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

<ol>
	<li>Aniento, F., Sanchez de Medina Hernandez, V., Dagdas, Y., Rojas-Pierce, M., Russinova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+endomembrane+trafficking+in+plants.">Molecular mechanisms of endomembrane trafficking in plants.</a> Plant Cell. 34 (1), 146-173 (2022).</li><li>Sigismund, S., 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=Endocytosis+and+signaling:+Cell+logistics+shape+the+eukaryotic+cell+plan.">Endocytosis and signaling: Cell logistics shape the eukaryotic cell plan.</a> Physiological Reviews. 92 (1), 273-366 (2012).</li><li>Lyu, Z., Genereux, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodologies+for+measuring+protein+trafficking+across+cellular+membranes.">Methodologies for measuring protein trafficking across cellular membranes.</a> ChemPlusChem. 86 (10), 1397-1415 (2021).</li><li>Rodriguez-Furlan, C., Raikhel, N. V., Hicks, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merging+roads:+Chemical+tools+and+cell+biology+to+study+unconventional+protein+secretion.">Merging roads: Chemical tools and cell biology to study unconventional protein secretion.</a> Journal of Experimental Botany. 69 (1), 39-46 (2017).</li><li>Foissner, I., Sommer, A., Hoeftberger, M., Hoepflinger, M. C., Absolonova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+wortmannin-induced+reorganization+of+the+trans-Golgi+network+the+key+to+explain+charasome+formation.">Is wortmannin-induced reorganization of the trans-Golgi network the key to explain charasome formation.</a> Frontiers in Plant Science. 7, 756 (2016).</li><li>Qi, X., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+and+environmental+signals+guiding+stomatal+development.">Hormonal and environmental signals guiding stomatal development.</a> BMC Biology. 16 (1), 21 (2018).</li><li>Han, S. K., Kwak, J. M., Qi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+lineage+control+by+developmental+program+and+environmental+cues.">Stomatal lineage control by developmental program and environmental cues.</a> Frontiers in Plant Science. 12, 751852 (2021).</li><li>Bharath, P., Gahir, S., Raghavendra, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abscisic+acid-induced+stomatal+closure:+An+important+component+of+plant+defense+against+abiotic+and+biotic+stress.">Abscisic acid-induced stomatal closure: An important component of plant defense against abiotic and biotic stress.</a> Frontiers in Plant Science. 12, 615114 (2021).</li><li>Becklin, K. M., Ward, J. K., Way, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Photosynthesis, Respiration, and Climate Change., 1st edition. , (2021).</li><li>Yang, M., Sack, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+too+many+mouths+and+four+lips+mutations+affect+stomatal+production+in+Arabidopsis.">The too many mouths and four lips mutations affect stomatal production in Arabidopsis.</a> Plant Cell. 7 (12), 2227-2239 (1995).</li><li>Hara, K., Kajita, R., Torii, K. U., Bergmann, D. C., Kakimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretory+peptide+gene+EPF1+enforces+the+stomatal+one-cell-spacing+rule.">The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule.</a> Genes &amp; Development. 21 (14), 1720-1725 (2007).</li><li>Hara, K., 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=Epidermal+cell+density+is+autoregulated+via+a+secretory+peptide,+EPIDERMAL+PATTERNING+FACTOR+2+in+Arabidopsis+leaves.">Epidermal cell density is autoregulated via a secretory peptide, EPIDERMAL PATTERNING FACTOR 2 in Arabidopsis leaves.</a> Plant &amp; Cell Physiology. 50 (6), 1019-1031 (2009).</li><li>Hunt, L., Gray, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signaling+peptide+EPF2+controls+asymmetric+cell+divisions+during+stomatal+development.">The signaling peptide EPF2 controls asymmetric cell divisions during stomatal development.</a> Current Biology. 19 (10), 864-869 (2009).</li><li>Sugano, S. S., 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=Stomagen+positively+regulates+stomatal+density+in+Arabidopsis.">Stomagen positively regulates stomatal density in Arabidopsis.</a> Nature. 463 (7278), 241-244 (2010).</li><li>Kondo, T., 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=Stomatal+density+is+controlled+by+a+mesophyll-derived+signaling+molecule.">Stomatal density is controlled by a mesophyll-derived signaling molecule.</a> Plant &amp; Cell Physiology. 51 (1), 1-8 (2010).</li><li>Hunt, L., Bailey, K. J., Gray, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signalling+peptide+EPFL9+is+a+positive+regulator+of+stomatal+development.">The signalling peptide EPFL9 is a positive regulator of stomatal development.</a> New Phytologist. 186 (3), 609-614 (2010).</li><li>Shpak, E. D., McAbee, J. M., Pillitteri, L. J., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+patterning+and+differentiation+by+synergistic+interactions+of+receptor+kinases.">Stomatal patterning and differentiation by synergistic interactions of receptor kinases.</a> Science. 309 (5732), 290-293 (2005).</li><li>Lin, G., 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=A+receptor-like+protein+acts+as+a+specificity+switch+for+the+regulation+of+stomatal+development.">A receptor-like protein acts as a specificity switch for the regulation of stomatal development.</a> Genes &amp; Development. 31 (9), 927-938 (2017).</li><li>Lee, J. S., 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=Direct+interaction+of+ligand-receptor+pairs+specifying+stomatal+patterning.">Direct interaction of ligand-receptor pairs specifying stomatal patterning.</a> Genes &amp; Development. 26 (2), 126-136 (2012).</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=The+manifold+actions+of+signaling+peptides+on+subcellular+dynamics+of+a+receptor+specify+stomatal+cell+fate.">The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate.</a> Elife. 9, e58097 (2020).</li><li>MacAlister, C. A., Ohashi-Ito, K., Bergmann, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+control+of+asymmetric+cell+divisions+that+establish+the+stomatal+lineage.">Transcription factor control of asymmetric cell divisions that establish the stomatal lineage.</a> Nature. 445 (7127), 537-540 (2007).</li><li>Pillitteri, L. J., Sloan, D. B., Bogenschutz, N. L., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Termination+of+asymmetric+cell+division+and+differentiation+of+stomata.">Termination of asymmetric cell division and differentiation of stomata.</a> Nature. 445 (7127), 501-505 (2007).</li><li>Ohashi-Ito, K., Bergmann, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+FAMA+controls+the+final+proliferation/differentiation+switch+during+stomatal+development.">Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development.</a> Plant Cell. 18 (10), 2493-2505 (2006).</li><li>Kanaoka, M. 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=SCREAM/ICE1+and+SCREAM2+specify+three+cell-state+transitional+steps+leading+to+Arabidopsis+stomatal+differentiation.">SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to Arabidopsis stomatal differentiation.</a> Plant Cell. 20 (7), 1775-1785 (2008).</li><li>Bergmann, D. C., Lukowitz, W., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+development+and+pattern+controlled+by+a+MAPKK+kinase.">Stomatal development and pattern controlled by a MAPKK kinase.</a> Science. 304 (5676), 1494-1497 (2004).</li><li>Wang, H., Ngwenyama, N., Liu, Y., Walker, J. C., Zhang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+development+and+patterning+are+regulated+by+environmentally+responsive+mitogen-activated+protein+kinases+in+Arabidopsis.">Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis.</a> Plant Cell. 19 (1), 63-73 (2007).</li><li>Ho, C. M., Paciorek, T., Abrash, E., Bergmann, D. 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。

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

714 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  

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

与气孔导度，以叶功能性状

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.

SCAP分析<em&gt;ñ</em&gt; -glycosylation和贩卖人体细胞

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

方法来研究上皮转运蛋白功能和表达在纯肠和Caco-2细胞生长在3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine members that can monomethylate or symmetrically/asymmetrically dimethylate arginine residues. Several antibodies recognizing different types of arginine methylation of various proteins are available; thus, providing tools for the development of PRMT activity biomarker assays. PRMT antibody-based assays are challenging due to overlapping substrates and motif-based antibody specificities. These issues and the experimental setup to investigate the arginine methylation contributed by individual PRMTs are discussed. Through the careful selection of the representative substrates that are biomarkers for eight out of nine PRMTs, a panel of PRMT activity assays were designed. Here, the protocols for cellular assays quantitatively measuring the enzymatic activity of individual members of the PRMT family in cells are reported. The advantage of the described methods is their straightforward performance in any lab with cell culture and fluorescent western blot capabilities. The substrate specificity and chosen antibody reliability were fully validated with knockdown and overexpression approaches. In addition to detailed guidelines of the assay biomarkers and antibodies, information on the use of an inhibitor tool compound collection for PRMTs is also provided.

These protocols provide the methodology used to assess the enzymatic activity of individual members of the protein arginine methyltransferase (PRMT) family in cells. Detailed guidelines on assessing PRMT activity using endogenous and exogenous biomarkers, methyl-arginine recognizing antibodies, and inhibitor tool compounds are described.

研究蛋白质精氨酸甲基转移酶1-9细胞活性的方法

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine ...