Name: temporal quantification of mapk induced expression in single yeast cells
Uploaded: 2013-10-04T23:00:00
Description: Watch this Scientific Journal Video about Temporal Quantification of MAPK Induced Expression in Single Yeast Cells at JoVE.com

作者感谢马蒂亚斯彼得和他的小组在生物化学研究所在ETH苏黎世这些方法已被开发的地方。这项工作已经由瑞士国家科学基金会的支持。

1。显微镜测量<ol><li>接种5毫升合成培养基中的酵母。 </li><li>细胞生长于30℃过夜。 </li><li>测量过夜培养物的OD 600，第二天早上。 </li><li>稀释过夜培养在5毫升合成培养基，以OD 600 0.05。 </li><li>生长的稀释的培养在30℃下为至少4小时。 </li><li>制备3倍浓缩在合成培养基（0.6 M）的NaCl溶液。 </li><li>准备1毫克/毫升的溶液刀豆蛋白A（刀豆）的PBS中。 </li><li>滤液〜200微升的溶液刀豆入?...

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显微镜井经ConA的治疗是一个重要的步骤，以确保电池的正确成像。因为刀豆具有低溶解度的在PBS中的（5毫克/毫升）中，过滤过程可以去除大的聚集体存在于未过滤溶液，并干扰成像。细胞相对强烈地附着到被处理的表面和在加入诱导溶液不应妨碍细胞的定位，从而允许连续跟踪之前和之后的刺激。这个处理也可以应用到流动通道或微流体装置进行的细胞进行的恒定流量7,15...

通过转导级联信号常常达到高潮蛋白的表达。这种表达谱中的表征对于理解生物学途径的功能的关键因素。上调的蛋白及其活化的动态频谱的识别可以通过例如微阵列，RNA印迹或蛋白质印迹1-3的各种技术来实现。然而，这些技术平均细胞的全体人民的响应。了解蛋白质的表达的精细调节，理想的是收集的测量在单细胞水平。理想情况下，这些测量还应提供定量数据服从发展的基本途径的数学模型。 显微镜和流式细胞仪两种技术，这是非常适合提供这样的定量单细胞测量。蛋白质与荧光蛋白质的内源性标记可以bÉ用来量化其4表达水平。然而，由于在该蛋白质的末端增加了一个大的荧光部分可以使其不起作用的，它往往是更可取的，以产生根据一个启动子驱动的荧光构建体的表达特异性表达记者。此报告蛋白是外生给蜂窝系统，因此不影响正在发生在细胞内信号转导事件。 既显微镜和流式细胞仪已经被广泛应用于酵母的信令字段。作为例子;科尔曼-雷纳和同事相关的，在单个细胞中，配合特定的和组成性表达，记者通过显微镜来量化在酵母交配信号转导级联5的噪声的表达，阿贾尔和使用流式细胞仪来研究调控网络的同事控制的GAL基因6的表达。在先前的研究7，我们已经使用了一个化合物-关于这两种技术从高渗透压甘油（HOG）途径在芽殖酵母研究的表达输出。这有丝分裂原激活的蛋白激酶（MAPK）途径是由超渗透胁迫触发。它导致MAPK Hog1，这转位到细胞的核的活化，诱导产生大约300个基因的表达的转录程序。为了研究这个过程，我们设计了一个基于该STL1启动子（响应Hog1活动8特异性诱导基因）驾驶一辆四金星荧光蛋白（P STL1-QV）的表达表达记者。流式细胞术测量露天细胞的两个群体的存在下，在中间应力水平（0.1M NaCl）中以表达荧光报道人口的一小部分。我们用显微镜来进一步研究这个问题，并发现这种噪声的蛋白质表达，内在因素9管辖。我们凑LD进一步观察到细胞Hog1活动的一个类似的水平可能显示明显不同的表达效果。这两种技术的结合允许我们来演示如何的应激反应基因的慢重塑的影响表达产物在单细胞水平7。 在本文中，我们使用p STL1-QV记者通过超渗透休克诱导如通过显微镜蛋白表达的定量的一个例子中的表达和流式细胞术。一脉相承受到0.2M的NaCl胁迫，研究了这两种技术。这将允许我们强调在这两个高度互补的技术，一些关键的差别。

<table border="1">
		<tbody>
		 <tr>
			 <td></td>
			 <td></td>
			 <td></td>
			 <td>Reagent</td>
		 </tr>
		 <tr>
			 <td>Yeast Nitrogen Base</td>
			 <td>ForMedium</td>
			 <td>CYN6202</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>CSM amino-acid mix</td>
			 <td>ForMedium</td>
			 <td>DCS0011</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Concanavalin A</td>
			 <td>GE Healthcare</td>
			 <td>17-0450-01</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Cycloheximide</td>
			 <td>Fluka Aldrich Sigma</td>
			 <td>C7698</td>
			 <td>Toxic</td>
		 </tr>
		 <tr>
			 <td>ySP9</td>
			 <td></td>
			 <td></td>
			 <td>W303Mata leu2::LEU2-pSTL1-quadrupleV enus</td>
		 </tr>
		 <tr>
			 <td>pSP34</td>
			 <td></td>
			 <td></td>
			 <td>pRS305 pSTL1 (-800 -0) - quadruple V enus</td>
		 </tr>
		 <tr>
			 <td></td>
			 <td></td>
			 <td></td>
			 <td>Material</td>
		 </tr>
		 <tr>
			 <td>Microscope</td>
			 <td>Nikon</td>
			 <td>Ti-Eclipse</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Microscope control software</td>
			 <td>Micro-manager</td>
			 <td>Ver 1.4.11</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Incubation chamber</td>
			 <td>LIS</td>
			 <td>The Box</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Fluorescence light source</td>
			 <td>Lumencor</td>
			 <td>SpectraX</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Camera</td>
			 <td>Hamamatsu</td>
			 <td>Flash 4.0</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Flow cytometer</td>
			 <td>BD</td>
			 <td>FACS calibur</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Sonicator bath</td>
			 <td>Telesonic</td>
			 <td>TUC-150</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>8-well-slide </td>
			 <td>Thermo Lab-tek</td>
			 <td>155409</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>96-well-plate</td>
			 <td>Matrical bioscience</td>
			 <td>MGB096-1-2-LG</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>FACS tube</td>
			 <td>BD Falcon</td>
			 <td>352054</td>
			 <td></td>
		 </tr>
		</tbody>
 </table>

temporal quantification of mapk induced expression in single yeast cells

基因表达在单细胞水平进行定量揭示了掩盖在在群体水平上进行测量，新的监管机制。基于显微镜和流式细胞仪两种方法都表现出这样的数据怎么可以被收购。荧光报道诱导活化时的高渗透压甘油MAPK途径在酵母中的表达被用来作为一个例子。每种方法的具体优点突出。流式细胞术测量大量细胞（10,000），并提供了直接的措施独立的荧光蛋白的慢动力学成熟的蛋白表达的动力学。通过显微镜活细胞成像是通过对比限于记者在较少的细胞的成熟形式的测量。然而，通过这种技术产生的数据集可以是非常丰富得益于多个记者的组合，以及在空间和时间信息从单个细胞中获得。这两种测量方法的结合，可以通过信号传导通路兑现蛋白表达的调控新的见解。

显微镜酵母细胞轴承表达式记者p STL1-QV（ySP9 7）被附连到良好的滑动件的底部并在显微镜下放置。将细胞通过加入应力介质的成像会话的过程中刺激直接进入井。这使我们能够通路诱导前获得细胞的一些图片，并按照他们的命运刺激后。在本案中，随访细胞为〜2小时，以10分钟的时间间隔。 图1A是诱导和图1B前非荧光的细胞的图像显示2小...

Watch this Scientific Journal Video about Temporal Quantification of MAPK Induced Expression in Single Yeast Cells at JoVE.com

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

基于流式细胞术和显微术的两个互补的方法给出，使定量基因表达的诱导通过MAPK途径在酵母的激活的动力学，在单细胞水平。

在单酵母细胞MAPK诱导表达的时空定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

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Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

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

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