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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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulators+of+stomatal+lineage+signal+transduction+alter+membrane+contact+sites+and+reveal+specialization+among+ERECTA+kinases.">Modulators of stomatal lineage signal transduction alter membrane contact sites and reveal specialization among ERECTA kinases.</a> Developmental Cell. 38 (4), 345-357 (2016).</li><li>Le, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin+transport+and+activity+regulate+stomatal+patterning+and+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+GNOM+ARF-GEF+mediates+endosomal+recycling,+auxin+transport,+and+auxin-dependent+plant+growth.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+regulation+of+stomatal+differentiation+potential+by+EPF1+and+ERECTA-LIKE1+ligand-receptor+signaling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction-resolution+fluorescence+imaging+with+conventional+fluorescent+probes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+super-resolution+imaging+techniques.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinning-disk+confocal+microscopy:+Present+technology+and+future+trends.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+column+and+whole-brain+imaging+with+molecular+contrast+and+nanoscale+resolution.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+confocal+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-photon+microscopy.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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. et al. 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。

Membrane traffic is a conserved cellular process that distributes membrane components (also known as cargoes), including proteins, lipids, and other biological products, between different organelles within a eukaryotic cell or across the plasma membrane to and from the extracellular space1. This process is facilitated by a collection of membranes and organelles named the endomembrane system, which consists of the nuclear membrane, the endoplasmic reticulum, the Golgi apparatus, the vacuole/lysosomes, the plasma membrane, and multiple endosomes1. The endomembrane system enables the modification, packaging, and transport of membrane components using dynamic vesicles that shuttle between these organelles. Membrane trafficking events are crucial to cell development, growth, and environmental adaptation and, thus, are under stringent and complex regulation2. Currently, multiple approaches in molecular biology, chemical biology, microscopy, and mass spectrometry have been developed and applied to the field of membrane trafficking and have greatly advanced the understanding of the spatiotemporal regulation of the endomembrane system3,4. Molecular biology is used for classical genetic manipulations of the putative players involved in membrane trafficking, such as altering the gene expression of the protein of interest or labeling the protein of interest with certain tags. Tools in chemical biology include the usage of molecules that specifically interfere with the traffic of certain routes4,5. Mass spectrometry is powerful for identifying the components in an organelle that has been mechanically isolated by biochemical approaches3,4. However, membrane traffic is a dynamic, diverse, and complex biological process1. To visualize the membrane trafficking process in live cells under various conditions, light microscopy is an essential tool. Continuous progress has been made in advanced microscope techniques to overcome the challenges in measuring the efficiency, kinetics, and diversity of the events4. Here, this study focuses on the widely adopted methodologies in chemical/pharmacological biology, molecular biology, and microscopy to study membrane trafficking events in a naturally simplified and experimentally accessible system, the stomatal developmental process.
Stomata are micropores on plant aerial surfaces that open and close to facilitate gas exchange between the inner cells and the environment6,7,8. Hence, stomata are essential for photosynthesis and transpiration, two events that are crucial for plant survival and growth. Stomatal development is dynamically adjusted by environmental cues to optimize the plant&#39;s adaptation to the surroundings9. Dating back to studies in 2002, the identification of the receptor protein Too Many Mouths (TMM) opened the door to a new era of investigating the molecular mechanisms of stomatal development in the model plant Arabidopsis thaliana10. After just a few decades, a classical signaling pathway has been identified. From upstream to downstream, this pathway includes a group of secretory peptide ligands in the epidermal patterning factors (EFP) family, several cell-surface leucine-rich-repeat (LRR) receptor kinases in the EREECTA (ER) family, the LRR receptor protein TMM, a MAPK cascade, and several bHLH transcription factors including SPEECHLESS (SPCH), MUTE, FAMA, and SCREAM (SCRM)11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26. Previous work indicates that one of the receptor kinases, ER-LIKE 1 (ERL1), demonstrates active subcellular behaviors upon EPF perception20. ERL2 also dynamically traffics between the plasma membrane and some intracellular organelles27. Blocking the membrane trafficking steps causes abnormal stomatal patterning, resulting in stomatal clusters on the leaf surface28. These results suggest that membrane traffic plays an essential role in stomatal development. This study describes a protocol to spatiotemporally investigate the ERL1 dynamics using protein-protein subcellular co-localization analysis combined with pharmacological treatment using some membrane trafficking inhibitors.

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  

基于图像的方法研究气孔谱系细胞中的膜运输事件

This technique studies the membrane trafficking events in stomatal development, the micropores on plant aerial surface, to facilitate photosynthesis and the transpiration. Various techniques are used to study plant stomatal development. This includes molecular biology, cell biology or microscopy, chemical biology, mass spectrometry, biochemistry, and structural biology.Individual stomata form asynchronously. Each stoma is formed by three transcend cell fate transitions. So collecting enough stomata lineage cells at a particular cell fate is one of the big challenges.Membrane trafficking is essential for cell survival and growth. Stomata lineage cell is no exception. This protocol addresses the commonly used approach to study membrane trafficking events in stomata lineage cells.Plants face challenging environment in the face of climate change. Stomata are passages for plant gas exchange, and are highly flexible to environmental stress for adaptation. We are interested in studying how abiotic stress affects the stomata development by focusing on the membrane trafficking of certain proteins in stomata lineage cells.

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

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。

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

550 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  

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

科研

生物学

﻿Preparation of Dual-Colored F1 Transgenic Arabidopsis thaliana

Preparation of Dual-Colored F1 Transgenic Arabidopsis thaliana  

To begin, grow homozygous transgenic Arabidopsis thaliana plants bearing ERL1-YFP and a red fluorescent protein, or RFP-labeled marker protein RFP ARA7 side-by-side in a growth room until flowering. On the young inflorescence of plant A, remove all the older flowers and calyx, except for one recently opened flower. Carefully remove the younger flowers and floral merostems.In the recently opened flower, remove the sepals, petals, and stamen using a pair of sharp tweezers, leaving only the pistil on the inflorescence. From plant B, take mature stamen from an opened flower and deposit the pollen grains into the stigma of the dissected pistil of plant A.Label this manually crossed flower, indicating its parents and the date of the genetic cross. When the calyx turns yellow or brown, harvest the F1 seeds to check for a successful F1 plant having yellow fluorescent protein, or YFP, and RFP signals.

双色F1转基因拟南芥的制备

Pharmacological Analysis of Dual-Colored F1 Transgenic Arabidopsis thaliana

Pharmacological Analysis of Dual-Colored F1 Transgenic Arabidopsis thaliana   

To begin the pharmacological analysis of dual-colored F1 Arabidopsis thaliana, dissect and remove the cotyledons of seven-day old transgenic seedlings. Add 500 microliters of a 30-micromolar Brefeldin A, or BFA solution, in a 1.5-milliliter micro-centrifuge tube. Immerse the dissected seedlings in it.Tightly attach a 10-milliliter syringe to the micro-centrifuge tube and apply a vacuum for one minute. Remove the syringe before incubating the sample in the solution for 30 minutes before imaging. Next, immerse the dissected seven-day old seedling into 25-millimolar wortmannin.Apply the vacuum for one minute before incubating the immersed sample for 30 minutes. Examining ERL-1 trafficking routes in transgenic plants showed that 30 minutes of exposure to Brefeldin A solution resulted in ERL-1-YFP detection in large compartments known as BFA bodies. It indicated that Brefeldin A could block ERL-1 trafficking suggesting recycling or endocytosis of ERL-1.When transgenics were treated with wortmannin for 30 minutes, ring-like structures called WM bodies were highlighted by ERL-1-YFP. It showed that ERL-1-YFP were transported to multivesicular bodies indicating that ERL-1 followed the route to vacuoles for degradation.

双色F1转基因拟南芥的药理分析

Sample Preparation and Confocal Imaging of Dual-Colored F1 Transgenic Arabidopsis thaliana

Sample Preparation and Confocal Imaging of Dual-Colored F1 Transgenic Arabidopsis thaliana   

To begin the sample preparation, dissect the true leaf from the pharmacologically treated Arabidopsis thaliana transgenic sample using a sharp razor blade. Place the true leaf on a glass slide in a drop of water with the abaxial side up. Slowly cover the true leaf with a cover slip without air bubbles.For imaging, set up the beam path by selecting a 514-nanometer laser for yellow fluorescent protein, or YFP excitation. Obtain high image quality by using high laser power. Turn on PMT or HYD and define the upper and lower emission band thresholds based on the spectrum for the YFP fluorophore.Set a detection window of 530 to 570 nanometers to collect the YFP signal. for the sequential image of the second color, click on the seek button and add a new channel. In seek two, select a 555-nanometer laser for the red fluorescent protein, or RFP excitation, and set a detection window of 570 to 630 nanometers to collect the RFP signal.Select a 63X, 1.2W core lens on the system for imaging the subcellular membrane traffic activity within stomatal precursor cells. Start the format with an image size of 1024 by 1024 pixels, then optimize it based on the zoom factor and the objective settings for a good resolution. Next, set up the speed of the scan head, starting with a speed of 400 hertz, and optimize this based on the specific situation of the samples.To zoom on a region of interest without changing the objective lens, enter a zoom factor of one and optimize this based on the specific needs of the experiment. The line average refers to the number of times each X line will be scanned to obtain an average result. Start with a 2X line average For imaging the membrane trafficking events and optimize as needed.select the XYT scan mode in the acquisition mode to enable the time series utility. Next, select the waiting period between the time points under time interval. Define the number of frames to be collected by selecting frames.Use the Z-stack scan mode with the maximum intensity projection to collect the three-dimensional information regarding the membrane trafficking event within The entire cell. Then select the XYZ scan mode in acquisition mode and then the Z-stack utility will become accessible. Select the Z wide option.Under the scan mode, define the Z stack's top and bottom images using the begin and end buttons. Next, define the thickness of the Z step by clicking on the Z step size. To process the image, click on the primary process tab in Confocal software.Under the open projects tab on the far left side, select the interested Z-stack file. Then, in the middle tab named process tools, select the projection function. Choose maximum in the dropdown panel of method and click on the apply button.A Z-stack projection image will be generated under the open projects tab. To generate the video from the time series images, click the file tab in Fiji software and open function to open all the time series images one by one in the correct order. Alternatively, drag the images onto image J to open the data in the correct order.Then find the stack function in the image tab and choose images to stack to generate a video. Once done, save the file in the avi format with the desired frame rate. In the true leaf of seven-day-old transgenic plants bearing ERL1 promoter ERL1 YFP, scattered cells were highlighted by the YFP signal where ERL1 YFP labeled the plasma membrane.Multiple punctate signals were also observed, suggesting that ERL1 was also localized to the endosomes. RFP Ara7 highlighted multiple punctate signals in almost all the cells, indicating the multivesicular bodies, or MVBs, in these cells. When RFP Ara7 merged with the ERL1 YFP signal, most punctates overlapped, suggesting that some ERL1 YFP molecules were transported to the MVBs.Time series of true leaves bearing ERL1 YFP and MVB marker protein RFP Ara7 showed that YFP-labeled endosomes moved together with RFP Ara7 within the stomatal lineage cells. It confirmed that ERL1 YFP was localized to the multivesicular bodies.