We thank Vladimir Polejaev (Yale West Campus Imaging Core) for the design and construction of the polarized TIRF microscope, David Baddeley (Yale University) for help with two-color detection instrumentation, and James E. Rothman (Yale University) and Ben O'Shaughnessy (Columbia University) and members of their groups for stimulating discussions. EK is supported by a Kavli Neuroscience Scholar Award from the Kavli Foundation and NIH grant 1R01GM108954.

作者宣称，他们没有竞争的经济利益。

成功地实施这里所描述的SUV-SBL融合法的关键取决于几个关键步骤，例如蛋白质的功能性重组到脂质体中，获得良好的质量SBLs，并选择合适的摄像参数，以检测单个分子。虽然这可能需要一些时间和努力才能成功，一旦试验成功实施，它提供了无法从简介中讨论的任何其他体外实验融合了丰富的有关融合过程的信息。率在该越野车停靠到和保险丝与SBL，对接至融合时间的分布，脂类扩散率，每熔断脂质体的大小和脂质释放对于给定的融合事件是否是弥散或孔控制的罐来确定。如果释放慢得多比从扩散预期，假定孔闪烁模型underlies脂质释放产量的相位差，P 0，时间的分数的孔隙我聚变27期间开放。 以前的工作探测单一的SUV，SUV融合18-20,28或脂质体和病毒颗粒的融合15-17,21,22,33,44染料分子之间依靠FRET和自我淬火SBLs。相比之下，我们在很大程度上避免染料脱猝灭探测脂质标签转移动力学入SBL，有两个原因。首先，FRET效率是脂质释放的一个高度非线性的记者。小的变化的染料间距离d导致在FRET效率大的变化，但只有一个有限的范围D / R 0的值，其中，R 0是福斯特距离。即，实际的染料稀释，并在脱猝灭信号的观察到的增加，可能会出现，由于下降到接近至R 0值所需的时间之间的延迟时间。同样，在脱猝灭信号变化不大，一旦染料密度充分降低后的脂质释放，使得<em&gt; D / R 0 <img alt="比...更棒" src="/files/ftp_upload/54349/greater.jpg" /> 1.4，即使染料显著量仍停留在囊泡。这些特性使得脱猝灭的好工具，以获得秒杀的信号，以检测融合事件，但使用高时间分辨率的脂质释放定量动力学分析复杂化。与此相反，在强度由于偶极再取向和渐逝场的效果从SUV到SBL荧光团转移的变化线性相关染料在SUV仍然保留的级分。第二，对于自猝灭需要高浓度的类脂的标签可能影响融合过程本身。 对于偏光TIRF激励，一个自制的设置， 如图4的成本效益，并提供了偏振特性良好的控制。然而，一个自制的设置是没有必要的，因为至少有一些商业的TIRF显微镜使用偏振光激发和关闭的，现成的双色发射分光模块可用。使用的偏振波保持（PM）光纤耦合偏振光激发光从激光器进入显微镜新兴商业显微镜成功先前25-27使用。在这样一台仪器，极化可以简单地通过旋转光纤输入到显微镜的TIRF单元PM旋转。与此相反，一些其它商业设置可以采用在TIRF单元的偏振分束器，使得偏振调整更加困难或不可能。不幸的是，往往不用于商业系统指定的偏振特性。因此，它是不希望构建一个自制的系统来讨论偏振特性，并在特定的商业选择投资之前尝试演示单元谁用户的关键。 脂质和可溶性货物放行的同时监测提供了融合进程的进一步信息。许多毛孔似乎RELE后重新密封ASING仅可溶性标签的一小部分封装入一个SUV 27。通常所有的脂质标签由孔隙重新密封时间发布，所以毛孔再密封不是单从监控脂质释放先前怀疑。标记的脂质的确，单独监测血脂发布，基斯林等人 29曾建议转移到SBL通过整个SUV膜进入SBL以极快的速度发生塌方。 后的内容部分释放在脂质体 - 拴系的双层补丁融合测定先前观察到的熔化再密封气孔。罗尔等人由〜8纳米长的DNA衬垫拴到盖玻片的54准备独立双层补丁。然后，他们引入脂质体，其融合到双层补丁被锚定到脂质体和修补膜的互补单链DNA脂质缀合物杂交来驱动。所有事件的12％是瞬时融合，产生部分的脂质体内容物释放IAL。脂质体爆裂事件，在以前的一些SUV-SBL融合试验44所示，是非常罕见的。作者建议了〜8nm的空间的双层补丁的下方，防止双层底物相互作用，也可能允许的脂质体内容的非泄漏转移到双层下方的空间。另外，所释放的可溶性标签扩散不显著在平面双层与玻璃基板之间的空间位阻。在此处所描述的测定中，平面双层类似地由聚（乙二醇） - 脂质缀合物的存在下保持〜5纳米从盖玻片路程。 可溶性和脂质货物双色监控所提供的附加信息带有一些取舍。首先，准备与两个标签的SUV是有点比单独包括脂质标签更多地参与。第二，有一些不可避免的损失信号对噪声在t从脂质标签监测信号时他同时采集速率为单色脂质释放实验（每帧15-20毫秒）。这部分损失是由于做了事实，同时作为脂质标签与可溶性SRB标记，是不是在单色实验中使用LR-PE是光明的。额外的损失是由于更高的背景，由于同时双激光激发和在光路中的其他部件（ 图4）。到目前为止，较低的信号 - 噪声从单一的可靠跟踪防止美国并在两色实验标签。这反过来又防止了深入的分析，以提取可以与单色的LR-PE测量的所有信息。这一挑战可能在将来通过优化获取条件和测试其他对兼容标签与​​改进的性质来克服。 同时监测可溶性和脂质货物释放也可以检测半融合，其中，所述近侧小叶已经融合的状态下，但远端小叶没有。实际上，这种双色实验可以提供半融合的体外融合实验18,20的存在下，在其他的唯一结论性的评估。然而，半融合能够可靠地在单色SUV-SBL实验监测血脂单独22,44释放检测。在这样的实验中，约1/2的SUV的初始脂质标记强度保持在融合点。当远端小叶以后熔化一定时间，如果该点还没有被漂白剩余强度可能会消失。 PEG化脂质的使用介绍SBL和玻璃之间支持一个软垫一直在重现胞外分泌的SNAP25要求的关键。在聚乙二醇的协议结果刷覆盖的面向流路以及所述SBL的一侧。这具有一些好处，例如减少的SUV和SBL之间的非特异性相互作用，如在引言说明。如果用的磷脂相互作用的蛋白质SBL引入测定中，然而，所述PEG刷子将提出对这种相互作用的立体屏障。因此，对于一些应用，这将是理想的不对称包括所述PEG层，只SBL和玻璃盖玻片之间。然后SBL的面向流路侧将能够与磷脂结合蛋白，例如突触结合蛋白相互作用。这可以通过使用可共价连接到盖玻片在一端双官能PEG链和携带在其他的脂质锚来实现。然后朗缪尔-布洛杰特法可用于沉积脂质单层上的PEG层55。脂质体上面与它该疏水表面保险丝引入，形成双层56。希望这种做法将在未来微流控相结合。 可以预期的是，这里提出的SUV-SBL测定将在探索与SNARE蛋白相互作用的其他蛋白质的作用的帮助，如Munc18 / SEC1家族，钙传感器突触-1，和complexin。

膜融合为脂质和蛋白质，分泌，施肥，发展的细胞内运输所需的通用生物过程，和包膜病毒进入宿主生物体1-3。对于大多数细胞内聚变反应包括通过胞吐的激素和神经递质的释放，融合2脂质双层的能量由形成同源可溶性N-乙基马来酰亚胺-敏感因子附着蛋白受体 （SNARE）的蛋白质之间的四螺旋束的提供，锚定在囊泡（v-SNARE）和分别的靶膜（叔SNARE）4，。突触小泡胞吐作用是最严格调控的聚变反应和动作电位1,4,5到货后一毫秒内发生。的融合孔，两个熔断隔间之间的初始连接，可以闪烁打开和再密封或不可逆5-7扩大之前关闭多次。前者结果在短暂的"亲吻与运行"的融合，而后者导致完全融合。理事融合这两种模式和调节孔径闪烁机制之间的平衡因素不能很好地理解5,8。 SNARE蛋白所需的胞吐作用;突触囊泡融合是由神经毒素9时SNARE蛋白裂解取消。使用小单层囊泡（SUV的）堆积聚变实验表明，斯耐尔不仅必需的，但也足以驱动膜融合10。在此批量测定中，重构用v-SNARE蛋白（V-SUV）的SUV被掺杂有荧光磷脂（N - （7-硝基2-1,3-苯并恶二唑-4-基）-phosphoethanolamine（NBD-PE）和（ N - （丽丝胺罗丹明B磺酰基）-phosphoethanolamine（LR-PE）和含叔SNARE蛋白（T-SUV）最初的v型的SUV NBD-PE的荧光被荧光共振能量转移淬无标签的小泡混合（FRET ），以LR-PE作为实验室ELED v型越野车与未标记叔SUV的融合，在现在结合膜的荧光面密度降低，在NBD-PE的荧光所产生的增加报告脂质混合10的程度。象散法是容易设置和分析，它已被广泛用来研究SNARE介导的融合10-14的机制。然而，它有一些限制，如低灵敏度和时间分辨率差。最重要的是，作为一个集合进行测定，平均结果​​在使半融合中间体难以检测对接和融合区分，以及所有事件。 在过去的十年几组，包括我们，已经开发了新的分析方法来监控在单级囊泡融合15-27事件。厦和他的同事用拴在表面上的V-SUV和监控他们与免费的T SUV的融合18,19。脂质混合是一对脂相连的荧光团之间的FRET使用EM监视层状分别在v型和叔越野车，使用全内反射荧光（TIRF）显微镜18。后来，Brunger的实验室使用单一脂质标签的物种连同内容标记物的脂质和内容混合20,28同时检测。既脂质和内容标记被包括在高，自猝灭浓度;融合SUV的未标记导致荧光脱猝灭20,28。 其他人已经融合v型越野车与T-SNARE蛋白15-17,21-27,29重组平面双层。目标（含T-SNARE）双层更好地模拟与一台质膜小，高度弯曲的囊泡的生理融合过程中的平面形状。该斯泰纳姆组采用与T-SNARE蛋白重组孔跨越膜，悬浮在多孔氮化硅基板，并检测使用激光共聚焦显微镜23个人的V型SUV的融合。其他˚F使用V型越野车与T-SNARE蛋白重组平面双层，支持在玻璃基板上15-17,21,22,24-27,29。使用支持的双层（SBLs）的巨大优点在于，TIRF显微镜可以用于检测具有优良信噪比并且在不脱离自由V形的SUV干扰对接和融合事件，虽然使用微流体使用还提供了单事件分辨率标准的远场荧光显微镜24。 一个主要问题是底物的相互作用双层是否以及如何影响支持双层质量和融合的过程。早期工作取得使用该直接支持的玻璃或石英衬底上15-17平面SBLs的。这些SBLs被吸附制成，爆裂，扩散和在衬底上叔SUV膜融合。它很快实现，然而，省略了密钥t-SNARE组分，SNAP25，从以这种方式制备SBLs导致V形的SUV对接和融合动力学indistinguishable使用完整的T-17 SNARE蛋白获得的。因为SNAP25 在体内 30,31绝对必需的融合，这些早期的尝试生理相关投入的问题。塔姆的研究小组通过使用更好的控制支持双层形成21战胜了这一挑战。它使用朗缪尔-布洛杰特沉积为SBL的无蛋白质的第一小叶，接着用叔-越野车21，其单层融合。这导致SNAP25依赖性融合。 以避免与直接支撑在玻璃基板上，而不需要使用朗缪尔-布洛杰特法的双层相关的潜在假象，Karatekin 等人介绍了柔软，水合聚（乙二醇）（PEG）的双层和基板24之间的缓冲。该变形也导致SNAP25依赖性融合24。垫着柔软的聚合物层上双层一直被称为更好地保存跨膜蛋白质的流动性和功能32，并与病毒融合33研究中使用。此外，聚乙二醇化双层似乎保留一定的能力进行自我修复，而且非常坚固34,35。首先，商购，脂质连接PEG链的一小部分被包括在叔SUV膜。当这些叔SUV的突发，并形成在玻璃基板上的平面双层，一个PEG刷覆盖平面双层的两个小叶。因为平面双层形成通过叔SUV的周围到所述亲水玻璃表面的PEG链的粘附驱动，脂质体破裂和平面双层形成相对不敏感，以所使用的脂质组合物。然而，当包括大量的胆固醇的，增加了越野车的粘结性时，越野车可以不自发破裂。如果是这种情况，可以采用渗透休克或二价离子，以帮助平面双层形成25。 如在本AP如上所述，proach的PEG刷覆盖所述平面的两侧，支持双层。面对微流体流动通道的刷有助于防止其也通常覆盖有聚乙二醇层传入v型越野车的非特异性粘附。 v型和叔SNARE复合体的形成从膜远侧N-末端并且前进朝向膜近端域36阶段开始。为V形的SUV与叔SBL中，v型和叔SNARE N-末端需要突出的PEG刷，这似乎是在测定条件下，上述情况下进行交互。刷高度可适于通过改变PEG化脂质的密度和PEG链长度37,38学习比斯耐尔其它蛋白质。所述PEG刷子覆盖定影双层的近侧表面中的另一个好处是，它们模仿它们填充有每平方微米39 30,000-40,000整合膜蛋白生物膜的拥挤的环境。就像在此测定的PEG链，所述REPU覆盖生物膜lsive蛋白层需要被推开以允许两个磷脂双层之间的接触为发生融合。 微流体流动通道在该测定中使用的，因为它们提供独特的优点。首先，微流体流使T-的SUV更均匀的沉积扩散和融合形成了T-SBL。其次，小声道音量（&lt;1微升）减少样品消耗。第三，所需的小体积允许下恒定流量进行的整个实验。流动弱除去，大概非特异性，从​​SBL 16附着v型越野车。它还维护V型的SUV的T-SBL以上，简化动力学分析17的密度不变。最后，停靠囊泡容易从由流25进行自由的区分。第四，多个微流体通道可以在相同的盖玻片一起使用，每个探测不同的条件。这使得杜里条件比较纳克相同的实验运行。类似的方法已被用于面包车Oijen组研究流感病毒和缓冲SBLs 33之间的融合。 在TIRF显微镜，渐逝场的指数衰减（以衰减常数〜100nm）的界限荧光激发到那些在玻璃 - 缓冲界面的非常接近的分子。这最小化是更远离荧光分子的贡献，增加信噪比，并且允许用10-40毫秒的帧的曝光时间单分子灵敏度。渐逝场也导致在融合的信号增加:从SUV标记的脂质转移到SBL，他们发现自己，平均而言，在更强的激励场。这增加了荧光较大的脂质体更强。 如果偏振光是用来产生渐逝场，附加效果有助于对T中的荧光变化从SUV到SBL标签的转让（BOT）。某些脂质染料具有与相对于它们所嵌入的双层的优选平均角度定向的跃迁偶极。这将在通过荧光团时，他们在SUV 兑 SBL发射的荧光的量的差异，由于偏振光分束将在两个膜激发染料不同。对于前者，激发光束将与周围的球形的SUV取向过渡偶极子相互作用，而对于后者，偶极取向将由扁平SBL几何局限。例如，当S偏振入射的光（偏振光垂直于入射平面）时，激发是更有效率的，当染料在SBL比在SUV脂质染料跃迁偶极方向平行于膜29,40一（如直接投资收益还是没41-43）。掺杂了这样的荧光团SUV似乎暗淡时，码头上SBL（ 图7，Representat<html>香港专业教育学院结果）。作为融合孔打开，并连接在SUV和SBL膜，荧光探针扩散到SBL和变得更容易被s偏振光渐逝场25,27,29激发。因此，周围的融合位点整合的荧光信号从所述的SUV染料转移到SBL 27（ 图3和图7）期间急剧增加。有助于信号伴随融合脱猝灭的荧光标记的时转移到SBL因为它们稀释变化的另外一个因素。相比，在这里描述的测定消逝场衰减和极化效应脱猝灭的贡献通常是次要的，因为只有一小部分（ <img alt="式（1）" src="/files/ftp_upload/54349/54349eq1.jpg" /> ）脂质的标记。 在融合信号增加可以利用通过比较时间来推断融合孔性质，1"SRC ="/文件/ ftp_upload / 54349 / 54349eq2.jpg"/&gt;，对于脂质通过的孔是自由可渗透脂质实际释放时间逃逸要求<img alt="式（1）" src="/files/ftp_upload/54349/54349eq3.jpg" /> 。如果两个时间尺度是可比，将它的结论是，孔呈现给脂质流动阻力小。然而，如果实际的释放时间是显著比时间为扩散限制释放更长的时间，这将表明一个过程中，如孔隙抖动，延迟脂质释放。扩散限制释放时间， <img alt="式（1）" src="/files/ftp_upload/54349/54349eq2.jpg" />取决于定影脂质体和脂质扩散的大小;其估计需要这两个参数来量化。测定的单分子灵敏度允许脂质扩散到其释放到SBL为每个聚变事件26之后追踪几个单脂的荧光团测定。每一熔断囊泡的大小可通过将（i）的单脂质染料的强度，（ii）在所有荧光团在融合转移到SBL中围绕一个停靠站点的总荧光变化，（iii）该已知标记的SUV的密度来估计27脂质，和（iv）每脂质的区域。对于许多融合事件中，发现了实际脂质释放时间要慢得多比由扩散控制的释放27预期，如先前指出的假定均匀的SUV大小44。假设脂质释放迟缓是由于毛孔闪烁，定量模型允许"毛孔开放"的估计，时间的部分孔隙依然融合27期间开放。 每当实际，以测试同时使用脂质和可溶性内容标签融合的机制是很重要的。例如，脂质释放可以通过比孔隙闪烁等工艺，如由SNARE蛋白是环绕吨脂质扩散的限制被延迟他孔隙。如果是这样的话，然后松开的内容将先于脂质标签释放，所提供的孔径是大到足以允许可溶性探针通道。在方法的一个更基本的缺陷可能是通过一个狭窄的融合孔的SBL连接到囊泡已经基本上保留了其融合前形状发生标记的脂质转移到SBL的假设。脂质转移到SBL也可以从融合孔与随之而来的，超极本的SUV进入SBL膜的迅速崩溃的快速散瞳造成的，正如上面所提到仅仅基于29脂质发布的数据。同时监测血脂和内容同时发布，发现许多气孔释放他们所有的脂质标签后重新密封，但保留了他们的一些可溶性的货物27。这表明，至少一些脂质体不塌陷到SBL融合之后，并且该脂质染料转移到SBL通过融合孔发生。此外，LIPID和内容，同时发布27发生，使得它不可能脂质释放迟缓，是由于受到周围孔45 SNARE蛋白阻碍脂质扩散的。 未监测溶于内容发布一款SUV-SBL融合协议之前由Karatekin和罗斯曼25公布。在这里，最近的事态发展都包括在内，脂质即同步监测和内容发布和SUV，脂质，并融合孔特性27的推定。该协议开始于制备微流体细胞，通过结合的聚（二甲基硅氧烷）制成（PDMS）弹性体包含块槽用玻璃盖玻片25的指令。接着，制备V-SUV的与两个脂质和内容标记进行说明。第4和5用于组装微流体细胞， 原位形成的SBLs并检查缺陷和流动性，引入VS的提供指令UVS到流动细胞和融合事件的检测。部分6提供用于数据分析的指令。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Milli-Q (MQ) water</td><td>Millipore</td><td></td><td></td></tr><tr><td>KOH</td><td>J.T. Baker</td><td>3040-05</td><td></td></tr><tr><td>Ethanol 190 Proof</td><td>Decon</td><td></td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Chemical</td><td>A416P4</td><td></td></tr><tr><td>HEPES</td><td>AmericanBio</td><td>AB00892</td><td></td></tr><tr><td>Sodium Cholride (KCl)</td><td>AmericanBio</td><td>AB01915</td><td></td></tr><tr><td>Dithiothreitol</td><td>AmericanBio</td><td>AB00490</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-[2-hydroxyethyl] piperazine-&lt;em&gt;N&#039;&lt;/em&gt;-[2-ethanesulfonic acid] (HEPES)</td><td>AmericanBio</td><td>AB00892</td><td></td></tr><tr><td>EGTA</td><td>Acros Organics</td><td>409911000</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Buffers&lt;/strong&gt;</td></tr><tr><td>HEPES-KOH buffer (pH 7.4)</td><td></td><td></td><td>25 mM HEPES-KOH, 140&amp;nbsp;mM KCl, 100&amp;nbsp;&amp;mu;M EGTA, 1&amp;nbsp;mM DTT</td></tr><tr><td colspan="4">&lt;strong&gt;Solvents&lt;/strong&gt;</td></tr><tr><td>Chloroform&amp;nbsp;</td><td>J.T. Baker</td><td>9180-01</td><td>in glass bottle, CAUTION, wear PPE</td></tr><tr><td>Methanol</td><td>J.T. Baker</td><td>9070-03</td><td>in glass bottle, CAUTION, wear PPE</td></tr><tr><td colspan="4">&lt;strong&gt;Liposome preparation&lt;/strong&gt;</td></tr><tr><td>Gastight Hamilton syringe</td><td>Hamilton</td><td>var. sizes</td><td>only use glass sringe with solents (Chlorophorm/ Methanon, 2:1, v/v)</td></tr><tr><td>Glass tubes Pyrex Vista 11 ml, 16x100 mm screw cap culture tube</td><td>Pyrex&amp;nbsp;</td><td>70825-16</td><td>clean thoroughly, rinse with chloroform</td></tr><tr><td>1-palmitoyl-2-oleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine, 16:0-18:1 PC (POPC)</td><td>Avanti Polar Lipids</td><td>850457</td><td>Lipids come dissolved in CHCl&lt;sub&gt;3&lt;/sub&gt; or as lyphilized powder in sealed vials. Aliquot upon opening. Store extra as dried lipid films under inert atmosphere at -20 &amp;deg;C. Keep stocks in CHCl&lt;sub&gt;3&lt;/sub&gt;/MeOH (2:1, v/v) at -20 &amp;deg;C. let come to RT before opening</td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phospho-L-serine (sodium salt), 18:1 PS (DOPS)</td><td>Avanti Polar Lipids</td><td>840035</td><td>Same as above.</td></tr><tr><td>1-stearoyl-2-arachidonoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine, 18:0-20:4 PE (SAPE)</td><td>Avanti Polar Lipids</td><td>850804</td><td>Same as above.</td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt), Brain PI(4,5)P&lt;sub&gt;2&lt;/sub&gt;</td><td>Avanti Polar Lipids</td><td>840046</td><td>Same as above.</td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine-&lt;em&gt;N&lt;/em&gt;-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt), 18:1 NBD PE</td><td>Avanti Polar Lipids</td><td>810145</td><td>Same as above.</td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine-&lt;em&gt;N&lt;/em&gt;-[methoxy(polyethylene glycol)-2000] (ammonium salt), 18:1 PEG2000 PE</td><td>Avanti Polar Lipids</td><td>880130</td><td>Same as above.</td></tr><tr><td>cholesterol (ovine wool, &gt;98%)</td><td>Avanti Polar Lipids</td><td>700000</td><td>Same as above.</td></tr><tr><td>DiD&#039; oil; DiIC18(5) oil (1,1&#039;-Dioctadecyl-3,3,3&#039;,3&#039;-Tetramethylindodicarbocyanine Perchlorate)</td><td>Molecular Probes</td><td>D-307</td><td></td></tr><tr><td>Rotavapor R-210</td><td>Buchi</td><td>R-210</td><td>heat bath above Tm of lipids used</td></tr><tr><td>OG n-Octyl-&amp;beta;-D-Glucopyranoside</td><td>Affymetrix</td><td>0311</td><td>store at -20 &amp;deg;C, let come to RT before opening</td></tr><tr><td>Shaker - Eppendorf Thermomixer R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Slide-A-Lyze Dialysis Cassettes, 20k MWCO, 3&amp;nbsp;ml</td><td>life technologies</td><td>66003</td><td></td></tr><tr><td>Bio-Beads SM-2 Adsorbents</td><td>Bio-Rad</td><td>1523920</td><td></td></tr><tr><td>OptiPrep&amp;nbsp;Density Gradient Medium</td><td>Sigma-Aldrich</td><td>D1556</td><td></td></tr><tr><td>Ultracentrifugation tube, Thinwall, Ultra-Clear, 13.2&amp;nbsp;ml, 14 x 89 mm</td><td>Beckman Coulter</td><td>41121703</td><td></td></tr><tr><td>Beckman SW41 Ti rotor</td><td></td><td></td><td></td></tr><tr><td>SuflorhodamineB</td><td>Molecular Probes</td><td>S-1307</td><td></td></tr><tr><td>Econo-Column&amp;nbsp;Chromatography Columns, 2.5 &amp;times; 10 cm</td><td>Bio-Rad</td><td>7372512</td><td></td></tr><tr><td>Sepharose CL-4B</td><td>GE Healthcare</td><td>17-0150-01</td><td></td></tr><tr><td>SYPRO Orange Protein Gel Stain</td><td>Molecular Probes</td><td>S-6650</td><td>5,000x Concentrate in DMSO</td></tr><tr><td colspan="4">&lt;strong&gt;PDMS block&lt;/strong&gt;</td></tr><tr><td>Sylgard 184 Silicone elastomer kit, PDMS</td><td>Dow Corning</td><td>3097358-1004</td><td></td></tr><tr><td>Pyrex glass petri dish, 150 x 20 mm, complete with cover</td><td>Corning</td><td>3160-152</td><td></td></tr><tr><td>Hole puncher - Reusable Biopsy Punch, 0.75&amp;nbsp;mm</td><td>World Precision Instruments</td><td>504529</td><td></td></tr><tr><td>Manual Hole Punching Machine&amp;nbsp;</td><td>SYNEO</td><td>MHPM-UNV</td><td></td></tr><tr><td>Drill .035 x .026 x 1.5 304 SS TiN coated round punch</td><td>SYNEO</td><td>CR0350265N20R4</td><td>drill diameter: 0.9 mm, punch bore size: 0.74 mm</td></tr><tr><td>Tygon Microbore tubing, 0.25 mm ID, 0.76 mm OD</td><td>Cole-Parmer</td><td>06419-00</td><td>0.010&quot; ID, 0.030&quot; OD</td></tr><tr><td>Silicone Tubing (0.51 mm ID, 2.1 mm OD</td><td>Cole-Parmer</td><td>95802-00</td><td>0.020&quot; ID, 0.083&quot; OD</td></tr><tr><td colspan="4">&lt;strong&gt;Cover glass - cleanroom cleaned&lt;/strong&gt;</td></tr><tr><td>Schott Nexterion cover slip glass D</td><td>Schott</td><td>1472305</td><td></td></tr><tr><td>plasma cleaner</td><td>Harrick</td><td>PDC-32G</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;pTIRF setup and accessories&lt;/strong&gt;</td></tr><tr><td>IX81 microscope body</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>EM CCD camera</td><td>Andor</td><td>ixon-ultra-897</td><td></td></tr><tr><td>Thermo Plate, heated microscope stage</td><td>Tokai Hit</td><td>MATS-U52RA26</td><td></td></tr><tr><td>1 ml hamilton glass syringes (4x)</td><td>Hamilton</td><td>81365</td><td></td></tr><tr><td>syringe pump</td><td>kd Scientific</td><td>KDS-230</td><td></td></tr></tbody></table>

snare-mediated fusion of single proteoliposomes with tethered supported bilayers in a microfluidic flow cell monitored by polarized tirf microscopy

在膜融合的泛在处理的融合孔的开口在两台以前分开的隔室之间的第一个连接。在通过胞外分泌神经递质或激素释放的融合孔可以瞬时打开和关闭多次，规范货物释放动力学。孔动力学也确定囊泡循环的模式;在短暂的"吻了就跑"融合不可逆转再加盖印章的结果，而扩张，从而达到完全融合。为了更好地了解哪些因素支配孔隙动力学，我们开发了一种测定中使用偏振全内反射荧光（TIRF）显微镜用单分子灵敏度和〜15毫秒的时间分辨率在体外系统中的生物化学良好定义并监控膜融合。融合的荧光标记的含V-SNARE蛋白（V-越野车）与平面双层轴承T-SNARE蛋白小单层囊泡，支持软聚合物垫（T-SBL，T-支持双层），被监视。该法采用微流体流动通道，以确保最少的样品消耗，同时提供越野车的密度不变。利用在熔化过程中从SUV脂质标签转移到SBL的快速信号增强，脂质染料转移的动力学进行监测。 TIRF显微镜的灵敏度可跟踪单个荧光脂质标签，从中脂质扩散和SUV的大小可以推断为每个融合事件。脂质染料释放时间可以更长比预期通过永久开放的毛孔畅通的通道。使用该假设脂质释放的相位差是由于孔隙闪烁，细孔"开放"，该时间孔隙保持熔融时打开的分数的模型，可以进行估计。可溶性标记可以在SUV的脂质和可溶性货物放行的同步监测进行封装。这种测量表明失去可溶性货物的一小部分经过一些孔隙可以重新密封。

Watch this Scientific Journal Video about SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy at JoVE.co

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

在这里，我们提出了一个协议来检测使用偏振TIRFM脂质体和微流体通道支持双层之间的单，SNARE介导的融合事件，与单分子敏感性和〜15毫秒的时间分辨率。脂质和可溶性货物放行可同时检测。脂质体大小，脂质扩散率，和融合孔性质被测量。

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

snare-mediated-fusion-single-proteoliposomes-with-tethered-supported

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.8K 次观看.  Yale University School of Medicine. 在这里，我们提出了一个协议来检测使用偏振TIRFM脂质体和微流体通道支持双层之间的单，SNARE介导的融合事件，与单分子敏感性和〜15毫秒的时间分辨率。脂质和可溶性货物放行可同时检测。脂质体大小，脂质扩散率，和融合孔性质被测量。 

视频: SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

无洗涤剂超快重组膜蛋白成脂双层使用融合补充充电 Proteoliposomes。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

科研

生物化学

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

形成免疫突触和Kinapse研究的支持平面双层膜

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

生物学

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

重建线粒体膜动力学的模型膜平台

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

生物工程

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

聚合物拥挤脂质膜上的单分子扩散和组装

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

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 ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

荧光寿命成像显微镜观察细胞内圈套的贩运

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

免疫与感染

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

SNARE蛋白介导的膜融合酶的细胞融合含量的分析

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

在支持的脂质双层上重建膜系的最小肌动蛋白皮质

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics

Source: Curtis, B. N. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/64090">Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.</a> J. Vis. Exp. (2022)
In this video, we demonstrate a procedure to investigate septin organization on a glass-supported lipid bilayer in vitro using total internal reflection fluorescence microscopy.

平面支持的脂质双层测定:一种基于显微镜的体外技术，用于研究生物膜模拟物上的 septin 组装

Source: Curtis, B. N. et al. Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022)
In this ...