+Sites+of+Cell-Substrate+Adhesion.">Differential Requirements for Clathrin-dependent Endocytosis at Sites of Cell-Substrate Adhesion.</a> Molecular Biology of the Cell. 21 (17), 3070-3079 (2010).</li><li>Mattheyses, A. L., Atkinson, C. E., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Single+Endocytic+Events+Reveals+Diversity+in+Clathrin,+Dynamin+and+Vesicle+Dynamics.">Imaging Single Endocytic Events Reveals Diversity in Clathrin, Dynamin and Vesicle Dynamics.</a> Traffic. 12 (10), 1394-1406 (2011).</li><li>Aguet, F., Antonescu, C. N., Mettlen, M., Schmid, S. L., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+analysis+of+low+signal-to-noise+images+link+dynamin+and+AP2+to+the+functions+of+an+endocytic+checkpoint.">Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint.</a> Dev Cell. 26 (3), 279-291 (2013).</li><li>Boucrot, E., Howes, M. T., Kirchhausen, T., Parton, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+of+caveolae+during+mitosis.">Redistribution of caveolae during mitosis.</a> J Cell Sci. 124, 1965-1972 (2011).</li><li>Bhaya, D., Davison, M., Barrangou, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+in+bacteria+and+archaea:+versatile+small+RNAs+for+adaptive+defense+and+regulation.">CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation.</a> Annual review of genetics. 45, 273-297 (2011).</li><li>Boch, J., Scholze, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+the+Code+of+DNA+Binding+Specificity+of+TAL-Type.">Breaking the Code of DNA Binding Specificity of TAL-Type.</a> III Effectors. Science. 326 (5959), 1509-1512 (2009).</li></ol>

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。这些现有的方法已经在鉴定蛋白质所必需的CME，但缺乏空间和时间分辨率相匹配CME或其他动态膜运输过程的生理尺度是非常有用的。这是很重要的，因为最近的证据表明，关键控制点是在异质性的蛋白质组成和动力学9-12，随着CME很可能迅速在空间上离散的方式调节。活细胞TIRFM提供给分析CME在空间上分离的单CCP的电平，在真实时间，在活细胞中的能力。 成功应用这一强大的分析都依赖于两个关键因素:细胞的健康和CME的参数的深入分析。对于标记的蛋白质和显微技术最大限度地减少光毒性前，适当的表达是至关重要的。细胞系中稳定表达的目的蛋白质是理想的，因为表达水平能够可靠地估计。我们标记的受体有两种N末端 ​​标记抗原决定簇或SPH标签21-25。虽然不是严格必要的，这两个标签系统促进CME的TIRFM因为受体可以选择性地在质膜上，在实验的开始进行检测。所需的额外的内吞组分也可以瞬时转染入这些稳定细胞株。在TRansfection协议这里应当指出使用TIRFM成像CME被最优化。第一，它是专为偶数和中等程度的表达。差表达式就必须较高的激光功率进行检测，并增加了风险为光毒性和光漂白。另外，由于该试验中记录了泪点为内化的CCP的消失，低信号和光漂白不利也分析。我们认为，在这种情况下，总的持续时间和/或成像的频率被降低。第二，对细胞的转染试剂的短潜伏期，转染和实验，并转染细胞的新鲜通路到成像之前盖玻片之间的间隔，都保证了成像的细胞处于最佳健康状态。第三，这确保了绝大多数网格蛋白结构是关键控制点，和网格蛋白或网格蛋白斑块不平坦的薄片。第四，本协议最小化的过表达转染CME组件，这一直显示Ñ ​​改变CME动态20,26。 用于成像具有低光毒性，它以优化成像系统获得最佳的分辨率和最大灵敏度是重要的。物镜和相机检测器尺寸的放大倍数应匹配以最小化不足或过采样和空的放大率。高数值孔径（1.45以上）的TIRF物镜应该用于收集光的最大可能的。我们获得的图像用电子倍增电荷耦合器件相机对高量子效率，低的曝光时间，以及快速读出速度。此外，为了确保该细胞的健康，他们被成像在Leibovitz的媒体，它是缓冲独立的CO 2，并补充了10％FBS，在一个完全封闭的腔室设置为37℃。因为TIRFM是高度敏感的，并使用能够检测甚至最小的环境光的高数值孔径物镜，关键是要创造一个封闭的成像环境自由从外部光和振动对可能扰乱细焦平面。 需要注意的是绝对的寿命和CME的大多数部件的动力学而变化很大程度上依赖于细胞类型，蛋白质的表达水平镀覆后，温度，天是很重要的，并在培养条件7,10,14,17,20其他变化， 25,27。这反映在使用这种技术，该测定法的一个明确的限制，在时间尺度和不同的组之间观察到的分布的巨大变化。另外，可能的是在细胞内，在成像的TIRFM的底表面的CCP，从顶表面23,25有不同的行为。虽然这是值得商榷的哪个面准确地代表了"典型"的细胞通过细胞外基质成分在组织包围，看到这种技术共产党动态的解释需要考虑这个电位差。该法的真正实力，因此，在于比较的变化在D关键控制点在相同的培养条件ynamics，在相同的细胞，急性操作，包括货物分子的聚集，例如响应于激活的GPCR之后。该比较允许对变化到相同的细胞正常化，从而控制了所有上面提到的可能诱发变异中共行为的参数。 我们通常采用手动和自动分析，如上所述，分析内吞事件的持续时间:人工验证，并使用了Imaris图像分析软件的目标识别。手工验证是劳力和时间密集的，因为它是由CCP的数目，用户可以计数的限制，但它被认为是最准确的技术资料。一个训练有素的人眼可以很容易地继续通过细胞的运动跟踪中国共产党，并改变形状或重点，准确地排除斑和缺陷像素。它也可以检测出中共形态变化的指示完成前夕NTS，如图所示为图3B和3C。这是必要的，然而，该分析仍然客观地这些限制范围内。这需要被一致地使用在所有的数据集的特定准则的定义。此外，我们通常分析以双盲的方式，其中所述图像的名称被加扰，以使条件仍然未知的分析图像的人的图像。自动检测， 例如使用"点"和"音轨播放时间"在了Imaris算法，可以用来检测大多数的内吞斑点在一个给定的电影，产生高的样本号。虽然许多选项，包括高度复杂的定制算法3,14,23,26,27，正在产生，这些方法的可靠性，检测正确的内吞事件，同时避免虚假检测仍然是最大的担忧。例如，在了I​​maris，细胞运动产生明亮的前缘，斑块，并出对焦框可以轻松甩开点检测算法，因为它有一种倾向，发现明亮的结构所组成完全的斑点。为了防止这些斑点从被连接到一个轨道，它们都必须被移除。单异常景点不在大结构，如斑可以使用编辑除去景点标签，而连接景点以后可能会使用编辑音轨选项卡中删除。对异常点也可以用自定义过滤器除去。例如， 图3D示出了用于限制避免包括在中共检测斑块的亮度滤波器。质量过滤器是用于删除错误的斑点另一个非常有用的过滤器。 "质量"是亮度的累积测量和形状，通过取点和高斯滤波的荧光强度的光点半径20的长度的¾找到。的质量曲线中发现的选定的过滤器的下方。它描述了斑点的检测数（y）的当质量一定值（x）。质量越低，越斑点检测。如果质量过滤太低，斑点将被检测到在没有可见荧光，反之，如果质量过滤器太高，只有最亮的斑点被检测到。沿走向更大的值（x）的曲线跟随;检测点的数量（Y）通常会大幅度下降。移动底部阈这一点。这是最低点以放置底部阈值。其他主要贡献者在TIRF场假点是移动到的小区的周边，并输入TIRF领域内体。一个强大的标准消除这些是点的移动， 即点随时间的位移。 CCP的移动速度非常小，除非作为整个单元的运动的一部分，而胞内体显示出快速的布朗或定向运动。较新的算法，而显著好转，仍在细化和优化3,14,23,26,27。由于这些会晤部门首长的日益成熟和可靠的，我们可以分析数百点或上千，而不必在每个点手工验证。然而，目前，我们认为这两项分析一起使用，以相互配合的精度。 我们所描述的协议，可以很容易地适应回答有关货物的内吞作用的许多问题。它可以被用来执行简单的共定位货物与CME部件，以及用于显示对细胞内吞机器由于特定激励特定组分的变化。该法还可以很容易地适应任何内吞过程中，如小窝28，即表明货物和上装部件的离散浓度，并在专门的细胞类型这些基本过程的研究。在未来，随着基因组编辑变得更加主流的20,29,30，我们预计这将成为标记组件的首选方法，以及动态和行为Ø˚F各种部件将被进一步细化。考虑到我们的细胞过程的认识已基本由基因，蛋白质修饰和interactomes的识别驱动的，我们在这里所描述的实验代表了查询，即下一个前沿领域之一。这些过程和机制的时​​空动力学的定义。

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><tbody><tr><td>DMEM/High Glucose with L-glutamine and sodium pyruvate</td><td>Fisher Scientific&amp;nbsp;</td><td>SH3024301</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS), no calcium, no magnesium</td><td>Gibco, by Life Technologies</td><td>21600-010</td><td></td></tr><tr><td>EDTA Free Acid</td><td>Amresco</td><td>0322-500G</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco, by Life Technologies</td><td>10437-028</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium, no phenol red</td><td>Gibco, by Life Technologies</td><td>21083-027</td><td></td></tr><tr><td>Opti-MEM</td><td>Gibco, by Life Technologies</td><td></td><td></td></tr><tr><td>HEPES</td><td>CellPURE by Fisher Scientific</td><td>BP2937-100</td><td></td></tr><tr><td>Effectene</td><td>Qiagen</td><td>301425</td><td>Transfection reagent</td></tr><tr><td>25 mm coverglass</td><td>Fisher Scientific</td><td>12-545-86</td><td></td></tr><tr><td>Corning cell culture treated flasks, 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Fisher Scientific</td><td>10-126-28</td><td></td></tr><tr><td>Cell culture 6-well plate</td><td>Greiner Bio-One, by VWR</td><td>82050-896</td><td></td></tr><tr><td>Monoclonal ANTI-FLAG M1</td><td>Sigma Aldrich</td><td>F3040-5MG</td><td></td></tr><tr><td>[D-Ala&lt;sup&gt;2&lt;/sup&gt;, N-Me-Phe&lt;sup&gt;4&lt;/sup&gt;, Gly&lt;sup&gt;5&lt;/sup&gt;-ol]-Enkephalin acetate salt (DAMGO)</td><td>Sigma Aldrich</td><td>E7384-5MG</td><td></td></tr><tr><td>Alexa Fluor 647 Protein Labeling Kit</td><td>Life Technologies</td><td>A20173</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>http://rsb.info.nih.gov/ij/</td></tr><tr><td>Imaris Image analysis software</td><td>BitPLane</td><td></td><td>http://www.bitplane.com/imaris/imaris, for automated analysis</td></tr><tr><td>Nikon Eclipse Ti inverted microscope and required accessories including filter cubes and filters</td><td>Nikon</td><td></td><td></td></tr><tr><td>Nikon TIRF arm with required adapters for Nikon Eclipse Ti</td><td>Nikon</td><td></td><td>For adjusting angle of incidence</td></tr><tr><td>iXon+ EMCCD camera and adapters</td><td>Andor</td><td></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，并保持另有均匀地分布在质膜。着眼于贴壁细胞质膜是坐在盖玻璃在激光共聚焦模式导致了填充有暗淡荧光的细胞。这是由一个聚焦于细胞，其中所述质膜仅看作是细胞周围荧光的环的中心不同。比较左面板和右面板中的图2A中 。切换到常规落射荧光或TIRF用不正确的角度将导致失焦荧光在同一细胞中变得显而易见， 如图2B所示 。当全内反射荧光的角度是正确的质膜非常鲜明，清晰，明亮，失焦荧光不再破坏了形象。比较图2C到图2A和2B。 图像清晰和尖锐，因为MOR是在粘合质膜，这在很大程度上是内TIRF字段。相反，β-抑制蛋白保留在细胞质池时尚未募集到内吞泪点，并且，因为它不是在TIRF字段出现混浊和失焦。一旦MOR由激动剂活性，β-抑制蛋白被募集到质膜，和两个β-抑制蛋白与受体重新分配到的CCP（ 图3A和电影1<html> β-arrestin的绿色，铁道部为红色，叠加为黄色）。 这些簇的寿命可以手动进行定量使用三个参数完成的内吞作用， 如图3B中所描绘。这些点表示关键控制点可以完全消失（也见图3C），开启和关闭"眨眼"或"夹断"更大的结构。后两种情况代表了在空间上太接近被解析为单独的事件的事件。在了Imaris斑点检测算法也是有用的量化关键控制点，如在图4中概述。 图3D表示使用了Imaris，过滤以除去非动态斑块结构之后的CCP检测。覆盖白色的球体代表检测点。不能被他们的整个生命周期检测调光点也被排除在"质量"过滤器。 "&gt; <img alt="图1" src="/files/ftp_upload/51805/51805fig1highres.jpg" /> 图1:转染过程的流程图。 （一）首先，播种各种稀释液（1:50 1:25 1:16，1:10），到12孔板。让细胞生长过夜。第二天，寻找一个很好的为约70％汇合，并均匀地分布，如图所示。这是将被转染的井（B）加入60微升的EC缓冲液和400纳克DNA导入微量离心管中。涡旋10秒，该液体旋到管的底部。加入2.4微升增强的。涡流，持续2秒和液体旋到管的底部。在室温下孵育3分钟。加入6微升的Effectene的。涡流，持续2秒和液体旋到管的底部。在室温下孵育20分钟，（C）慢慢混合0.8毫升DMEM中含有10％FBS的转染。加入细胞之前选择的好。孵育细胞5小时，在37℃。更换新鲜DMEM含10％FBS。 <img alt="图2" src="/files/ftp_upload/51805/51805fig2highres.jpg" /> 图2:寻找TIRFM领域。 （一）细胞膜成像在焦。左侧面板描绘重点放在了小区的中心细胞的共焦。质膜只看作是一个"环"的单元格周围。聚焦下到基质膜，毗邻盖玻片导致细胞填充在昏暗的荧光。质膜"环"不应该在较薄的TIRF字段可见（B）该基质膜以较浅的TIRF角度穿过样品制备常规落射荧光照明。这个照射从该单元的顶部的整个细胞和多聚焦荧光的存在（C）中的TIRF质膜。重点丝状伪足，有助于找到这架飞机。请注意，它是光滑平面上。比例尺是10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51805/51805fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/51805/51805fig3highres.jpg" /> 图3:可视化和量化的全内反射荧光吞事件。 （一）前，除了诱导细胞内吞作用的兴奋剂后表示μ-阿片受体（MOR）和β-休止的细胞。激动剂导致两个蛋白的内吞泪点的重新分配。比例尺为10μm（B）三种类型的内吞事件，可见通过可视休止动力学，与先前所描述的形态一致的。 "稳"，一是稳定的信号，即迅速消失。这是主要的类型，这表明中共出芽和离开TIRF字段。 "闪烁"，即闪烁的信号<html>以较低的信号，并重新出现在同一地点由于中共第二组装在同一地点。 "掐断"，管状突起脱落的斑点，当两个关键控制点太接近对方被解析为不同的点，一个是内吞。该框是2×2微米（C）可视MOR的网格蛋白介导的内吞作用在单事件分辨率。给出的值是表示对MOR内吞事件的主要部分的两个例子。框架是每3秒。该框是2×2微米（D）检测使用了Imaris个体内吞的事件。白球表示检测到景点的CCP。使用的是"质量"过滤器检测点进行了优化。而无法对他们的一生的全部检测到的调光斑点也被排除在外的质量过滤器。这被示为未检测到大斑块结构分别用"强"过滤器被删去。ig3large.jpg"目标="_ blank将"&gt;请点击这里查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/51805/51805fig4highres.jpg" /> 图4:采用了Imaris内吞活动的动态目标识别。 （一）调整显示窗口，用于在步骤4.1中所述来调整图像的显示。（二）开放的"点"算法建设者。构建器将显示在下面的窗口（C）的斑点算法构建的第一步。检查"跟踪景点（一段时间）。"检查"利益分部只是一个区域，"如果需要的话。（四）投资回报率选择画面。请参阅步骤4.2的 ​​详细信息（E）来定义一个测量点的直径需要在多个窗口。小组表示:切换使用的导航超越切片模式gation栏上方，点击一个点，在这里显示的测量直径，通常在最右边，在那里输入被测直径的极性端。见步骤4.3-4.4。（f）在4.5-4.5.3描述的现场质量过滤器。在4.5.7中所述（G）编辑点窗口。（H）测量跟踪和观察轨道窗口，在4.6中描述。（一） 4.7滤波道窗口描述。（J）编辑跟踪4.7.1中描述的窗口。在4.7.2中所述（K）保存数据画面。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51805/51805fig4large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51805/Movie1.avi" target="_blank">电影1</a> 。 <strong&gt;铁道部集群和内吞作用的活细胞全内反射荧光显微镜为例影片。β-arrestin的显示为绿色，铁道部显示为橙色。无论是蛋白定位到关键控制点的铁道部激动剂刺激前。一旦MOR被激活时，这两个β-休止和MOR集群中的CCP，视作不同的泪点可以分别分析了它们的性质。

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

Universidad San Sebastian

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

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

科研

生物工程

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

免疫与感染

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

神经科学

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

生物化学

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

使用全内反射荧光（TIRF）显微镜对脂质双层中的横向迁移率和离子通道活性进行单分子成像

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

用荧光显微镜分析膜受体扩散和簇簇的图像处理协议

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

生物学

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Source: Marie-Ana&#239;s, F., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54470">&#34;Phagosome Closure Assay&#34; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM).</a> J. Vis. Exp. (2016).
This video demonstrates a high-resolution total internal reflection fluorescence (TIRF) microscopy technique for real-time visualization of phagosome formation and closure during macrophage-mediated phagocytosis of IgG-opsonized red blood cells (RBCs) attached to the surface of a glass bottom dish. The macrophages extend pseudopodia around the RBCs and engulf them inside the phagosomes, detaching them from the glass surface.

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

摘要

探索更多视频

此视频中的章节

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

受体动力学通过单分子荧光显微镜的高分辨率时空分析

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学

使用全内反射荧光（TIRF）显微镜对脂质双层中的横向迁移率和离子通道活性进行单分子成像

用荧光显微镜分析膜受体扩散和簇簇的图像处理协议

利用pHluorin标记的受体监视亚细胞定位和贩运

基于 TIRF 显微镜的吞噬体形成和闭合可视化

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

摘要

探索更多视频

此视频中的章节

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

受体动力学通过单分子荧光显微镜的高分辨率时空分析

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

使用全内反射荧光（TIRF）显微镜对脂质双层中的横向迁移率和离子通道活性进行单分子成像

用荧光显微镜分析膜受体扩散和簇簇的图像处理协议

利用pHluorin标记的受体监视亚细胞定位和贩运

基于 TIRF 显微镜的吞噬体形成和闭合可视化

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

通过全内反射荧光（TIRF）显微镜在体外可视化肌动蛋白和微管耦合动力学