ai_agent

这项工作得到了美国国家科学基金会（IOS-2217757）（X.Q.）和阿肯色大学医学科学（UAMS）布朗森基金会奖（H.Z.）的支持。

1. 溶液的制备
<ol><li>通过将 15 mL 漂白剂与 35 mL 蒸馏水和 50 μL Triton X-100 混合来制备种子灭菌溶液。</li>
	<li>通过将BFA粉末溶解在乙醇中至终浓度为10mM（储备液）来制备布雷菲尔丁A（BFA）溶液。通过将Wm粉末溶解在DMSO中至终浓度为10mM（储备液）来制备麦芽汁（Wm）溶液。</li>
</ol>2. 播下种子
<ol><li>将来自每个所需转基因植物的 10-50 颗种子等分到 1.5 mL 微量离心管中。向每个管中加入1mL种子灭菌溶液，并通过在摇床上轻轻倒置试管10分钟来彻底混合。</li>
	<li>弃去种子灭菌溶液，用1mL高压灭菌的蒸馏水洗涤种子五次。</li>
	<li>向每个试管中加入 300 μL 高压灭菌的蒸馏水，并将种子播种在含有 1% （w/v） 蔗糖和 0.75% （w/v） 琼脂的半强度 MS 培养基上。根据需要用相应的抗生素补充培养基。</li>
	<li>将板倒置在4°C下在黑暗中2天以同步发芽。</li>
	<li>2天后，将板转移到22°C下具有16小时光照/ 8小时黑暗循环（80μmol/ m2 /s 1）的生长室中。 这被认为是发芽后的第一天（1 dpg）。</li>
	<li>将幼苗移植到10 dpg的土壤中以进一步生长。</li>
</ol>3. 双色F1转基因植物的制备
<ol><li>并排生长带有 ERL1-YFP （植物A）的纯合转基因植物和带有RFP标记标记蛋白 RFP-Ara7 （植物B）20 的纯合转基因植物，直到在22°C下在生长室中开花16小时光照/ 8小时黑暗循环（80μmol/ m2 / s1）。</li>
	<li>从植物A中选择一个年轻的花序，保留一朵即将在花序上开放的花朵进行遗传杂交。去除所有较老的花朵和长方形。此外，小心地去除年轻的花朵和花分生组织，以避免将来混淆。</li>
	<li>用锋利的镊子去除萼片、花瓣和雄蕊，轻轻解剖未开封的花朵。只将雌蕊留在花序上。</li>
	<li>从植物B上开放的花中取出成熟的雄蕊，并将花粉粒沉积在植物A上解剖的雌蕊的柱头上。</li>
	<li>通过指出其父亲、母亲和遗传杂交的日期来标记这种手动杂交的花。让花成熟，并在长方形变成黄色/棕色时收获 F1 种子（杂交后~20 天）。检查同时具有 YFP 和 RFP 信号的成功 F1 工厂。</li>
</ol>4. 药物治疗
<ol><li>对于BFA处理，去除7日龄幼苗的子叶，将其余幼苗浸入模拟溶液（0.3%乙醇）或30μM BFA溶液中，真空应用1分钟，并保持样品浸入30分钟前成像。</li>
	<li>对于麦芽汁酶处理，去除7天龄幼苗的子叶，将其余幼苗浸入0.25%DMSO溶液或25mM麦芽甘素中，真空应用1分钟，并在成像前保持样品浸泡30分钟。</li>
	<li>对于样品的真空处理，在1.5 mL微量离心管中，加入500μL相应的药物溶液，并将解剖的幼苗浸入溶液中。将10 mL注射器紧紧连接到微量离心管上，并真空1分钟（图1）。取出注射器，并将样品在溶液中保持所需的时间。轻轻取出幼苗进行成像准备。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65257/65257fig01.jpg"> 图 1:简单的真空装置。 将 10 mL 注射器连接到 1.5 mL 微量离心管上进行真空处理。 <a href="https://www.jove.com/files/ftp_upload/65257/65257fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
5. 成像样品制备
<ol><li>使用锋利的剃须刀片从处理过的样品中解剖真实的叶子。轻轻地将真叶放入载玻片上的一滴水中，并保持远轴面朝上。用盖玻片慢慢覆盖真叶，同时避免捕获任何气泡。</li>
</ol>6. 共聚焦成像
注意:在这项工作中，使用徕卡SP8倒置扫描共聚焦显微镜对样品的荧光信号进行成像。
<ol><li>光束路径设置<ol><li>选择 514 nm 激光器进行 YFP 激发。使用高激光功率来增加信号强度，从而获得高图像质量。但是，激光功率高于5%存在光漂白的风险，并且样品的健康可能会受到影响。如果样品信号不太暗，则从低激光强度开始。</li>
		<li>打开PMT/HyD进行检测，并根据YFP荧光基团的光谱定义发射带上限和下限阈值。设置530-570nm的检测窗口以收集YFP信号。</li>
		<li>第二种颜色的顺序成像:单击Seq按钮，然后添加新通道。默认情况下，先前设计的YFP光束路径将为Seq 1。在序列2中，选择561 nm激光器进行RFP激发，并设置570-630 nm的检测窗口以收集RFP信号。</li>
	</ol></li>
	<li>扫描条件设置（图2）<ol><li>要对气孔前体细胞内的亚细胞膜交通活动进行成像，请在系统上选择63x/1.2 W Corr透镜进行成像。</li>
		<li>格式是指以像素为单位的图像大小。从 1,024 像素 x 1,024 像素开始，然后根据缩放系数和物镜设置对其进行优化，以获得良好的出版质量分辨率。</li>
		<li>速度是指激光经过每个像素时扫描头的速度。虽然较低的扫描速度通常会导致更好的信噪比，但它们可能无法有效地捕获快速变化的膜运输事件。从 400 Hz 的速度开始，并根据样本的具体情况进行优化。</li>
		<li>变焦系数用于在不更改物镜的情况下放大感兴趣区域。从缩放因子 1 开始，并根据实验的特定需求对其进行优化。</li>
		<li>线 平均值 是指扫描每条X线以获得平均结果的次数。较大的线平均值会降低结果图像中的噪声，但也会增加扫描时间和对激光的曝光时间。要对膜贩运事件进行成像，请勿使用高线平均值。从 2 倍线平均值开始，然后根据需要进行优化。</li>
	</ol></li>
	<li>收集时间序列 注意:在研究膜运输事件时，通常需要一个时间序列来记录亚细胞内体的快速运动。<ol><li>在 采集模式下，选择 xyt 扫描模式以启用时间序列实用程序。</li>
		<li>在 时间间隔下确定时间点之间的等待期。或者， 单击最小化以 立即一个接一个地拍摄图像。在以下时间序列实验中使用了7 s时间间隔。</li>
		<li>选择 持续时间，然后输入实验运行的总时间。或者，通过选择帧来定义要收集的 帧 数（图3）。</li>
	</ol></li>
	<li>要收集有关整个细胞内膜运输事件的三维信息，请使用Z-stack扫描模式。Z 堆栈图像的最大强度投影通常用于分析。<ol><li>在采集模式下选择 xyz 扫描模式，然后 Z-Stack 实用程序将变得可访问。</li>
		<li>选择 Z 宽度 选项。在 扫描模式下，使用" 开始 "和 "结束 "按钮定义 Z 堆栈的顶部图像和底部图像。</li>
		<li>通过单击 z 步长来定义 z 步长的粗细。或者，定义要拍摄的图像数量以覆盖 Z 堆栈的整个范围。为确保样本之间的一致性，请定义 z 步长（图 4）。</li>
	</ol></li>
	<li>图像处理<ol><li>z-stack图像的最大强度投影由共聚焦软件（http://www.leica-microsystems.com）生成。在主"进程"选项卡中，首先在最左侧的"打开项目"选项卡下选择感兴趣的 z 堆栈文件。然后，切换到名为"过程工具"的中间选项卡，选择"投影"功能，在"方法"的下拉面板中选择"最大值"，然后单击"应用"按钮。z 堆栈投影图像将在"打开的项目"选项卡下生成。</li>
		<li>时间序列图像的视频由斐济（https://imagej.net/Fiji）生成。在文件 选项卡中，使用 打开 函数以正确的顺序打开所有时间序列图像。在" 图像 "选项卡中，找到 "堆栈 "功能，然后选择 "要堆叠的图像 "以生成视频。以具有所需帧速率（ 视频 1 为 5 帧/秒）的 .avi 格式保存文件。</li>
	</ol></li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65257/65257fig02.jpg"> 图 2:XY 维度的扫描模式面板。 扫描模式面板用于设置图像扫描条件。 <a href="https://www.jove.com/files/ftp_upload/65257/65257fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65257/65257fig03.jpg"> 图 3:xyt 扫描模式下的时间序列实用程序。 时间序列实用程序用于设置成像条件以连续收集一系列图像。 <a href="https://www.jove.com/files/ftp_upload/65257/65257fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65257/65257fig04.jpg"> 图 4:xyz 扫描模式下的 z 堆栈实用程序。 z 堆栈实用程序用于设置成像条件以在 z 轴上收集一系列图像。 <a href="https://www.jove.com/files/ftp_upload/65257/65257fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>

通过共聚焦成像对 ERL1 动力学进行时空研究

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<li>Le, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Auxin+transport+and+activity+regulate+stomatal+patterning+and+development%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Auxin transport and activity regulate stomatal patterning and development.</a> Nature Communications. 5, 3090(2014).</li>
<li>Geldner, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Arabidopsis+GNOM+ARF-GEF+mediates+endosomal+recycling%2C+auxin+transport%2C+and+auxin-dependent+plant+growth%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth.</a> Cell. 112 (2), 219-230 (2003).</li>
<li>Qi, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autocrine+regulation+of+stomatal+differentiation+potential+by+EPF1+and+ERECTA-LIKE1+ligand-receptor+signaling%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Autocrine regulation of stomatal differentiation potential by EPF1 and ERECTA-LIKE1 ligand-receptor signaling.</a> Elife. 6, 24102(2017).</li>
<li>Heilemann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subdiffraction-resolution+fluorescence+imaging+with+conventional+fluorescent+probes%5BTitle%5D)+AND+%22Angewandte+Chemie%22%5BJournal%5D)">Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.</a> Angewandte Chemie. 47 (33), 6172-6176 (2008).</li>
<li>Leighton, R. E., Alperstein, A. M., Frontiera, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+super-resolution+imaging+techniques%5BTitle%5D)+AND+%22Annual+Review+of+Analytical+Chemistry%22%5BJournal%5D)">Label-free super-resolution imaging techniques.</a> Annual Review of Analytical Chemistry. 15 (1), 37-55 (2022).</li>
<li>Oreopoulos, J., Berman, R., Browne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinning-disk+confocal+microscopy%3A+Present+technology+and+future+trends%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Spinning-disk confocal microscopy: Present technology and future trends.</a> Methods in Cell Biology. 123, 153-175 (2014).</li>
<li>Gao, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+column+and+whole-brain+imaging+with+molecular+contrast+and+nanoscale+resolution%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution.</a> Science. 363 (6424), (2019).</li>
<li>Nwaneshiudu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Introduction+to+confocal+microscopy%5BTitle%5D)+AND+%22Journal+of+Investigative+Dermatology%22%5BJournal%5D)">Introduction to confocal microscopy.</a> Journal of Investigative Dermatology. 132 (12), (2012).</li>
<li>Sanderson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-photon+microscopy%5BTitle%5D)+AND+%22Current+Protocols%22%5BJournal%5D)">Multi-photon microscopy.</a> Current Protocols. 3 (1), 634(2023).</li>
</ol>

作者声明不存在利益冲突。

内膜系统将真核细胞的细胞质分离成不同的区室，从而使这些细胞器具有专门的生物学功能。为了在正确的时间将货物蛋白和大分子运送到它们的最终目的地，许多囊泡被引导到这些细胞器之间穿梭。高度调控的膜运输事件在细胞的活力、发育和生长中起着重要作用。调节这一关键而复杂的过程的机制仍然知之甚少。
这里已经描述了几种方法，用于观察膜蛋白的亚细胞定位?...

膜运输是一种保守的细胞过程，它将膜成分（也称为货物），包括蛋白质、脂质和其他生物产物，在真核细胞内的不同细胞器之间或穿过质膜进出细胞外空间1。该过程由称为内膜系统的膜和细胞器的集合促进，内膜系统由核膜、内质网、高尔基体、液泡/溶酶体、质膜和多个内体组成1.内膜系统能够使用在这些细胞器之间穿梭的动态囊泡对膜组件进行修饰、包装和运输。膜运输事件对细胞发育、生长和环境适应至关重要，因此受到严格而复杂的监管2。目前，分子生物学、化学生物学、显微镜和质谱的多种方法已被开发并应用于膜运输领域，并极大地促进了对内膜系统时空调控的理解3,4。分子生物学用于对参与膜运输的假定参与者进行经典的遗传操作，例如改变目标蛋白质的基因表达或用某些标签标记感兴趣的蛋白质。化学生物学中的工具包括使用专门干扰某些路线4,5的交通的分子。质谱法对于鉴定通过生化方法机械分离的细胞器中的成分非常强大 3,4.然而，膜交通是一个动态、多样和复杂的生物过程1。为了可视化各种条件下活细胞中的膜运输过程，光学显微镜是必不可少的工具。先进的显微镜技术不断取得进展，以克服测量事件的效率、动力学和多样性的挑战4.在这里，本研究侧重于化学/药理生物学，分子生物学和显微镜中广泛采用的方法，以研究自然简化和实验可访问的系统（气孔发育过程）中的膜运输事件。
气孔是植物空中表面上的微孔，它们打开和关闭以促进内部细胞与环境之间的气体交换6,7,8。因此，气孔对于光合作用和蒸腾作用至关重要，这两个事件对植物的生存和生长至关重要。气孔发育通过环境线索动态调整，以优化植物对周围环境的适应9.追溯到2002年的研究，受体蛋白太多嘴（TMM）的鉴定为研究模式植物拟南芥10气孔发育的分子机制打开了新时代。仅仅几十年后，一种经典的信号通路就被确定了。从上游到下游，该途径包括表皮模式因子（EFP）家族中的一组分泌肽配体，EREECTA（ER）家族中的几种细胞表面富含亮氨酸重复（LRR）受体激酶，LRR受体蛋白TMM，MAPK级联反应和几种bHLH转录因子，包括SPEECHLESS（SPCH），MUTE，FAMA和SCREAM（SCRM）11,12,13,14， 15,16,17,18,19,20,21,22,23,24,25,26.先前的工作表明，其中一种受体激酶ER-LIKE 1（ERL1）在EPF感知20时表现出活跃的亚细胞行为。ERL2还在质膜和一些细胞内细胞器之间动态运输27。阻断膜运输步骤会导致气孔图案异常，导致叶片表面出现气孔簇28。这些结果表明，膜交通在气孔发育中起着至关重要的作用。本研究描述了一种使用蛋白质 - 蛋白质亚细胞共定位分析结合使用某些膜运输抑制剂的药物治疗来时空研究ERL1动力学的方案。

<table><tbody><tr><td>10 mL syringes</td><td>VWR</td><td>BD309695</td><td>Vacuum samples</td></tr><tr><td>Brefeldin A (BFA)</td><td>Sigma</td><td>B7651</td><td>membrane trafficking drug</td></tr><tr><td>Confocal Microscope</td><td>Leica</td><td>Lecia SP8 TCS with LAS-X software package</td><td>Imaging</td></tr><tr><td>Dissecting Forceps</td><td>VWR</td><td>82027-402</td><td>Genetic cross</td></tr><tr><td>Fiji</td><td>NIH</td><td>https://imagej.net/Fiji</td><td>Image processing</td></tr><tr><td>Leica LAS AF software</td><td>Leica</td><td>http://www.leica-microsystems.com</td><td>Image processing</td></tr><tr><td>transgenic seeds of ERL1-YFP</td><td></td><td></td><td>Qi, X. &lt;em&gt;et al&lt;/em&gt;. The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate. Elife. 9, doi:10.7554/eLife.58097, (2020).</td></tr><tr><td>transgenic seeds of RFP-Ara7</td><td></td><td></td><td>Ebine, K. et al. A membrane trafficking pathway regulated by the plant-specific RAB GTPase ARA6. Nat Cell Biol. 13 (7), 853-859, doi:10.1038/ncb2270, (2011).</td></tr><tr><td>Wortmannin (Wm)</td><td>Sigma</td><td>W1628</td><td>membrane trafficking drug</td></tr></tbody></table>

image-based methods to study membrane trafficking events in stomatal lineage cells

在真核细胞中，膜成分（包括蛋白质和脂质）在时空上运输到内膜系统内的目的地。这包括新合成的蛋白质向细胞表面或细胞外部的分泌运输，细胞外货物或质膜成分进入细胞的内吞运输，以及货物在亚细胞器之间的循环或穿梭运输等。膜运输事件对于所有真核细胞的发育、生长和环境适应至关重要，因此受到严格的监管。细胞表面受体激酶感知来自细胞外空间的配体信号，经历分泌和内吞转运。本文介绍了使用质膜定位的富含亮氨酸重复受体激酶ERL1来研究膜运输事件的常用方法。这些方法包括植物材料制备、药物治疗和共聚焦成像设置。为了监测ERL1的时空调控，本研究描述了ERL1与多泡体标志蛋白RFP-Ara7之间的共定位分析，这两种蛋白质的时间序列分析，以及用膜运输抑制剂brefeldin A和wortmannin处理的ERL1-YFP的z-stack分析。

先前的一项研究表明，ERL1是一种经历动态膜运输事件的活性受体激酶20。ERL1是质膜上的跨膜LRR受体激酶。内质网中新合成的ERL1在高尔基体中处理并进一步运输到质膜。质膜上的ERL1分子可以使用其细胞外LRR结构域18感知EPF配体。在被抑制性EPFs（包括EPF1）激活后，ERL1被内化到内体中，运输到多泡体（MVB），然后运输到液泡中降解以终止信号20。...

Watch this Scientific Journal Video about Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells at JoVE.com

作者聚焦:研究气孔谱系细胞中膜运输事件的基于图像的方法  

这里介绍了几种常用的方法来研究质膜受体激酶的膜运输事件。本手稿描述了详细的方案，包括植物材料制备、药物治疗和共聚焦成像设置。

基于图像的方法研究气孔谱系细胞中的膜运输事件

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

该技术研究气孔发育中的膜运输事件，植物空中表面的微孔，以促进光合作用和蒸腾作用。各种技术用于研究植物气孔发育。这包括分子生物学、细胞生物学或显微镜、化学生物学、质谱、生物化学和结构生物学。单个气孔异步形成。每个造口由三个超越细胞命运的转变形成。因此，在特定细胞命运中收集足够的气孔谱系细胞是最大的挑战之一。膜运输对细胞的存活和生长至关重要。气孔谱系细胞也不例外。该协议解决了研究气孔谱系细胞中膜运输事件的常用方法。面对气候变化，植物面临着具有挑战性的环境。气孔是植物气体交换的通道，对环境压力具有高度的灵活性以适应。我们有兴趣通过关注气孔谱系细胞中某些蛋白质的膜运输来研究非生物胁迫如何影响气孔发育。

image-based-methods-to-study-membrane-trafficking-events-stomatal

Research

JoVE Journal

Biology

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

1.3K 次观看.  Rutgers University. 该技术研究气孔发育中的膜运输事件，植物空中表面的微孔，以促进光合作用和蒸腾作用。各种技术用于研究植物气孔发育。这包括分子生物学、细胞生物学或显微镜、化学生物学、质谱、生物化学和结构生物学。单个气孔异步形成。每个造口由三个超越细胞命运的转变形成。因此，在特定细胞命运中收集足够的气孔谱系细胞是最大的挑战之一。膜运输对细胞的存...

视频: 作者聚焦:研究气孔谱系细胞中膜运输事件的基于图像的方法  

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

用于植物组织中基因表达研究的机器人和动态图像分析

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

科研

生物学

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

拟南芥子叶表皮发芽过程中的长期、高分辨率共聚焦延时成像

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像拟南芥子叶表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells

<a href='https://app.jove.com/v/63375/studying-membrane-protein-trafficking-drosophila-photoreceptor-cells'>Source: Wagner, K. et al., Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins.&#160;J. Vis. Exp. (2022).</a>This video demonstrates the visualization of membrane protein trafficking in Drosophila photoreceptor cells using eGFP-tagged TRPL proteins. It details the preparation of transgenic flies for imaging, the capture of fluorescence images of photoreceptor cells, and the quantification of TRPL trafficking by measuring relative eGFP fluorescence in the rhabdomeres.

研究果蝇感光细胞中的膜蛋白运输

Source: Wagner, K. et al., Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins.&#160;J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neuromuscular Disorders

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

使用 pTIRFM 对质膜变形进行成像

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

使用可变角度落射荧光显微镜对植物细胞表面动力学进行实时成像

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

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

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

使用烟草瞬时表达系统对 MAPK 信号通路进行空间重组成像

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 ...

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

拟南芥幼苗糖溶液浸泡处理成簇气孔诱导系统

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

拟南芥幼苗糖溶液浸入治疗的聚集性植物诱导系统

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

环境科学

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

使用扫描电子显微镜可视化膜褶皱的形成

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

使用扫描电子显微镜可视化膜褶皱形成

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

医学

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.

使用 eGFP 标记蛋白研究果蝇感光细胞中的膜蛋白运输

Here, non-invasive methods are described for localization of photoreceptor membrane proteins and assessment of retinal degeneration in the Drosophila compound eye using eGFP fluorescence.

使用eGFP标记的蛋白质研究 果蝇 光感受器细胞中的膜蛋白运输

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

通过表皮表型评分鉴定参与气孔发育的基因

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

通过表皮表型评分 鉴定 参与气孔发育的基因

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

基于图像的方法研究气孔谱系细胞中的膜运输事件

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Summary

Explore More Videos

机器人技术和植物组织的基因表达研究的动态图像分析

从长期来看，高解析度激光共聚焦时间流逝成像拟南芥子叶表皮萌发过程中

研究果蝇感光细胞中的膜蛋白运输

成像质膜变形随着pTIRFM

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

拟南芥幼苗糖溶液浸入治疗的聚集性植物诱导系统

使用扫描电子显微镜可视化膜褶皱形成

使用eGFP标记的蛋白质研究果蝇光感受器细胞中的膜蛋白运输

通过表皮表型评分鉴定参与气孔发育的基因

基于图像的方法研究气孔谱系细胞中的膜运输事件

Transcript

Summary

Explore More Videos

机器人技术和植物组织的基因表达研究的动态图像分析

从长期来看，高解析度激光共聚焦时间流逝成像拟南芥子叶表皮萌发过程中

研究果蝇感光细胞中的膜蛋白运输

成像质膜变形随着pTIRFM

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

拟南芥幼苗糖溶液浸入治疗的聚集性植物诱导系统

使用扫描电子显微镜可视化膜褶皱形成

使用eGFP标记的蛋白质研究 果蝇 光感受器细胞中的膜蛋白运输

通过表皮表型评分 鉴定 参与气孔发育的基因

使用eGFP标记的蛋白质研究果蝇光感受器细胞中的膜蛋白运输

通过表皮表型评分鉴定参与气孔发育的基因