We thank Dr. Gaudenz Danuser and members of his lab for creating the plusTipTracker software and for helpful discussion regarding using the software, in particular Maria Bagonis and Sebastien Besson. We especially thank the Boston College Media Center for their assistance and support in the creation and editing of the video. We also thank members of the Lowery Lab for useful discussions and constructive criticism, and Abigail Antoine for proof-reading the manuscript. This work was funded by an NIH R00 MH095768 award to LAL.

该协议与视频是为了作为一个伴侣到原来的文件，说明更详细地4软件包，以及附带的软件下载Danuser实验室网站上的技术报告。我们鼓励读者如果有使用该软件对其他问题仔细审查这些文件。 1，在此之前的图像分析<ol><li>每个定时影片转换成TIFF（标记图像文件格式）图像文件的顺序。如果有多个生长锥/单元在给定的电影，第一茬每一个生长锥/格打造自己的图像序列。 注意:这是没有必要的，因为各个区域，其中感兴趣区（ROI），可以在plusTipTracker来选择。然而，使用较小的图像尺寸增大了运算处理的速度，所以此步骤，建议如果在图像中显著空白空间。 </li><li>保存每个系列的TIFF在自己的在MATLAB中设置访问路径中被称为"图像"文件夹（注意，"图像"是区分大小写）。要添加一个新的路径，浏览到相关的文件目录中的"当前文件夹"窗口，在该目录图标上单击右键，选择"添加到路径 - 选定的文件夹和子文件夹"。重要的是，该plusTipTracker软件文件夹被添加到路径，以及。 </li></ol> 2 plusTipGetTracks 注:在图像分析中的第一步骤是检测EB1-GFP彗星，彗星链接到轨迹，并确定微管动力学的参数。这是获得与命令"plusTipGetTracks"4。 <ol><li>要开始分析，开放式的MATLAB应用程序，并输入"plusTipGetTracks"进入命令行窗口。这将导致出现一个新的对话框。 </li><li>点击"设置新项目"，然后选择一个（或MOR先前的TIFF图像序列，通过选择适当的"图像"文件夹（或包含"图片"文件夹）目录E）。一旦这一步完成后，文件目录（roi_1）将被创建（在保存"图像"在同一个文件夹），将包含未来的数据文件。注:在"设置新项目"中的步骤可以提前完成，一个单独的会话中。 </li><li>一个新的窗口将出现:"选择一个多边形，在最后一点上单击右键，然后单击"创建规则&#39;"。点击"确定"。所选择的图像序列的第一个图像将被显示出来。使用鼠标点击，并创建一个多边形，它包括生长锥的全部内容。双击鼠标，关闭该多边形。 </li><li>一旦多边形被关闭，一个对话框将会出现:"你想选择其他的投资回报率？"如果图像有另一个生长锥来分析，选择"是";否则Š选"否"。 </li><li>选择要立即分析该项目。点击"选择项目"，然后选择文件夹（roi_X）来分析。 </li><li> àlistSelectGUI画面会出现。选择从屏幕的左侧的项目（次）和将它们移动到右侧的画面。点击"确定"。选择一个位置保存项目列表，然后单击"保存"。 </li><li>选择"检测"，"跟踪"和"后处理"。一旦这些选择已经作出，右侧的对话框将成为可配置的。配置每个选项。 <ol><li>这些参数被用于检测的彗星链接到MT的轨道。在选择这些控制参数进行跟踪详情载于随附的软件下载技术报告的PDF 9-10页;仔细阅读本报告，如果遇到问题。跟踪EB1-GFP的彗星在Xenop的目的我们蟾生长锥，使用下面的跟踪设置:搜索半径范围（像素）5-12，最小分履带长度（帧）3;最大间隙长度（帧）8;最大收缩率因数0.8，最大倾角前50，最大角度10落后，波动半径2.5。这些设置被显示在图1。 </li></ol> 注:最大收缩率设定为减少的检测"向后差距"的数目，如"向后差距"不在的生长锥的上下文来分析是有用的，给定的拥挤状况和可能的错误在轨道之间的联系。此外，无论是最大前进角和波动半径设定为相对较高，为生长锥的MT表现除了生长和收缩率小的频繁易位，并增加这些控制设置允许在连接这一步增加运动。 <ol><li>在后处理设置填充取决于所需的特定图像采集的设置。 </li></ol> &lt;/ LI&gt; <li>一旦设置已配置，点击"开始"。取设定已选择的软件的运行。这可能需要几分钟到几小时，这取决于所选择的项目和它们的大小的数目。在命令窗口中会显示剩余的每个函数的估计时间。当plusTipGetTracks步骤完成后，在命令窗口中会显示"完成！" 注:MT动力学具体参数现在已经计算了该软件的长名单（见参考文献4的细节），但对于MT动态的生长锥的分析，最有用的参数来检查是MT增长的轨道速度（以微米/分钟），增长的轨道寿命（以秒为单位），并且增长的轨道长度（微米）。 </li></ol> 3 plusTipSeeTracks 注意:我们的微管轨道已经被定义，则函数"plustipSeeTracks"用于轨道可视化4。此功能可以提供用于可视化多个输出时，包括空间MT动力学的地图和速度的电影，但这里的重点是仅仅使用"音轨影"以显示叠加在生长锥图像MT轨道。虽然plusTipGetTracks可以同时分析多个电影，plusTipSeeTracks只能分析一次一个电影。 <ol><li>输入"plusTipSeeTracks"进入命令行窗口。 </li><li>在对话框中加载后，点击"选择项目"。选择包含​​该项目的可视化，并单击"选择文件夹"的父目录。一个新的窗口将出现"选择您希望显示的项目"。选择可视化，并单击"确定"的文件。 </li><li>接下来，点击"选择保存的投资回报率。"导航到同一个文件夹roi_X在上一步中选择一个，并选择名为"roiYX"的文件。 </li><li>点击"选择输出目录"指定瓦特这里的MATLAB将节省的轨道可视化文件。注意:我们建议您使用包含数据的其余部分相同的文件夹中。 </li><li>选择"Make轨道电影"，并在屏幕中会显示所有与+提示计算彗星轨道plusTipGetTracks。这一步可以节省的跟踪时间序列在电影格式，文件"allTracks_X_X_X"中。有用于保存电影为AVI选项，否则默认格式是作为Quicktime.mov文件。 </li></ol> 4 plusTipGroupAnalysis 注意:此最终功能是用于创建动画的群体进行分析和他们的MT轨道参数的比较。 <ol><li>输入"plusTipGroupAnalysis"进入命令行窗口。要手动选择组进行比较，首先取消选择"自动组从层次结构"。然后，点击"选择项目"。导航到包含所有roi_X文件夹的父目录来分析。 </li><li> àlistSelectGUI画面会出现。选择所有的项目中的基团，从屏幕的左侧包括与它们移动到右侧的画面。点击"确定"。选择一个位置保存项目列表，然后单击"保存"。 </li><li>将出现一个窗口:"请从列表中选择第一集团"。点击"确定"。该listSelectGUI窗口将再次显示。这一次，只选择那些符合应当汇集起来的第一批文件。点击"确定"。 </li><li>然后，输入组名称，然后单击"确定"。将出现一个窗口:"选择另一个组？"答案据此，继续选择组。将出现一个窗口:"选择一个位置来保存您的群组列表"。导航到位置，然后单击"保存"。 </li><li>点击"选择输出目录"，选择其中的输出文件夹将存储。 </li><li>选择要进行哪种类型的组的分析 - MT的磁道是否应该合并的每个组或每个细胞分析应该执行。推荐的统计测试已经指定。以包含所有的分析磁道，取消选择"删除在电影的开始/结束磁道"。否则，有此复选框选中删除所有MT增长的轨道是在过程的影片开始或结束。 </li><li>本集团的分析做出选择后，选择"比较组"。 </li></ol>

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The authors declare that they have no competing financial interests.

PlusTipTracker提供了一个简单的，图形化的用户界面，可以快速自动检测几乎所有可见的EB1-GFP彗星在一个细胞或生长锥中，彗星链接进入轨道，并计算吨参数。其它出版物都报道了相似类型的软件的设计（例如，马克思等人的标签EB1动力学在生长锥18也用于定量分析）。但是，这个软件似乎是在其四通八达的交通网络独特的，因为它是自由的Danuser实验室的网站，专门设计的开放源代码可下载的交钥匙软件有用的细胞生物群落。而获得的MATLAB是必需的，一个不需要充分熟悉，以便利用该软件在此计算机的应用程序。不过，也有需要的易用性来解决的几个问题。 首先，使用该SOF时出现的最常见的问题之一tware和计算机应用首次被相关的文件路径。如果出现此错误（与提示"错误使用CD - 参数必须包含一个字符串中的错误formatPath ..."），那么最简单的解决方案是确保plusTipTracker软件，以及该目录与所有的"象"子目录，都在相同的"MATLAB"文件路径。最好是，如果这些都没有在"Program Files"目录，因为它已被认为在"程序文件"名字空间可能是一个问题。与此相关，值得注意的是，plusTipTracker保存时首先计算plusTipGetTracks分析这是所使用的文件的路径，并且同样地，该文件的路径，必须保持当此数据被访问，并且通过使用另外的软件组件使用的是非常重要的。功能plusTipGetTracks，plusTipSeeTracks和plusTipGroupAnalysis所有使用原来保存的文件路径，并且因此，尝试到c所有这些功能对于一个给定的电影，将文件移动到其他路径后，会导致错误。 在跟踪过程中plusTipGetTracks步骤失败发生在分析过程中另一个常见的​​错误是。这会发生，如果在图像序列的帧中没有检测到的彗星。这将彻底停止了分析，并不会出现后期处理。一个简单的办法来规避这个问题，让分析着手，就是在它不会被错误地连接到任何实际轨道的空间，创造了图像上的模拟彗星。这不会影响最后的轨道参数，作为不连续帧的最小数量存在任何彗星将被过滤掉的最终分析。 可能出现的一个其它的问题是有故障的彗星检测。这通常可以固定通过提高感兴趣区域的选择在步骤2.3。它紧密绘制所述感兴趣区域的细胞周围和未画出重要的是AW伊德尔区域比是必要的。该软件使用这个区域来确定彗星探测过程中使用的背景。如果彗星检测仍是次优的默认设置，这些设置可以在plusTipGetTracks窗口（在步骤2.7）进行调整。 任何分析后，重要的是通过肉眼进行验证的自动轨道键，使用plusTipSeeTracks。跟踪设置可能需要进行修改，以减少假阳性或假阴性彗星键的号码。看到原来plusTipTracker文件4，以及伴随软件下载的优化设置的详细信息的技术报告的PDF。这个软件相比手工跟踪的性能以前在非神经元细胞4进行了测试。生长锥构成一个稍微不同的挑战，但是，由于生长锥微管呈现出频繁易位在所有方向17，除了MT增长和收缩。一个ISSU未找到是一个主要关注e是在生长锥的紧凑的MT是否对跟踪的困难17。 MT的，因为只有一个子集，是在不断发展的阶段，EB1-GFP的目的，解决和跟踪个人EB1-GFP的彗星是不是有问题。然而，应该指出的是，使用非洲爪蟾生长锥这些先前的研究，这是特别选择，因为它们相对较大的生长锥的尺寸（大约10微米），比其他脊椎动物的生长锥。使用这些较大的生长锥可进行更精确的EB1-GFP的彗星分析。 以评估该软件用于分析EB1-GFP轨道在爪蟾生长锥的实用性和准确性，我们比较了使用plusTipTracker用手工跟踪一个相同数据系列的经验（数据未显示）。所花费的时间手工轨道EB1-GFP的彗星在39彗星轨道（平均生长锥在1分钟的时间推移SERIES，与各帧之间2秒）为两小时以上，相比之下，两分钟的软件。用两种方法得到的参数是（用于自动跟踪与每分钟7.0微米的手工跟踪每分钟7.4微米）相似吨增长速度。然而，对于生长寿命和长度，软件分析导致显著较短轨迹（约一半的时间和距离）。这是由于生长的轨道被分成由软件如果彗星进入和移出焦点随着时间的推移。而人眼能很容易地识别出它是同一颗彗星，该软件不会。此问题是不存在问题不过，如果一个人使用该软件以比较多个条件。因为相同的跟踪参数用于所有条件（并假设彗星以相同的速率在多个条件出入焦点），则相对寿命和长度仍然比较相当有用的测量。至于自动分析错误鼠ES，这些极大地依赖于图像的质量。在高的信号 - 噪声电影，错误结合或不正确轨迹的百分比是在个位数。即使在低质量的电影（其中个体彗星仍然由眼清晰可见，但在背景噪声大值），错误率仍然足够低（5-15％），该保存用的软件的显著时间是值得的成本中的错误。这一点尤其在分析数百生长锥（每种条件60至八个生长锥是在先前的研究中17分析的）的情况。 要注意的是这个软件被设计用于检测+ TIP彗星只绑定到生长的MT端，如EB1-GFP是很重要的。除了不断增长的两端鉴于联动和跟踪算法期望彗星只存在于微管的聚合，用这个软件来分析荧光标记+提示，结合缩水MT的动力结束会导致不正确的信息关于计算的吨增长速度。 一个这样的软件相对于其他单颗粒跟踪软件的独特的特点，是它考虑到已知的MT行为来计算不仅聚合参数，而且收缩参数。它通过将一个EB1-GFP的彗星已经消失了一个新已经在同样的轨迹形成的正后方它这样做（这就是所谓的backgap，或bgap轨道）。同时，该算法可以很好地用于某些类型的细胞，例如HeLa细胞4，它是一种不太有效的特征在生长锥分析MT动力学时。这是因为MT轨迹经常彼此跟随沿着在生长锥完全相同的路径（通常是沿F-肌动蛋白束以下），因此它通常是不可能分辨bgap键是否正确。出于这个原因，它是不推荐使用在生长锥的bgap数据输出。 尽管有这些微不足道的瑕疵和问题必须使用plusTipTracker时必须考虑（和几乎所有的自动图像处理程序），该软件可用于分析数以千计的EB1-GFP彗星在一段相对较短的量非常有用的工具。

该方法的目的是获得关于微管（MT）的量化信息加端跟踪蛋白（+ TIP）中活的生长锥动力学。 MT +技巧是一组定位于微管9,10的加末端蛋白。他们进行了一系列的功能来调节吨动力不稳定性11，包括聚合，灾难和救援率的参数。一个很好的方法，用于分析MT动力学是跟踪+ TIP EB1，其特异性地结合到生长的MT加结束1,12的行为。 EB1是众所周知的招募其他几种蛋白不断增长的MT加端13,14，和最近被确立为MT成熟因子15，推动两吨增长和灾难频15,16。 在生长锥的MT动力学许多研究都采用手工跟踪方法来测量时间的推移1-3改变EB1-GFP的动态，EB1 localizati上到MT加-端可被用作一个标记为MT聚合。审查E​​B1-GFP的彗星为吨增长了代理的一个关键好处是，MT动力甚至可以在显著吨重叠区域进行测量。而手跟踪EB1-GFP彗星的方法提供了有用的见解MT行为1-3，这是耗时的，并且可以被偏置。此外，作为异常的生长锥的行为很可能在细胞骨架动力学分钟的变化，分析MT的仅一小部分的结果（通常是在有需要时手动跟踪）可能会错过显著信息。 因此，我们测量使用的软件包，plusTipTracker 4全球MT动力学参数，采集的高分辨率，标记EB1在培养胚胎生长锥5直播之后的图像。这个软件在Danuser实验室开发的，已被用于一些研究中的各种细胞类型6-8表征MT动力学。它是一个开源的，我们ER型，基于MATLAB软件包，包括自动检测，跟踪，可视化和分析的荧光标记+小费电影。 MT的动力的具体参数是由该软件计算出的长长的名单（见参考文献4的细节），但在生长锥的MT动态分析，最有用的参数是MT增长的轨道速度（微米/分钟），增长的轨道寿命（以秒为单位），并且增长的轨道长度（微米）。该软件可以直接从Danuser实验室网站下载（下称"软件"）。而Danuser实验室目前支持+提示跟踪分析，这是纳入到一个软件包，称为U-轨道2.0，原始的，独立的软件将继续提供一个较新的接口。两个程序之间的底层算法是相同的（至少为2014），具有接口和数据输出，仅相差。对于小MATLAB和/或计算分析experi初级用户ENCE，plusTipTracker有更多人性化的功能，包括自动统计参数输出。 在这里，我们描述了用于分析在培养的爪蟾生长锥EB1-GFP的动态图像的步骤。该协议被用在最近的一篇论文研究的MT动力17。也为表达对EB1-GFP的培养生长锥的详细说明，请参阅洛厄里等人 2012 5。虽然本文主要侧重于审查中的生长锥EB1-GFP动力学，相同的方案可以用于其他类型的细胞17。对于所有类型的细胞，帧之间的时间间隔应为0.5-2秒，最优+ TIP跟踪之间。最多为4秒的帧之间的时间间隔是可能的，但这种增加的间隔时间导致额外的跟踪误差。

<table><tbody><tr><td>plusTipTracker software</td><td>Danuser Lab</td><td>&lt;a href=&quot;http://lccb.hms.harvard.edu/software.html&quot;&gt;http://lccb.hms.harvard.edu/software.html&lt;/a&gt;</td><td>This software may be hosted by another website in the future.&amp;nbsp; If the listed site does not exist, search &quot;Danuser Lab Software&quot; on a web search engine to find the site.</td></tr><tr><td>MATLAB software</td><td>Mathworks</td><td>&lt;a href=&quot;http://www.mathworks.com/products/matlab/&quot;&gt;http://www.mathworks.com/products/matlab/&lt;/a&gt;</td><td></td></tr></tbody></table>

using plustiptracker software to measure microtubule dynamics in xenopus laevis growth cones

微管（MT）的正端跟踪蛋白（+ TIPS）定位于微管的日益加端和规范的MT动力1,2。其中最知名的和广泛使用的+小技巧分析MT动力学是最终结合蛋白EB1，这一切结合不断增长的MT加末端，因此，是一个标志吨聚合1。内生长锥EB1行为的许多研究已经使用费时和偏置计算机辅助，手跟踪方法来分析单个微管1-3。我们的方法是用量化的软件包，plusTipTracker 4吨动力学全局参数，继收购高分辨率，标记EB1在培养胚胎生长锥5实时图像。这个软件是一个基于MATLAB的，开源的，用户友好的，结合了自动检测，跟踪，可视化和分析的荧光标记+小费电影包。在这里，我们提出的协议使用plusTipTracker为荧光标记+提示彗星在培养非洲爪蟾生长锥的分析。但是，此软件还可以用于在各种细胞类型6-8来描述MT动力学。

使用如下所述的提供信息的几个文件，量化+动态提示在活细胞中该软件。 函数plusTipGetTracks标识（使用图1中所示的示例设置）磁迹，然后提供关于+提示轨道参数。要查看该软件已经获得的信息，进入了在步骤2.2中创建的roi_X目录。的"壮举"文件夹包含"overlayImages"，这是一系列的图像显示检测到的彗星。检查用图像分析软件可以显示彗星检测的精度这些图像。 "元"文件夹还包含有关+提示彗星统计数据的详细信息，其中包括"projData"的文件，以及在"统计"的文件。要查看"统计"的文件，将其拖动到电子表格应用程序的开放工作。此文件包含计算ð微管参数每部电影（ 图2）。如上所述，MT的动力具体参数一长串的计算由本软件（见参考文献4的详细信息），包括MT增长的轨道速度（微米/分钟），增长的轨道寿命（以秒为单位），以及增长的轨道长度（微米）。 该功能plusTipSeeTracks保存电影的追踪彗星，它可以通过打开文件"allTracks_X_X_X"（ 图3）进行审查的。 功能plusTipGroupAnalysis将多个单独的数据集划分为组，并创建文件夹（命名perCell或pooledData，这取决于所选择的分析），包含组参数数据，包括直方图，图，并为每个组内比较组和各个参数的电子表格（ 图4）。 <img alt="图1" fo:content-WIDTH ="6英寸"SRC ="/文件/ ftp_upload / 52138 / 52138fig1highres.jpg"WIDTH ="600"/&gt; 用于在爪蟾生长锥EB1-GFP彗星图1 PlusTipGetTracks设置，该图显示了可用于EB1-GFP的分析，"检测"，"跟踪"和"后处理"的步骤的特定的设置彗星在非洲爪蟾生长锥。该plusTipParamSweepGUI工具，该plusTipTracker包内可用，可用于优化其它模式生物和/或细胞类型7的跟踪设置。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/52138/52138fig2highres.jpg" width="500" /> 图2:屏幕截图plusTipGetTracks分析得到的MT参数。"元"的文件夹，运行plusTipGetTracks创建，包含了关于+提示C分享欧米特统计。通过拖动"统计"文件到一个电子表格应用程序，微管动力学参数进行检查。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/52138/52138fig3highres.jpg" width="600" /> 图3的屏幕截图从plusTipSeeTracks分析得到的MT轨迹的电影。PlusTipSeeTracks不但允许微管轨道的可视化，但也允许用户观看从plusTipGetTracks获取的数据的有效性可作为验证的工具。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/52138/52138fig4highres.jpg" width="500" /> 图4屏幕截图plusTipGroupAnalysis得到吨的参数。PlusTipGroupAnalysis为用户提供了一种简单的方法来比较组和betwee各个参数通过组合多个单独的数据集，并生成统计输出，从而可以在电子表格应用程序进行检查n各自组内。

Watch this Scientific Journal Video about Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones at JoVE.com

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

该基于MATLAB的，开源软件包，plusTipTracker，可用于分析图像序列的荧光标记+小费量化微管动力学。

使用plusTipTracker软件测量微管动力学<em&gt;非洲爪蟾</em&gt;生长锥

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...

using-plustiptracker-software-to-measure-microtubule-dynamics-xenopus

Nanyang Technological University

Research

JoVE Journal

Biology

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

10.3K 次观看.  Boston College. 该基于MATLAB的，开源软件包，plusTipTracker，可用于分析图像序列的荧光标记+小费量化微管动力学。 

视频: Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

科研

生物化学

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

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

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

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

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

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

神经科学

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion.

Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.

分段微管的制备研究运动驱动的微管拆解完

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

生物学

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

肌动蛋白动力学、离合器耦合和牵引力分析，促进生长锥体前进

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

高分辨率成像和芽殖酵母个人星体微管动力学分析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

通过旋转磁盘显微镜在单极式斜接主轴中测量微管动力学

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

使用活细胞显微镜测量扩散过程中的细胞边缘突起动力学

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...

使用plusTipTracker软件测量微管动力学

摘要

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此视频中的章节

通过TIRF显微镜在体外同时可视化交联和单微管的动力学

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

分段微管的制备研究运动驱动的微管拆解完

肌动蛋白动力学、离合器耦合和牵引力分析，促进生长锥体前进

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

高分辨率成像和芽殖酵母个人星体微管动力学分析

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

通过旋转磁盘显微镜在单极式斜接主轴中测量微管动力学

使用活细胞显微镜测量扩散过程中的细胞边缘突起动力学

使用plusTipTracker软件测量微管动力学

摘要

探索更多视频

此视频中的章节

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

分段微管的制备研究运动驱动的微管拆解完

肌动蛋白动力学、离合器耦合和牵引力分析，促进生长锥体前进

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

高分辨率成像和芽殖酵母个人星体微管动力学分析

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

通过旋转磁盘显微镜在单极式斜接主轴中测量微管动力学

使用活细胞显微镜测量扩散过程中的细胞边缘突起动力学

通过TIRF显微镜在体外同时可视化交联和单微管的动力学