这项工作得到了NIH的资助AI071815（光大集团）和路线图奈米发展中心奖PN2EY016586（MLD的）。

1。标签GP120 在本协议中使用的荧光染料是有效的蛋白质结合，是充分斯塔比尔照片，显微成像，符合我们的显微镜激光器的激发波长。这三个条件必须得到​​满足时，选择标记蛋白的荧光分子。 <ol><li>交换他的6个标记的DH12（礼物，爱荷华州立大学博士迈克尔赵）的gp120蛋白缓冲使用无菌PBS离心过滤装置（30 kDa的截留分子量）。确保gp120的浓度不超过1毫克/毫升。注：他的6个标签从其他X4或R5热带株的gp120的蛋白质也进行了测试，现在可以买到从免疫技术。 </li><li>添加碳酸氢钠的pH值9.0蛋白质溶液获得50MM终浓度碳酸氢钠〜pH值8.5。 </li><li>胺反应的Alexa Fluor 488（AF488）荧光染料，这是二在的水ssolved在10倍摩尔过剩。这种混合物在室温下在黑暗中孵育1-2小时。 </li><li>以除去多余的染料，像以前一样使用离心过滤装置，并补充新鲜无菌PBS列。重复此步骤，直到所有的自由染料被删除（3-4 4毫升PBS洗涤）。降速，直到gp120的集中约1毫克/毫升。注：免费染料去除透析或离心过滤装置，只有当染料溶于水，或其他可用于凝胶过滤。 </li><li>每分子标记的蛋白质（蛋白质的F / P）来衡量的荧光强度，确定在适当的波长（例如，在纳米488 AlexaFluor 488）和蛋白质的荧光染料浓度（单位为μm）（我们使用1的NanoDrop分光光度计使用的蛋白质和标签的设置），然后除以这两个数字给你的F / P比。相同的程序被用于其他蛋白质，如ICAM-1和Cy5染料。注：其他类型的spectroph可用于otometers获得的蛋白浓度和荧光染料浓度获得的F / P比值，如果不可用的NanoDrop。 </li></ol> 2。流动细胞大会和双层的制备流动细胞和双层编 ​​制的一般程序已在以前14的详细描述。在这里，我们勾勒出具体的协议，准备上Bioptech流动池包含他6标签的gp120 DH12双层。对于涉及感染病毒或受感染的细胞的研究和小卷是必要的，因为有限的试剂，一次性Ibidi的流动室（粘的幻灯片，我0.2鲁尔），可用于并遵循相同的双向层制备过程的步骤2.4-2.9。为Ibidi流商会的信息，可从该公司网站， <a href="http://www.ibidi.com/service/display_material/CA_8p_EN_150dpi.pdf">http://www.ibidi.com/service/display_material/CA_8p_EN_150dpi.pdf</a> </A&gt;。 <ol><li>准备Pirhana溶液（45毫升硫酸（微量金属品位98％）和15毫升30％的过氧化氢​​）14。用塑料夹子，浸泡盖玻片在15分钟的Pirhana解决方案。 </li><li>转移支付单充满了皮卡，纯净水的烧杯，洗下运行2分钟（每面一分钟）水的每一个。地方机架晾干。 </li><li>组装Bioptechs FCSII商会作为先前所描述的14。 </li><li>脂质体的混合物，含有12.5％的Ni 2 +的螯合血脂捕获，并提出他的6-gp120的双层表面上16。脂质体混合物1μL滴加入到microaqueduct幻灯片，而不触及尖端的玻璃。准备最多5％的流通池双层，重复4次以上，以便有五个1μL点以前14所示。 </li><li>将血脂上的盖子滑。盖上不锈钢CL白色扣环放大器的基本单元，翻转整个装置和密封。标记的双层标记的位置。在室温10分钟孵育。 </li><li>重视双向活塞左灌注管的管，如视频所示。生成与它的3路旋塞阀（管先前已与肝素钠缓冲液含有人血清白蛋白（HSA）（哈佛商学院/人血清白蛋白（HBS）的填充管端的积极半月板：50毫升哈佛商学院的10倍[10倍HEPES缓冲液：200毫米肝素钠，pH值7.2，1.37 M氯化钠，氯化钾50毫米，7毫米的Na 2 HPO 4，60毫米D-葡萄糖20毫升血清白蛋白25X，500μL1M 氯化钙 ，1毫升1个M氯化镁2卫生署2 0带来总量到500毫升和0.22μm的过滤器过滤器），是没有气泡。右灌注管和连接管轻轻一推，通过流动池的缓冲。 </li><li>座双层〜300酪蛋白含有100μM的氯化镍2μL填写1毫升注射器及 ​​所附的三路活塞开放部分。根TLY推缓冲区和脱离注射器之前关闭两个活塞。在室温30分钟孵育。 </li><li>他6标签AF488标记的gp120（浓度决定于16先前所述）。我们使用的gp120/μm2〜250分子模仿当地环境局助理秘书长集群上发现的HIV-1病毒颗粒表面17密度。同样，我们使用的双层此前报道14〜250分子/μm的ICAM-1 2。加载到流通池与酪蛋白。孵育30分钟在黑暗中，在室温下（包括带箔）。使用相同的程序纳入其他蛋白质，如他的12个标记Cy5标记ICAM-1上的双层。 </li><li>用5毫升缓冲双层。 </li></ol> 3。通过FRAP还原双层的质量控制在双层的蛋白质必须是横向扩散。加入到双层的细胞之前，重要的是要保证在准备双层蛋白质的流动。在这里，我们描述（FRAP还原力）最亮的TIRF，宽领域的萤光，或激光扫描共聚焦显微镜可进行手动的方法3漂白后荧光恢复。我们的目标是形象完全统一的荧光领域中的脂双层，漂白斑点，和淬灭分子的回报监察漂白面积。 50％在2分钟内复苏是可以接受的。所有主要的显微镜制造商（徕卡，尼康，奥林巴斯，蔡司）出售此应用程序的工作目标照明的TIRF系统。虽然每家公司提供的TIRF照明和其他业务功能的变化，基本上是相同的图像质量。 <ol><li>使用低激发强度，以尽量减少漂白。使用高数值孔径的物镜，如钠1.3至1.45 40X，60X或100X。当使用一个标准的荧光的TIRF显微镜，减少光intens的ITY，把激发光路径中的中性密度过滤器。大多数激光系统可设置低照度，低电压设置一个acusto光可调谐滤波器（AOTF的）或acusto光束分离器（AOBS）。 </li><li>记录“预漂白”的形象。如果使用冷却CCD相机，以弥补低光照条件下的分级，可设置为2x2或4x4的，可设置增益，可用于高，或长期暴露（例如以秒）。如果使用共聚焦，针孔孔径可以打开;增益设置高，慢扫描或平均使用。 </li><li>漂白斑点。如果使用标准的荧光的TIRF显微镜，删除所有ND过滤器允许的最大强度照明，关闭的视场光阑，并揭露了几秒钟的样本。如果使用激光扫描共聚焦激光功率最大的约4倍至8倍漂白斑点和变焦设置。一个警告：聚焦使用不太高放大，因为双层可能会损坏excessively集中的光子。 </li><li>在十到十五秒钟的间隔，记录图像使用相同的照明和录制设置在3.1和3.2。两到三分钟内当场强度接近“预漂白”的形象，应收回。快速恢复是一个成功的移动双层的迹象。如果当场仍然是黑暗的，尤其是如果边缘保留高对比度，荧光蛋白是在双层动，不应该被用于实验的。 </li></ol> 注：在我们目前的显微镜，我们可以提供0.9 mW的641 nm的光漂白荧光典型的浓度在不到4秒的ICAM面积为240微米2，一个圆形光斑。读者应该发现，漂白时间少于30秒，是足以在一个可重复的方式执行此法汞柱或氙灯的关键路线。我们也将参考您参考额外的DET 3苦恼。 4。注射细胞和免疫荧光染色<ol><li>热身流细胞至37°C（这是可以做到无CO 2在37℃孵化约30分钟）。 </li><li>加入1毫升注射器激活人体的CD4 + T细胞在哈佛商学院/ HSA〜400μL（2-5X10 6 /流动池）。让细胞附上〜45分钟，在37℃培养箱双层。 ，如果Ibidi室使用，提供额外的缓冲区每15-20分钟。 </li><li>注射2％多聚甲醛固定细胞，孵育10分钟，在37°C </li><li>房间温度用1ml PBS缓冲液洗3倍。在室温下5分钟注射0.1％的Triton X-100通透细胞。 </li><li>洗1毫升室温PBS缓冲液（磷酸化蛋白质的探测时，你可以添加在1mm的终浓度或PBS另一个磷酸酶抑制剂钒酸钠，以防止在加工过程中的磷酸盐去除）的3倍。块用5％山羊血清酪蛋白为在室温25分钟。动物血清型取决于二级抗体。 </li><li>洗室温PBS缓冲液1毫升的3倍。新增〜300μL30分钟缓冲/流细胞的主要抗体 - 在室温下1小时。检测到初始膜的近端激活信号，我们使用的磷酸化特异性抗体LCK（pLck），总LCK或菲英岛。 </li><li>洗室温PBS缓冲液1毫升的3倍。在缓冲液中添加适当的荧光标记的第二抗体为初级抗体。对于我们的实验中，我们使用的Alexa Fluor 568标记的羊抗兔二。在室温20分钟孵育。洗PBS缓冲液1毫升的3倍。 </li><li>注：这是必不可少的，只有二级抗体（无抗体）治疗控制双层。按照步骤4.1-4.5和4.6跳跃，然后继续进行步骤4.7。这将决定你的第二抗体结合非特异性水平。另外，可以直接使用的标签版的主要抗体。 </li></ol> 5。 TIRF显微镜图像采集 TIRF显微镜可以激发荧光信号限制在250 nm或更薄飞机在玻璃基板和电池接口。这保证图像从立即apposed细胞膜脂质双层和收购的唯一信号。因此，只有在突触的分子成像。因为的TIRF图像，只有在电池底部的膜近端的面积，内部或重新分配到背膜蛋白质会不会被发现。然后，它可能是重要的，此外收购标准广角或共聚焦显微镜，以确定积累或分布在突触区域的蛋白质相比，细胞的其他卷的图像。 所有主要的显微镜制造商（徕卡，尼康，奥林巴斯，蔡司）销售目标照明的TIRF系统，这个AP工作折叠术。虽然每家公司提供的TIRF照明和其他业务功能的变化，基本上是相同的图像质量。每个的TIRF显微镜有自己的操作细节，但以下的一般规则适用于以获取高分辨率成像。 <ul><li>照明应该是低，以避免漂白。 </li><li>相机应该有一个线性响应。 </li><li>相机应该是不饱和宽动态范围成像能力。 </li><li>所有的设置应设置常数的定量分析。 </li></ul> 6。数据分析研究细胞内的信号激活的CD4 + T与gp120的细胞在VS的相互作用的结果，完成的TIRF图像定量分析，以确定是否细胞内信号分子，如Lck和Fyn被激活，并招募到突触15。在这里，我们描述了一个简单的方法适用于大多数图像分析ImageJ的和Metamorph如应用程序包。我们已经使用ImageJ的，它运行在Windows，Macintosh和Linux，我们的方法，在这里12。 在分析 &gt; 设置测量选项卡，检查区的框和平均灰度值 。当你使一个地区的利益是一个多边形或写意跟踪封闭形状的图像，并做分析 &gt; 测量 ，软件会从这个测量成果表中的数据。该地区将是在像素数，或在2微米。中庸是在任意单位的平均强度。它必须从它的背景强度减去。面积乘以平均减去背景返回任意单位的蛋白质的综合强度。 <ol><li>设置测量平均和地区。 </li><li>打开一个实验条件下的图像。 </li><li>对于每一个细胞，跟踪和测量感兴趣的地区（平均），并跟踪和衡量一个地区以外的地区的利益（背景）。 </li><li>做了一个实验条件时，要么副本测量和粘贴到Excel或其他电子表格程序或保存测量。 </li><li>每个实验条件下，返回到步骤6.2。 </li><li>当测量完成后，计算的结果。计算公式如下： 平均强度=平均值 - 背景 综合强度=平均强度*面积</li></ol> 7。代表结果在VS gp120与互动的结果来衡量初始膜近端信号分子的Lck和Fyn激活和招聘，主要人类CD4 + T细胞被引入到轴承gp120和ICAM-1 15双层。介绍到双层仅ICAM-1的细胞，作为控制信号的定义的基础水平。 SPEcific荧光强度为gp120和ICAM-1含双层和在整个接触面积与ICAM-1的双层互动细胞的细胞内gp120的接触面积测量。平均荧光强度的增加是一个增强的迹象，招募和激活信号分子的VS。这将进一步证明了一个综合性的荧光强度可比增长。但是，如果没有集成荧光强度的变化，这表明信号分子的再分配。 后的细胞含有gp120和ICAM-1，总Lck和pLck（Y394）的双层互动招募到VS接口与gp120的共定位（图1及2）。 LCK总的平均强度（图1）是对含有gp120和ICAM-1的双层高于单独与ICAM-1，但综合强度水平（图1）是相似的，这表明，LCK被重新分配到后，CD4 + T细胞结合到gp120的中央集群。然而，量化pLck（Y394）（图2）表明，平均强度上的gp120和ICAM-1含双层高于ICAM-1型单双层，和综合强度与gp120和ICAM双层高-1比上ICAM-1型单双层。这表明，而LCK接口总水平在细胞类似的到gp120和ICAM-1和ICAM-1的单双层，增加gp120结合残留Y394磷酸化在LCK激活循环的坚持。相比之下，菲英岛不被招募到VS（图3），为更多的菲英岛是目前在细胞的接触面积，ICAM-1的比对含有gp120和ICAM-1的双层单双层。因此，我们得出结论，LCK，菲英岛，是积极的HIV-1的gp120诱导VS激酶。 数字：膜近端信号艾滋病毒-1的gp120诱导VS。图片上代表细胞显示的gp120 + ICAM-1的双层（顶部面板）和ICAM-1的双层（底板）。单个细胞的荧光强度进行量化作为细胞脚印手动追踪的领域内与图1中的黄线，标志着区域所示。 TIRF显微镜检测的平均水平和综合强度的量化，分别在左边和右边的图形。共有30至350个细胞进行量化为每个条件。酒吧= 5微米。从三个重复实验数据显示。 <img alt="图1" src="/files/ftp_upload/3757/3757fig1.jpg" /> 图1。CD4 + T细胞被引入到gp120和ICAM-1，ICAM-1的单双层含45分钟，然后固定和总LCK染色。 <img alt="图2" src="/files/ftp_upload/3757/3757fig2.jpg" /> 图2。CD4 + T细胞被引入到双层含gp120和ICAM-1或ICAM-1仅45分钟，然后固定和染色pLck（Y394）。 <img alt="图3" src="/files/ftp_upload/3757/3757fig3.jpg" /> 图3。CD4 + T细胞被引入到gp120和ICAM-1，ICAM-1的单双层含45分钟，然后固定和染色总菲英岛。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+cell+to+cell+transfer+across+an+Env-induced,+actin-dependent+synapse.">HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse.</a> J. Exp. Med. 199, 283-293 (2004).</li><li>Jolly, C., Mitar, I., Sattentau, Q. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+an+intact+T-cell+actin+and+tubulin+cytoskeleton+for+efficient+assembly+and+spread+of+human+immunodeficiency+virus+type+1.">Requirement for an intact T-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1.</a> J. Virol. 81, 5547-5560 (2007).</li><li>McDonald, D., Wu, L., Bohks, S. M., KewalRamani, V. N., Unutmaz, D., Hope, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+HIV+and+its+receptors+to+dendritic+cell-T+cell+junctions.">Recruitment of HIV and its receptors to dendritic cell-T cell junctions.</a> Science. 300, 1295-1297 (2003).</li><li>Pearce-Pratt, R., Malamud, D., Phillips, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+cytoskeleton+in+cell-to-cell+transmission+of+human+immunodeficiency+virus.">Role of the cytoskeleton in cell-to-cell transmission of human immunodeficiency virus.</a> J. Virol. , 682898-682905 (1994).</li><li>Rasband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li><li>Rizzuto, C. D., Sodroski, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+virion+ICAM-1+to+human+immunodeficiency+virus+infectivity+and+sensitivity+to+neutralization.">Contribution of virion ICAM-1 to human immunodeficiency virus infectivity and sensitivity to neutralization.</a> J. Virol. 71, 4847-4851 (1997).</li><li>Vardhana, S., Dustin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+Planar+Bilayers+for+the+Formation+of+Study+of+Immunological+Synapses+and+Kinapse.">Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse.</a> J. Vis. Exp. (19), e947-e947 (2008).</li><li>Vasiliver-Shamis, G., Cho, M. W., Hioe, C. E., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+type+1+envelope+gp120-induced+partial+T-cell+receptor+signaling+creates+an+F-actin-depleted+zone+in+the+virological+synapse.">Human immunodeficiency virus type 1 envelope gp120-induced partial T-cell receptor signaling creates an F-actin-depleted zone in the virological synapse.</a> J. Virol. 83, 11341-11355 (2009).</li><li>Vasiliver-Shamis, G., Tuen, M., Wu, T., Starr, T., Cameron, T., Thomson, R., Kaur, G., Liu, J., Visciano, M., Li, H., Kumar, R., Ansari, R., Han, D., Cho, M., Dustin, M. L., Hioe, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+type+1+envelope+gp120+induces+a+stop+signal+and+virological+synapse+formation+in+noninfected+CD4++T+cells.">Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells.</a> J. Virol. 82, 9445-9457 (2008).</li><li>Zhu, P., Liu, J., Bess, J., Chertova, E., Lifson, J., Grise, H., Ofek, G., Taylor, K., Roux, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+three-dimensional+structure+of+AIDS+virus+envelope+spikes.">Distribution and three-dimensional structure of AIDS virus envelope spikes.</a> Nature. 441, 847-852 (2006).</li></ol>

作者宣称没有利益冲突。

以前的研究已经在细胞与细胞的共轭系统的可视化VS，然而这些研究并没有提供足够高的分辨率的图像可视化在突触的超分子结构。在我们的实验室中，我们利用玻璃平面双层系统的代表表示病毒包膜gp120与细胞粘附分子ICAM-1感染的细胞表面。在TIRF显微镜，这从一个高信号噪声比的双层表面在100-200 nm的荧光信号检测的同时，我们能够探测到从在VS的ICAM-1的gp120的超分子隔离。此外，标准的染色方法可应用于双层系统，用于检测和量化的具体招聘LCK积极，但不是菲英岛，gp120的接触面积比15。因此，平面双层突触界面的高分辨率成像实验系统提供了一个在2D平面的TIRF微SCOPY，以及宽视场或聚焦照明方法。然而，该系统还具有调整配体的流动性，平面弯曲的限制，和生物膜的波动不是由平面双层转载。此外，这是在体外系统，因此有其他限制，如缺乏其他分子膜，这将是对目前受感染的细胞和细胞骨架的机械调节分子的流动性和细胞活力。此外，动态和分布的分子，如三聚体与单体的gp120可能无法代表生理上的双层。不过，即使有了这些限制，这个系统仍然是非常有价值的研究病毒细胞或细胞 - 细胞相互作用，以及这些方法可以作为一个有用的指南，研究人员正在寻求高分辨率图像检测超分子组织，是无法辨别在传统的细胞与细胞的共轭体系。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>他签的艾滋病毒的gp120 </td><td>赵博士的礼物</td><td></td><td> DH12 </td></tr><tr><td>他签的艾滋病毒的gp120 </td><td>免疫技术</td><td></td><td>各种x4或R5的热带</td></tr><tr><td>人血清白蛋白</td><td>威廉姆斯医学公司</td><td> 521302 </td><td>人血清白蛋白25％ </td></tr><tr><td> amicon超离心式过滤器</td><td> Millipore公司</td><td> UFC803024 </td><td> ultracel-30K </td></tr><tr><td> LCK兔单克隆抗体</td><td>细胞信号</td><td> 2787 </td><td></td></tr><tr><td> P-LCK兔PAB </td><td>圣克鲁斯生物技术公司</td><td> SC-101728 </td><td>酪氨酸394 </td></tr><tr><td>菲英岛兔单克隆抗体</td><td> Millipore公司</td><td> 04-353 </td><td></td></tr><tr><td>的Alexa Fluor 568 2°;抗体</td><td>分子探针</td><td> A11034 </td><td>山羊抗兔IgG（H + L） </td></tr><tr><td>的Alexa Fluor 488 </td><td>分子探针</td><td> -20000 </td><td>羧酸琥珀酰亚胺酯</td></tr><tr><td> FCS2商会</td><td> bioptechs </td><td> 060319-2-03 </td><td></td></tr><tr><td> Microaqueduct幻灯片</td><td> bioptechs </td><td> 130119-5 </td><td></td></tr><tr><td> 30毫米回合瓦特/孔</td><td> bioptechs </td><td> 1907-08-750 </td><td> 0.75毫米厚</td></tr><tr><td>长方形垫片</td><td> bioptechs </td><td> 1907-1422-250 </td><td>厚14x240.25毫米</td></tr><tr><td>聚乙烯管</td><td> bioptechs </td><td> 20202275 </td><td> 1/16“（25英尺） </td></tr><tr><td>三通阀</td><td> BioRad公司</td><td> 7328103 </td><td></td></tr><tr><td>两路阀</td><td> BioRad公司</td><td> 7328102 </td><td></td></tr><tr><td>我粘幻灯片0.2露儿</td><td> ibidi </td><td> 80168 </td><td></td></tr><tr><td>盖玻片</td><td> ibidi </td><td> 10812 </td><td></td></tr><tr><td> 1ml注射器</td><td>屋宇署</td><td> 309659 </td><td></td></tr><tr><td>狗 - NTA </td><td>阿凡提极性脂</td><td> 790404C </td><td></td></tr><tr><td> DOPC </td><td>阿凡提极性脂</td><td> 850375C </td><td></td></tr><tr><td>盖玻片</td><td> bioptechs </td><td> 40-1313-0319 </td><td> 40毫米</td></tr><tr><td>的TIRF显微镜</td><td>尼康</td><td></td><td></td></tr><tr><td> ImageJ的</td><td>国家卫生研究院</td><td></td><td> <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/~~V</a> </td></tr></tbody></table>

imaging of hiv-1 envelope-induced virological synapse and signaling on synthetic lipid bilayers

人类免疫缺陷病毒1型（HIV-1）感染发生最有效的传输2,10,11细胞通过细胞。这涉及形成一个病毒学突触（VS），这是F-肌动蛋白依赖的细胞与细胞接触被感染的细胞上的HIV-1的gp120信封后形成的交界处，与CD4和细胞之间的CD4 + T细胞的细胞转移（中九龙干线）的趋化因子受体CCR5或CXCR4对靶细胞8。此外gp120和它的受体，其他膜蛋白，特别是粘附分子LFA-1及其配体，细胞间粘附分子家庭，在VS的形成和病毒的传播发挥了重要作用，因为它们是病毒感染的供体细胞的表面上和靶细胞，以及对HIV-1病毒颗粒1,4,5,6,7,13的信封。也伴随着细胞内的信号事件gp120的聘用及其受体转VS形成。事实上，我们最近发现的CD4 &lt;SUP&gt; + gp120与细胞间的相互作用引起招聘的TCR signalosome包括LCK，CD3ζ，ZAP70，土地增值税，SLP-76，ITK，PLCγ15相关的信号分子的磷酸化。 在这篇文章中，我们提出了一个方法，可视化超分子的安排和膜近端信号VS形成过程中发生的事件。我们采取的玻璃支持作为还原模型来表示附有病毒包膜gp120与细胞粘附分子ICAM-1的感染艾滋病毒的细胞表面的平面双向层系统的优势。该协议描述为监测HIV-1的gp120诱导VS集会和信号激活的事件，包括我）双向层在流动单元的筹备和大会，二）注射细胞和免疫荧光染色检测细胞相互作用的细胞内信号分子的一般程序与HIV-1 gp120和ICAM-1的双层，III）TIRF显微镜图像采集，1ND IV）的数据分析。该系统生成VS超越与传统的细胞与细胞系统实现接口的高清晰度图像，因为它允许招募到这些子域与特定信号分子检测VS的单个分子组成不同的集群。

Watch this Scientific Journal Video about Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers at JoVE.com

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

本文介绍了一种方法，可视化的全内反射荧光显微镜（TIRF）HIV-1诱导信封上的玻璃支持平面双层病毒学突触的形成。该方法也可以结合免疫荧光染色检测艾滋病毒1信封引起的病毒学突触的形成过程中发生的信号分子的活化和再分配。

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

imaging-hiv-1-envelope-induced-virological-synapse-signaling-on

Research

JoVE Journal

Immunology and Infection

12.1K 次观看.  New York University Langone School of Medicine. 本文介绍了一种方法，可视化的全内反射荧光显微镜（TIRF）HIV-1诱导信封上的玻璃支持平面双层病毒学突触的形成。该方法也可以结合免疫荧光染色检测艾滋病毒1信封引起的病毒学突触的形成过程中发生的信号分子的活化和再分配。

视频: Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

科研

免疫与感染

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

使用基因型的方法预测HIV-1辅助用途（归经）的序列分析

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

在体外对HIV - 1核心脱壳

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a &#34;cell-like&#34; environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy.

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

HIV-1 Gag的可视化绑定到巨人的单片囊泡（GUV）膜

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.