We thank Yumeng (Helen) Xia (Rutgers University) for manuscript editing. The work on BASL polarity formation is supported by grants from the U.S. National Institute of General Medical Sciences to J.D. (R01GM109080) and Rutgers University.

1.质粒构建<ol><li>产生由如前所述1,6-克隆技术的构建体。 <ol><li>先用适当的引物高保真DNA聚合酶扩增BASL的编码序列，YDA和MPK 6和它们子克隆到条目的载体。查找7的详细协议。 </li><li>以生成（分别DNmpk6 8和DNyda 4）MPK 6和YDA的显性负版本，使用MPK 6和YDA的条目的质粒为模板，通过定点诱变方法9引入点突变。 </li></ol></li><li>要建立烟草瞬时测定的结构，进行标准的LR（attL位点¯x重组的attR），反应（7详细过程），以BASL融入pH35CG 10，DNyda和DNmpk6到pXNGW和pXCGW分别载体11。 <ol><li>确认通过酶切和测序产生的质粒。一旦ŝuccessful，变换二进制构建入根癌农杆菌菌株GV3101 12和在28℃生长他们选择平板2 - 3天。 </li></ol></li></ol> 2. 农杆菌渗透溶液的制备<ol><li>接种几新鲜转化的土壤杆菌的菌落到10ml的Luria-BERTANI（LB）具有适当的抗生素培养基（庆大霉素25微克/毫升，利福霉素25微克/毫升，壮观霉素100μg/ ml的对pH35CG，pXNGW和pXCGW构造;同样的浓度的庆大霉素和利福霉素与卡那霉素50微克/毫升为P19（表达抗转基因的沉默蛋白质）13。 </li><li>生长在28°C摇床农杆菌培养在200转的速度约16小时，然后测量外径600（估计达到1.5 - 1.8）（ 图2A-B）。降速文化在2500 XG 10分钟。重悬和WAsh的含MgCl 2的10mM的（渗透液）的细胞沉淀。 </li><li>再次降速培养并重新暂停与浸润溶液适量粒料达到最终OD 600 = 0.5（ 图 2C-D）。浸润植物之前，离开在室温下将细胞混合物为1 - 3小时。此步骤有助于维持细胞稳态。 </li></ol> 3.卷烟厂和叶注射液<ol><li>使用烟草（ 本生烟），增长4 -第5周龄在一个标准的植物生长室（23℃，16小时光照/ 8小时黑暗的循环）14叶注射液。 注意:在此阶段，烟草植物通常具有6叶（2大，2培养基，和2小）。这两个中等大小的叶子是最优化的叶注射液（这两个路数太旧了，而两个小的往往是太年轻）。 </li><li>浸润前一天，浇灌tobac合植物饱和的土壤中。 </li><li>在浸润天，预孵育在黑暗中的植物为1 - 3小时。此步骤允许气孔在表皮广开，渗透到叶表皮细胞时，极大地方便了根 。 </li><li>使用标有日期的颜色条，旗叶进行渗透。标记区域用黑色粗Sharpie笔渗入（可选的;渗入领域是被感染后可见）。 </li><li>取农杆菌再悬浮等体积并在通过温和涡旋（ 图2E）100×15毫米的培养皿混合。注:在我们的例子中，我们混合1毫升P19的1毫升CFP-BASL的1毫升DNyda-nYFP和1ml DNmpk-cYFP的。 </li><li>在浸润的过程中，填充渗透混合物3mL的无针注射器（1 - 2毫升）中，推入烟草叶（ 图2F）的底侧（背面）。而渗透，它是有帮助用一只手推，另一只手轻轻支撑上侧（正面，对抗推压力）。 注意:一旦渗透混合物被成功注入，受感染的区域将成为在表皮容易辨认。然而，渗透可能会失败，由于恶劣的注射液（组织损伤）或推不足（没有液体渗透）。对于每个测试，我们建议在两个独立的叶子至少两个注射（来自不同的植物）。 </li><li>放置感染植物放回生长室。一般来说，蛋白质的表达都是经过48小时以下的渗透检测。 </li></ol> 4.共焦成像<ol><li>在48小时后浸润，用打孔器从注射部位切除叶盘（通常为3 - 4个磁盘/可以生产区）（ 图2G）。使用镊子轻轻的叶盘水珠转移到幻灯片（远轴朝上），并安装它们。避免过压哈日在盖滑移ð因为挤压/受损细胞不会在共聚焦显微镜（ 图2H）下显示良好。 </li><li>共焦成像之前，扫描于外延荧光化合物显微镜安装样本，以检查的表达水平。注:根据我们的经验，这表明中间表达水平的细胞共聚焦显微镜下最好代表。 </li><li>开始对共聚焦显微镜的40X（NA 1.3）镜头。调节滑动到所需的位置，从而使表达荧光蛋白的中等水平的细胞保持在中心。对于CFP和YFP激发/发射光谱是458海里/ 480 - 500纳米和514纳米/ 520 - 540分别纳米。 </li><li>激活"实时"按钮来预览图像，然后调整激光强度和智能增益最均衡的荧光强度/噪音比。以改善成像质量，增加扫描像素和/或帧/线平均。最后，调整调焦旋钮，REACH中间焦平面并点击"捕获"采取的形象。 注意:CFP和YFP的图片可通过打开或关闭的"连续"扫描分别模式，选项检查被顺序地或同时捕获。 </li><li>当Z-投影期望的细胞的深入视图中选择"XYZ"摄像模式时，限定的扫描范围的深度（10 - 15微米），并收集从顶部图像到目标小区的底。编译通过在图像文件右键点击徕卡软件获得的图像，选择"重命名"，并导出图像为.tiff文件。 </li><li>图片30 - 40细胞定量分析。 </li></ol> 5.图像处理与定量分析<ol><li>使用斐济软件（http://fiji.sc/Fiji）来处理图像并进行量化。 <ol><li>绘制荧光强度图。 <ol><li>为了更好地可视化CFP / YFP表达是否均匀分布的，使用斐济证明CFP和YFP的信号强度沿着电池周边。要做到这一点，首先启动斐济，然后选择"文件"和"打开"开始图像。单击工具菜单中的"行"，并通过右键单击选择"分段线"。决定的感兴趣的区域，并绘制沿着电池周边的线迹在CFP或YFP的信号（ 图3A-C）。 注意:斐济测量沿着分段线的绝对荧光强度值。 </li><li>选择下拉菜单中的"分析"和"剧情资料"来生成与代表CFP / YFP沿分割线的水平位置和强度值的曲线图。如果需要的话，重复的强度值（通过选择菜单中的"复制"）到Excel或其它图形软件生成可比强度的曲线图。 </li></ol></li><li>量化极性程度。 <ol><li>收集大约20 represen从3重复实验对准焦点细胞，以评估CFP和YFP的烟草细胞的极性程度。计算在低荧光强度（定义为L）的基础上的高荧光强度（定义为H）的比率的极性程度。注意:定量方法在图3中说明。 <ol><li>为了测量H和L值，得出沿小区外围（如上所述）感兴趣区域分割的线，请从下拉菜单中选择"分析"，然后选择"测量"。当一个"结果"窗口弹出时，用"中庸"的价值观进行量化。 </li><li>从显示CFP和YFP， 例如相对均匀表达的细胞，CFP-BASL（ 图3A）或YFP的从DNyda-nYFP和DNmpk6-cYFP（ 图3B）的共表达补充，通过测量得到的H和L的值两个随机选择的圆周段具有相等的长度。 </li> &lt;li&gt;从呈现荧光蛋白的明显极性积累，在这种情况下，细胞，CFP-BASL，DNyda-nYFP和DNmpk6-cYFP（ 图3C），的共表达收集来自周边区域轰值与富集的荧光信号从低/无荧光积累区域LS。 </li><li>池H / L的所得比率从20个细胞一起并测试正态分布。通过计算学生的t检验的p值（正态分布）或柯尔莫哥洛夫-斯米尔诺夫（KS）测试（非正态分布）。 </li></ol></li></ol></li></ol></li></ol>

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没有利益冲突的声明。

一般使用和信号转导的烟草瞬时表达系统的可能的修改:荧光蛋白的过表达（FP）-tagged在烟草表皮蛋白已成功用于在植物细胞中蛋白的亚细胞定位的快速检测。然而，在植物中研究蛋白质复合物的动态信令分配是一项具有挑战性的主题，由于复杂的亚细胞环境中，瞬时蛋白质-蛋白质相互作用和信号转导事件。为了应对这一挑战，我们采取了附设系统的优势，研究MAPK信号的事件?...

蛋白质错综复杂的分层网络，并形成络合物的发挥，在一个不可预知的细胞环境发生的几乎所有的生物过程的关键角色中进行互动。然而，从植物发育生长的响应，所以一直缺乏方便的工具，不仅可以快速而且有效地在亚细胞水平识别和监控这些动态信号事件。 烟草叶表皮瞬时表达系统已经呼吁优势，为在活细胞中可视化的荧光蛋白质。这个系统提供了半体内条件允许翻译后蛋白质修饰和蛋白质定位快速检查。通过检查补充YFP信号时，双分子荧光互补（附设）测定通知蛋白质 - 蛋白质相互作用的在植物细胞中的可能性。相比其他方法， 如酵母双杂交（Y2H）和Co-immunoprecipitation（联合IP）附设还提供了可视化，其中在亚细胞水平，可能会发生蛋白 - 蛋白相互作用室的有力手段。 因为不对称细胞分裂（ACD）保存干细胞群的同时用于组织/器官形成产生新的细胞类型，它对于促进真核多细胞不可或缺的机制。 拟南芥气孔发展已被用作模型系统，用于在植物中学习的ACD。的前体细胞，拟分生组织母细胞，除以不对称产生两种不同的子细胞中，拟分生组织（经历终止于一对保卫细胞之前干细胞样师）和一个气孔谱系接地单元（SLGC）（可分裂并分化成路面细胞），分别为（ 图1）。在气孔ACD，在天堂气孔不对称的新型蛋白质断裂（BASL）被premitotically极化带动分裂不对称，这INC路得身体不对称和细胞命运的不对称1。该MAPKKK YODA和的MAPKs，MPK3和6组成的MAPK级联是中部的气孔师图案和命运采用2,3，4,5。 近日，张某等人 。联极性蛋白BASL在拟南芥的气孔ACD 6 YDA-MAPK信号通路。规范YODA-MAPK通路，通MPK3 / 6，磷酸BASL并激活它的两极分化。磷酸BASL用作脚手架和募集YODA（YDA）和MPK3 / 6，以形成蛋白复合物，并在细胞皮质6集中的信令。 MAPK信号分量和BASL和YDA-MAPK途径之间的正反馈环的极化表示用于在植物细胞中的蛋白质偏振一种新的机制。当地丰富的MAPK信号是假设紧扣细胞命运分化气孔ACD 6（ 图1）。一项所述的k的一个支持这种模式安永实验数据从表现在BASL 6的诱导表达的的MAPKs的空间再分配烟草检测出来。 在一般情况下，这是不容易来监控发生MAPK信号因为MAPK分子经常发现到处一个细胞的内部。在这项研究中，我们所用的拆分YFP系统可视化的上游激酶和下游的人之间的相互作用暗示其中信令中继发生。我们通过进一步延长使用附设系统的共表达的第三蛋白（CFP-标签）与分割YFP对（暗示蛋白质 - 蛋白质相互作用的）来可视化是否以及如何补充YFP可以由共进行空间调制CFP表达的蛋白质。通过这样做，我们证明在烟草表皮的细胞皮层诱导YDA和MPK 6，之间的相互作用空间重组从均匀分布到极化图案CFP-BASL的共表达人的细胞。因此，这种系统具有用于当细胞被内部或外部刺激挑战监控的条件下在植物细胞中动态信令事件待开发的潜力（ 例如，其它蛋白质，化学应用，病原体攻击，或者环境变化等的共表达。 ）。

<table><tbody><tr><td>Phusion DNA polymerase</td><td>New England Biolabs</td><td>M0530S</td><td></td></tr><tr><td>pENTR/D/TOPO</td><td>Life Technologies</td><td>K2400-20</td><td></td></tr><tr><td>QuickChange II XL Site-directed Mutagenesis Kit</td><td>Agilent Technology</td><td>200521</td><td></td></tr><tr><td>LB broth</td><td>AMRESCO</td><td>J106-2KG</td><td></td></tr><tr><td>Bacto Agar</td><td>AMRESCO</td><td>J673-1KG</td><td></td></tr><tr><td>Gentamycin</td><td>Sigma-Aldrich</td><td>G3632-1G</td><td></td></tr><tr><td>Rifampicin</td><td>Sigma-Aldrich</td><td>R3501-1G</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>K4000-25G</td><td></td></tr><tr><td>Spectinomycin</td><td>Sigma-Aldrich</td><td>S4014-5G</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266-100G</td><td></td></tr><tr><td>25 x 75 mm Slide</td><td>VWR</td><td>16004-382</td><td></td></tr><tr><td>24 x 50 mm Cover glass</td><td>VWR</td><td>48393-241</td><td></td></tr><tr><td>Laser scanning confocal microscope</td><td>Leica&amp;nbsp;</td><td>TCS SP5 II</td><td>LAS AF Lite software</td></tr><tr><td>40X objective lens</td><td>Leica</td><td>HCX&amp;nbsp;Plan APO&amp;nbsp;</td><td>HCX&amp;nbsp;Plan APO, NA 1.30</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>SORVALL 6+</td><td></td></tr><tr><td>Hole puncher</td><td>Staples</td><td></td><td></td></tr><tr><td>50 ml falcon tubes</td><td>Genesee Scientific</td><td>21-108</td><td></td></tr><tr><td>No. 5 Forceps</td><td>Canemco &amp;amp; Marivac</td><td>205EQA-Spec</td><td></td></tr></tbody></table>

imaging spatial reorganization of a mapk signaling pathway using the tobacco transient expression system

在活细胞中的动态信号事件的可视化一直是个挑战。我们扩大建立瞬时表达系统，在烟草表皮细胞的生物分子荧光互补（附设）测定中，从检测蛋白质 - 蛋白质相互作用，以监测在植物细胞中的信号转导的空间分布。在这个协议中，我们所用的附设测定以表明相互作用和拟南芥 MAPKKK YODA和MAPK6之间的信令发生在质膜。当支架蛋白BASL被共表达时，YODA-MAPK相互作用引入并与CFP-BASL空间共极化。此改性烟草表达系统允许信令本地化和动态变化（小于4天），可容纳的荧光蛋白的颜色（至少三个）多个快速检查。我们还提出了具体的方法来定量蛋白质分布（非对称的空间定位，或"两极分化";）在烟草细胞。这种先进的烟草表达系统具有用于在活的植物细胞动态信令事件的快速测试被广泛使用的潜力。

为了防止由过度活跃的MAPK信号诱导的细胞死亡，YDA（DNyda）和MPK 6（DNmpk6）的激酶非现行版本在烟草细胞共表达测定中使用。既不通过附设也不CFP-BASL本身产生不均匀分布图案（ 图3A-B）揭示DNyda和DNmpk6之间的相互作用。然而，当CFP-BASL引入DNyda-DNmpk6相互作用对，两者CFP和YFP信号被重新分配给一个高度偏振的方式（ 图3C）。这种重新分布表明BASL与YDA?...

Watch this Scientific Journal Video about Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System at JoVE.com

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

imaging-spatial-reorganization-mapk-signaling-pathway-using-tobacco

Research

JoVE Journal

Biology

9.7K 次观看.  Rutgers, The State University of New Jersey. At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells. 

视频: Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

环境科学

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-&#946; is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-&#946;/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-&#946; displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-&#946; signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-&#946; signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Here, we present a protocol for live cell imaging of TGF-&#946;/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

腺病毒报告系统 TGF β/Smad3 信号通路的活体细胞成像

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

癌症研究

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

烟草 Benthamiana叶在制备鳞片上的瞬态表达

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

生物化学

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

在细胞分化过程中光介导的可逆调节丝裂原活化蛋白激酶途径<em&gt;爪蟾</em&gt;胚胎发育

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

发育生物学

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

一种高效的植物多嵌合体荧光融合蛋白的协同表达

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

化学

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

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 ...

生物学

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

神经科学

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

一种用于阴南芥叶片安装的通用方法

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...