Name: using plustiptracker software to measure microtubule dynamics in xenopus laevis growth cones
Uploaded: 2014-09-07T15:00:00
Description: Watch this Scientific Journal Video about Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones at JoVE.com

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?...

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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?...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">plusTipTracker software</td>
			<td style="width:116px;">Danuser Lab</td>
			<td style="width:345px;"><a href="http://lccb.hms.harvard.edu/software.html">http://lccb.hms.harvard.edu/software.html</a></td>
			<td style="width:172px;">This software may be hosted by another website in the future.&#160; If the listed site does not exist, search &#34;Danuser Lab Software&#34; on a web search engine to find the site.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Matlab software</td>
			<td style="width:116px;">Mathworks</td>
			<td><a href="http://www.mathworks.com/products/matlab/">http://www.mathworks.com/products/matlab/</a></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?...

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 ...

Research

JoVE Journal

Biology

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

Plastic Embedding and Sectioning of Xenopus laevis Embryos

塑料嵌入和非洲爪蟾胚胎切片

科研

生物学

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

从海兔的神经细胞培养的高分辨率成像生长锥

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Obtaining Eggs from Xenopus laevis Females

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

生产<em&gt;热带爪蟾</em&gt;卵提取物识别微管相关的RNA

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Facial Transplants in Xenopus laevis Embryos

Facial Transplants in Xenopus laevis Embryos

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

剖析<em&gt;非洲爪蟾</em&gt;神经嵴的<em&gt;在体外</em&gt;外植体培养或<em&gt;在体内</em&gt;移植

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...