这项工作是支持由美国国立卫生研究院拨款AI57159（SCH）和AI72346（AMvO）。 SCH是霍华德休斯医学研究所的研究员。

玻璃盖滑官能在融合实验中使用的是平面双层支持的葡聚糖水合膜。葡聚糖作为一个平面双层玻璃表面之间的间隔。这可以防止成为停留在玻璃表面的膜元件，并提供病毒粒子的内容可以逃脱后，融合的空间。盖玻片官能通过与环氧硅烷，它允许我们化学债券右旋糖酐玻璃（Elender， 等，1996年）的治疗。 <ol><li>排列在陶瓷染色架25毫米的盖玻片，并将其放置到一个jar或烧杯机架。 </li><li>填充10％的实验室高档玻璃器皿洗涤剂溶液的容器中。将在30分钟的超声波清洗机的容器。 </li><li> 1M postassium氢氧冲洗去离子水的容器和盖玻片机架，和填充。返回容器浴30分钟sonicator。 </li><li>重复此清洗程序，使用丙酮和乙醇。 </li><li>冲洗水和干燥的coverlips。 </li><li>最后，清洁的盖玻片，用三分钟的氧等离子体脱衣舞。 </li><li>最后的清洁步骤氧化表面，使玻璃均匀的亲水性和反应在下列硅烷步骤。 </li><li>准备3 glycidoxypropyltrimethoxy硅烷在异丙醇0.2％的解决方案。浸泡5分钟，在此解决方案的盖玻片。 </li><li>丢弃硅烷溶液和异丙醇冲洗盖玻片几次。 </li><li>现在涂一层硅烷的盖玻片，但他们必须治愈让硅烷共价债券的玻璃。放在烤箱设置为80度摄氏一小时的盖玻片。 </li><li>虽然在盖玻片固化，掂量出右旋糖酐500，并准备了30％W / V解决方案。预热水将有助于溶解。我们将使用约1毫升每盖玻片的解决方案。一旦葡聚糖溶解，消泡在真空干燥器的解决方案。 </li><li>当硅烷盖玻片完成固化，从陶瓷机架中删除，并安排他们平面上的一个塑料冰柜盒里面。用1毫升吸管盖顶面〜1毫升右旋糖酐解决方案的每个盖玻片。关闭冷冻箱，并在一个安静的地方为24-36小时离开。 </li><li>在抓盖玻片，用塑料镊子倒出多余的葡聚糖和更换成陶瓷机架。在水冲洗盖玻片几次，让盖玻片，在水中浸泡48小时。的罐子可表上方肩轻轻地激动。 </li><li>在烤箱设置为80摄氏度，并储存在真空干燥器干燥的盖玻片。 </li></ol>脂质体的制备支持的脂质双层脂质体吸附葡聚糖官能盖玻片组成。吸附脂质体融合在一起海誓山盟，直到平在其表面的膜破裂和传播。 <ol><li>一个典型的脂质体配方，用于检测由1,2，dioleoyl - SN -甘油- 3 - phosphocholine（DOPC），1 -油酰- 2 - 棕榈酰- SN -甘油- 3 - phosphocholine（POPC），胆固醇，牛脑Disialoganglioside（GD 1A），和N -（（6 - （biotinoyl）氨基）hexanoyl）-1,2 - dihexadecanoyl - SN -甘油- 3 - phosphoethanolamine在X10 -5 4:4:2:0.1:5比例。所有组件都存储在氯仿或三氯甲烷和甲醇的解决方案。 </li><li>准备转移到玻璃试管中的每个组件数量，包含两个脂质微摩尔混合物。溶剂蒸发下氩气或氮气流。留在两个小时的真空干燥器的试管中取出剩余的溶剂。 </li><li> HNE缓冲区在400微升（5毫米HEPES，145毫米氯化钠，0.1毫米EDTA），重悬干脂质膜。 </li><li>暂停转移到塑料离心管。冻结暂停在液氮浴和解冻在温水澡。重复冻结/解冻周期的四倍。 </li><li>装配与100 nm的聚碳酸酯滤膜过滤的脂质挤出机，挤出的脂质悬液25倍。暂停挤压后会出现更加透明。 </li><li>脂质体应使用上，他们准备的一天。他们可在室温下保存，直至使用。 </li></ol>微流控流细胞制备一个简单的微流体流动细胞是由双节棍磁带夹在一个石英片和官能盖玻片。 <ol><li>获取一个20x20x3毫米的石英片。使用钻石毛刺对立面上钻两个1毫米的孔。 </li><li>剪下从双节棍带表的20平方毫米，并用刀片，切出15 x 2毫米的部分，从中心。 </li><li>揭去一面磁带备份，磁带和坚持的夸脱ž幻灯片。对方坚持一个官能盖玻片。 </li><li>聚乙烯管材削减两个20厘米的长度和它们在石英片上的孔插入。 </li><li> 5分钟环氧胶密封的流动池。 </li><li>胶水固化时，黄金的流动细胞和油管用缓冲液。 </li><li>一个受支持的脂质双分子层，可以形成在这个阶段，进入渠道的脂质体混悬液约80微升。 </li></ol>病毒粒子标签<ol><li> H3N2型流感A（X - 31）是通常用于融合实验，但已成功用于其他流感毒株和其他病毒。 </li><li>病毒是两种不同的荧光染料标记，以便检测hemifusion和孔隙形成。 </li><li>溶解在HNE缓冲区sulforhodamine B（SRB），20毫米的解决方案。结合病毒悬液10微升20微升的染料溶液，并在室温下20小时内离开。在此期间，染料会慢慢积累的病毒颗粒内。 </li><li>删除多余的染料，通过PD - 10脱盐柱的病毒悬液。病毒如果有足够的材料，色带可以看出，在列。这个频段包含标记的病毒，可以收集，因为它是洗脱。 </li><li>如果没有带是可见的，收集200微升的分数和确定分数包含标记病毒通过测量565 nm处的吸收分光光度计。病毒应在总量约0.8毫升洗脱。 </li><li>标签的SRB标记的病毒悬液加入13微升至2毫米的二甲基甲酰胺（DMF）十八烷基罗丹明110（Rh110C18）解决方案的病毒包膜。 </li><li>搅拌管连接到实验室肩暂停3个小时离开。 </li><li>使用前面所述的PD - 10脱盐柱净化过剩Rh110C18病毒。 </li></ol>显微镜配置配备高NA油浸物镜适合全内反射荧光显微镜荧光显微镜上进行实验。氩/氪气激光纳米线488和568，是用来激发Rh110C18和SRB标记的病毒。每个标签的同时成像是通过分裂与分色镜的绿色和红色荧光发射和独立集中到单独的一个电子倍增CCD相机半的图像。 执行检测执行涉及的融合实验流细胞内的生化成分逐步装配：装配平面脂双层，对接标记的病毒颗粒，涂层表面用荧光素，并开始与低pH值的缓冲（图1A）的反应。 <ol><li>山显微镜舞台上的流通池，流速度每分钟40微升注射器泵出口管连接。 </li><li>清除多余的脂质体的流通渠道流入300微升HNE。 </li><li>聚焦显微镜和调整激光功率350瓦/厘米2的488纳米的光，70瓦/厘米2 568纳米的光照亮表面。如果您使用的是一个60X NA 1.45油浸物镜，设置激光强度分别为100微瓦和20微瓦，。 </li><li>标记病毒（1 / 100稀释）泵入流通池。由于病毒扩散到流道底部表面，粒子将开始坚持在脂质双层中的神经节苷脂。 </li><li>当一个适合停靠的病毒颗粒密度已达到（到1000左右每视野颗粒）。开关的入口管的解决方案包含两个微克每毫升荧光标记的链霉。标记链霉素再加上双层脂质分子结合，并最终将会看到一个暗淡的绿色背景。 </li><li>洗流道与300微升的HNE。 </li><li>选择图像区域，聚焦显微镜和CCD相机准备时间已过图像采集。设定曝光时间为100 ms和收集率在10赫兹的帧。 </li><li>开始融合，在酸性缓冲液含有10 mM柠檬酸钠和140毫米氯化钠中流动。缓冲区的pH值应调整被检测的pH值低于该病毒的关键。 X - 31熔断器在pH低于5.5。 </li></ol>代表性的成果图1b显示了双层停靠融合病毒粒子之前和期间的快照。每个衍射极限点，代表一个单独的病毒粒子。红色和绿色荧光成像到CCD相机的顶部和底部的部分，从而使病毒包膜和内容标签的同步观测。通常有两倍多点相比，在绿色通道红色，这反映内容染料相比效率较低的标签，信封标签。从表面发射的约束荧光绿色背景荧光消失后的柠檬酸缓冲液的到来，并允许确定的核聚变反应的开始时间。病毒包膜弥漫在整个hemifusion秸秆淬火Rh110C18 Hemifusion事件阵阵绿色荧光检测，并在平面膜稀释。向外扩大荧光灯云可以看出hemifused颗粒周围，展现在平面膜染料的脂肪酰基相连的自由扩散。孔隙的形成是从SRB的红色荧光衰变被困在病毒颗粒的内部观察。融合孔的开放，使染料逃进下面的观察区域的平面双层和出的水空间。 每个粒子的荧光强度的轨迹，绘制从单个粒子的综合强度，促进hemifusion和孔隙的形成（图1C）倍的产量和决心。 Hemifusion和孔隙形成事件的时间是作为一个Rh110C18荧光爆裂或SRB信号衰减的最大坡度发生在哪个点确定。在一个成功的实验中，约50％Rh110C18产量dequenching信号，30％的SRB颗粒标记标记颗粒提供可用的信号。同时染料标记的颗粒，约10％，表明脂质混合孔隙形成的信号。 图2A - c显示hemifusion和孔隙形成的滞后时间从一个典型的实验的分布。图2d - F显示从五个相同的条件下进行的实验相结合的事件的事件分布。即使有一个温和的意见，直方图的形状是显而易见的。因为每一个实验是通过检测pH值下降时间同步，可以相结合，并多次实验，可以得到详细的限制中间步骤率的信息。 <img src="/files/ftp_upload/1484/1484fig1a.jpg" border="0" alt="图1" /> <img src="/files/ftp_upload/1484/1484fig1b.jpg" border="0" alt="图1" /> 图1实验设计。 A）病毒颗粒两种荧光染料标记，监察hemifusion和融合孔的形成的动力学。荧光是由高NA显微镜物镜收集到的CCD成像。二）荧光图像（右）前（左）和在个别病毒颗粒的融合。每幅图像的顶部和底部一半对应红色和绿色的荧光，分别支持双层面积相同〜50 × 100毫米2。三）红色SRB的病毒含量示踪（上部跟踪）的荧光强度，绿色Rh110C18膜染料（中等跟踪），和荧光pH传感器（低跟踪）提供之间的pH值下降，hemifusion，并融合后的确切时间。请<a href="https://www.jove.com/files/ftp_upload/1484/1484fig1.jpg">点击这里</a>看到图1的放大版本。 <img border="0" src="/files/ftp_upload/1484/1484fig2tmb.jpg" alt="图2" /> 图2荧光标记的病毒融合动力学。分布的pH值下降，hemifusion（A，D）和孔隙的形成（B，E）之间所经过的时间。上升和衰减的分布，表明存在多个中间步骤。 hemifusion为单个粒子的融合孔的开放之间的过渡，是指数分布，表明单一的限速步骤。小组交流展示一个单一的实验结果。面板的DF是由五个独立的实验，在相同条件下进行编译。请<a href="/files/ftp_upload/1484/1484fig2.jpg1484">点击这里</a>看大图图2。

<ol>
	<li>Elender, G., Kuhner, M., Sackmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalisation+of+Si/SiO2+and+glass+surfaces+with+ultrathin+dextran+films+and+deposition+of+lipid+bilayers.">Functionalisation of Si/SiO2 and glass surfaces with ultrathin dextran films and deposition of lipid bilayers.</a> Biosens Bioelectron. 11, 565-577 (1996).</li><li>Floyd, D. L., Ragains, J. R., Skehel, J. J., Harrison, S. C., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-particle+kinetics+of+influenza+virus+membrane+fusion.">Single-particle kinetics of influenza virus membrane fusion.</a> Proc Natl Acad Sci U S A. 105, 15382-15387 (2008).</li></ol>

流体和连续支持的脂质双层的准备工作都具有挑战性。微量污染物质或表面缺陷会阻止双层扩散。仔细清洗和沉积一层均匀的葡聚糖是必不可少的。 长时间的病毒颗粒成像可以漂白的荧光标记，或使他们成为灭活。在生物成像和单分子领域的照片这种损害是众所周知的，一般认为源于激发荧光基团的产生活性氧。为了最大限度地减少照片损伤，我们一般尽量减少激发激光功?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Corning cover glass squares, 25 mm</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>CLS286525</td>
<td>&nbsp;</td>
<tr>
<td>Fused silica slides</td>
<td>&nbsp;</td>
<td>Technical Glass</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Staining rack</td>
<td>&nbsp;</td>
<td>Thomas Scientific</td>
<td>8542E40</td>
<td>&nbsp;</td>
<tr>
<td>(3- glycidoxypropyl)trimethoxysilane</td>
<td>&nbsp;</td>
<td>Gelest</td>
<td>SIG5840.1</td>
<td>&nbsp;</td>
<tr>
<td>Dextran T-500</td>
<td>&nbsp;</td>
<td>Pharmacosmos</td>
<td>5510 0500 4006</td>
<td>&nbsp;</td>
<tr>
<td>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
<td>&nbsp;</td>
<td>Avanti</td>
<td>850375</td>
<td>&nbsp;</td>
<tr>
<td>1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC)</td>
<td>&nbsp;</td>
<td>Avanti</td>
<td>850457</td>
<td>&nbsp;</td>
<tr>
<td>Cholesterol</td>
<td>&nbsp;</td>
<td>Avanti</td>
<td>700000</td>
<td>&nbsp;</td>
<tr>
<td>N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (biotin-X DHPE)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>B1616</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin, fluorescein conjugate</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S-869</td>
<td>&nbsp;</td>
<tr>
<td>Disialoganglioside GD1a from bovine brain</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G2392</td>
<td>&nbsp;</td>
<tr>
<td>Mini Extruder</td>
<td>&nbsp;</td>
<td>Avanti</td>
<td>610000</td>
<td>&nbsp;</td>
<tr>
<td>0.1 micron polycarbonate membrane filters</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>800309</td>
<td>&nbsp;</td>
<tr>
<td>10 mm drain discs (membrane supports)</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>230300</td>
<td>&nbsp;</td>
<tr>
<td>Sulforhodamine b sodium salt</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>S1402</td>
<td>&nbsp;</td>
<tr>
<td>Octadecyl rhodamine 110 (Rh110C18)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>See Floyd et al. 2008 for synthesis protocol</td>
</tr>
<tr>
<td>PD-10 desalting columns</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>17-0851-01</td>
<td>&nbsp;</td>
<tr>
<td>Nikon fluorescence microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>TE2000-U</td>
<td>&nbsp;</td>
<tr>
<td>Plan Apo TIRF 60x 1.45 NA objective</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Innova 70C Spectrum Ar/Kr laser</td>
<td>&nbsp;</td>
<td>Coherent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>702213</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

method for measurement of viral fusion kinetics at the single particle level

膜融合是包膜病毒进入细胞的过程中必不可少的步骤。通常传统的融合检测报告，大量的融合活动，使其难以定量分析的分子步骤的顺序。我们已经开发出体外，双色荧光检测，监视与目标上基本上是流体支持双层的单个病毒颗粒融合动力学。 流感病毒颗粒，是培养一个绿色的染色膜和一个红色的亲水性荧光团染色的病毒内部的亲脂性荧光团。我们一个神经节苷脂含脂双层存款右旋糖酐functionilized玻璃表面上的流通池，孵化平面双层和图像100 x 100微米2区的荧光的病毒颗粒，包含几百个粒子在防治荒漠化公约“，摄像头。成像，红色和绿色的荧光，我们可以同时监控膜染料（绿色）和水含量的颗粒（红色）的行为。 在降低pH值低于融合的p​​H值值，颗粒会与膜的融合。 Hemifusion，合并外病毒膜与目标膜外单张单张，将作为一种绿色荧光粒子的突然变化可见。后，随后剩下的两个远端单张融合，将形成一个孔，将目标膜下释放病毒颗粒的红色发光的荧光基团。此事件会引起减少单个粒子的红色荧光。最后，从pH敏感的荧光基团，是嵌入在目标膜集成荧光报告的pH值下降的确切时间。 从三个荧光痕迹，所有的重要事件（pH值下降，脂质混合后hemifusion，混合孔隙形成后的内容）现在可以在一个简单的方式和单独的每个粒子中提取。通过收集直方图许多单个粒子的各种转换的运行时间，我们可以判断相应的中间体的寿命。甚至隐藏中间体，没有一个直接的荧光观察，可以直接从显示这些直方图。

Watch this Scientific Journal Video about Method for Measurement of Viral Fusion Kinetics at the Single Particle Level at JoVE.com

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

我们提出了一个<em在体外，</em&gt;两色荧光分析法的可视化与流体的目标双层单个病毒粒子的融合。通过标记荧光基团的差异染色病毒的细胞膜和其内部的病毒颗粒，我们是能够监测hemifusion和孔隙形成的动力学。

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

method-for-measurement-viral-fusion-kinetics-at-single-particle

Research

JoVE Journal

Biology

12.8K 次观看.  Harvard Medical School. 我们提出了一个

视频: Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.
For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as "single particles". Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule's 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.
In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution1 and random conical tilt (RCT) method2. In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method3.

This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).

单粒子电子显微镜外切体综合重建，使用随机的锥形倾斜方法

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

科研

生物学

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

测量单个活细胞中的mRNA转录的动力学

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

在单核级LINE-1逆转录转座子分析

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

小鱼肾注射液循环系统中微粒的简易有效管理与可视化

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb &#955; DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified &#955; DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &amp;#955; DNA at the Single Molecule Level

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified &#955; DNA substrate and a Quantum-dot labeled protein.

Qdot 标记蛋白与现场特异修饰的λ DNA 在单分子水平上的相互作用可视化

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

衣藻中心粒单粒子重建的分离与荧光成像

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...