这一项目得到了 NPU、LO1417 (捷克共和国教育部、青年和体育部) 的支持。

1. 生物材料的制备
<ol><li>制备生物材料, 表达荧光标记蛋白的兴趣, 以及细胞质标记。按照导言中提到的步骤操作。</li>
</ol>2. 共焦激光扫描显微术
<ol><li>用 FM 4-64 染料7染色1节所制备的材料。<ol>
<li>应用适合于所研究材料的染色协议。对于烟草 BY-2 细胞, 染色 200 ul 的细胞悬浮与 0.2 ul 10 毫米 FM 4-64 溶液在 dimethylsulfoxide。 注: dimethylsulfoxide 4-64 染料的典型染色浓度约为10微米, 采用10毫米的库存溶液。使用最低的调频4-64 浓度进行适当的染色, 不要在冰上孵化细胞 (fm 4-64 和低温会影响等离子膜的性能)。继续观察使用共聚焦显微镜立即。染色与图像采集之间的间隔不应长于10分钟;否则, 吞染料会影响调频4-64 染色。</li>
		<li>连续处理多个样品, 并在实验开始时不沾污所有样品。</li>
	</ol>
</li>
	<li>捕获一个赤道共焦部分每一个单元格。<ol>
<li>设置用于作为蛋白质标签 (必须在第一个通道中) 和 FM 4-64 (必须在第二个通道中) 的生色的连续双通道扫描。设置更高的光学分辨率 (10–20 px/微米)。一个例子设置: 63X 油浸泡目标, NA 1.3, 图像大小 1024 x 1024 像素。确保等离子体膜平面垂直于所采集图像中的共焦剖面平面。</li>
		<li>每个样本捕获至少20个单元, 以便有足够的数据进行统计评估 (取决于加工材料中的信号变异性)。</li>
	</ol>
</li>
</ol>3. 所需软件安装
<ol><li>安装斐济 ImageJ 分发8和所需的宏外围设备。<ol>
<li>从网站下载软件 https://fiji.sc/#download, 并通过拆箱安装。</li>
		<li>通过双击 "斐济. app" 目录中的 "ImageJ (.exe)" 文件, 启动斐济。</li>
		<li>从以下站点下载宏 "外围设备": http://kfrserver.natur.cuni.cz/lide/vosolsob/Peripheral/source/Peripheral_.ijm。</li>
		<li>在斐济, 选择插件 |宏 |安装...在主菜单中, 指定下载文件 "Peripheral_. ijm" 的路径。 注: 安装的宏 "外围设备" 的两个新工具将出现在斐济工具栏中: "采取配置文件工具" 和 "外围蛋白菜单"。</li>
	</ol>
</li>
	<li>安装 R 项目软件9和所需的软件包。<ol>
<li>按照站点 https://cloud.r-project.org/上的说明进行 r 项目安装。 注: 整体分析将在斐济进行。R 软件将只在内部由斐济宏 "外围设备" 在计算期间调用。</li>
		<li>运行 R GUI, 选择包 |安装软件包...在主菜单中, 并指定要安装的最近的存储库和包 "ggplot2" 。 注意: 或者, 键入 R 命令行: 安装. 软件包 ("ggplot2")。</li>
		<li>从目标下载 R 软件包外围设备: http://kfrserver.natur.cuni.cz/lide/vosolsob/Peripheral/source/peripheral_latest.tar.gz。通过选择软件包来安装它们|从本地文件安装软件包......。 注意: 或者, 键入到 R 命令行仅此命令: 安装. 软件包 ("http://kfrserver.natur.cuni.cz/lide/vosolsob/Peripheral/source/peripheral_latest.tar.gz", 回购 = NULL, 类型 = "源")。</li>
	</ol>
</li>
</ol>4. 斐济的图像分析
<ol><li>处理细胞质标记的共焦图像。<ol>
<li>使用插件将图像导入斐济|生物格式 |从斐济菜单中使用默认设置的生物格式导入程序。</li>
		<li>检查生物格式进口商是否正确识别了图像校准。左上角信息字段图像窗口中显示的图片维度必须等于原始图片尺寸。</li>
		<li>用不正确的维度单独处理不适当校准的图像,例如, 通过在对话框窗口中设置正确的参数分析 |设置比例...从斐济菜单: 将图像宽度以像素填充到距离 (像素) 字段中, 并将适当长度单位中的实际宽度输入到已知距离域中)。 注: 如果莱卡 LCS 文件的校准不正确, 请按照步骤4.2.1。</li>
	</ol></li>
	<li>运行分析设置的 "导入选项" 宏。以三种不同的方式激活宏: 选择插件 |宏 |导入选项 [f1]在主斐济菜单中, 在单击菜单工具外围蛋白菜单后选择同一项, 或按键盘短切 F1。在 "调用的宏" 对话框窗口中设置参数。<ol>
<li>在莱卡格式的情况下, 通过激活莱卡图像检查来解决不正确的校准。此选项正确地从文件中导入 "lei" 扩展名的图像维度。</li>
		<li>在字段高斯模糊半径 (px)中定义适当的值。这会影响图像平滑和降噪。低值 (1 像素) 足够, 不会导致图像信息丢失。</li>
		<li>在字段配置文件行宽 (px)中设置值。较粗的线条会导致轮廓曲线的平滑度更高。建议使用默认值 10 px。</li>
		<li>根据"示例分隔符"和"导出的项" 的定义设置图像标题分析。 注意: 图像标题 (不带扩展名的文件名,例如: "Experiment-1_line-02_cell-11") 将在字段"示例分隔符" (例如: "-_") 中列出的所有字符之间拆分 (如"-_") 到一组项目中 ("实验", "1", "行 "、" 02 "、" 单元格 "和" 11 "), 可以在以下分析中用作分组因素(示例、单元格、行或处理标识)。通过将其序列号 (第一项的 "0") 输入到已提交的"导出物料"中, 定义将使用哪些项目。使用空格分隔格式,例如"3 5"。在此示例中, 项目 "02" 和 "11" 将被称为 "Fac_0" 和 "Fac_1" 的分组因素。另外, 每个测量的标识都被称为 "i" 因子。</li>
		<li>或者, 如果可以保留完整的图像标题, 则保留示例分隔符为空, 并在导出的项中输入0。</li>
		<li>在 Windows 操作系统上, 检查在 R.exe 的字段路径中是否正确自动检测 R 软件的路径。否则, 请在系统中指定 R.exe 文件的完整路径 (例如, "C:\Program 文件 \r \r .exe")。</li>
		<li>单击"确定"。在下一个对话框窗口中检查标题分析, 然后单击"确定" 或 "返回" 重置。 注: 所有图片将自动平滑, 最终重新校准。将在 "结果" 窗口中显示所有导出分组因子的列表。</li>
	</ol></li>
	<li>在处理过的图像中对等离子膜区域进行线性选择, 并测量荧光剖面。<ol>
<li>从 "斐济" 工具栏中选择 "斐济工具"直线。单击图像并在等离子膜区域中拖动一条线。该线必须从细胞外空间开始, 并应垂直于等离子膜。选择具有较厚和更均匀的皮层细胞质层的区域。线性选择的最佳长度为 5-10 微米。</li>
		<li>在步骤 4.2 (快捷 "x") 中或单击 "斐济工具栏" 中的 "采用配置文件工具", 以类比方法运行 "带剖面" 宏。将执行两个通道中荧光剖面的自动测量, 数据将显示在结果窗口中。</li>
		<li>如果使用 "x" 键被认为是用户不友好的, 在文本编辑器中打开宏源文件Peripheral_. ijm , 在 "宏" 中替换 "x" 符号, 将配置文件 [x] "{" 与一个更有利的符号 (在斐济环境中未用作活动键盘短切) 并保存文件。重新安装宏 (步骤 3.1.4), 并从图像导入开始分析。</li>
		<li>根据单个信号的变异性, 取每个单元格的代表性数字。</li>
		<li>通过文件保存数据|另存为..。始终使用 "csv" 扩展;正确的数据格式是必要的。</li>
	</ol></li>
	<li>运行绘图剖面数据宏(短切 F5), 用于测量轮廓的图形可视化。在 "调用的宏" 对话框窗口中设置参数。<ol>
<li>是否对输入数据保留复选框使用筛选？未选中。</li>
		<li>通过单击相应的单选按钮, 指定是否应从结果窗口中的最近数据、单个 CSV 文件或指定目录中的所有 CSV 文件创建绘图。 注意: 如果绘图是从最近的结果创建的, 则首先保存数据 (另存为...对话框将自动激活)。</li>
		<li>通过选择适当的复选框, 指定将用于绘制绘图的分组因素。如果所有配置文件都应显示在唯一的图形中, 请选择 "i" 因子。如果所有数据都应在单个绘图中绘制, 则保留复选框未选中。 警告: 在处理的结果中选择不存在的分组因子会导致绘图创建过程中出现错误。</li>
		<li>当在字段输出文件前缀中保存绘图时指定文件名, 并在文件中定义图象尺寸, 绘制高和绘图宽度。 注意: 生成的绘图将作为 PNG 文件保存在源数据目录中。</li>
		<li>如果可以生成其他 pdf 输出, 请选中 " pdf 输出" 复选框。</li>
		<li>单击"确定"。指定在下一个对话框窗口中保存结果或最终进行数据导入的路径。在显示执行的 R 代码的对话框窗口中单击"确定"确认分析。 注意: 绘图将在新的图像窗口中打开, R 分析输出将在文本窗口中列出。</li>
	</ol></li>
	<li>运行配置文件筛选设置宏(短切口 F2), 如果某些绘制的配置文件在胞外空间 (标记蛋白) 或细胞内空间 (FM 4-64 信号) 中有过多信号。请按照 "调用的宏" 对话框窗口中的参数进行设置。 注意: 筛选允许根据指定的 x 坐标的最大允许强度阈值删除较差的配置文件。<ol>
<li>对于每个通道, 在文件删除测量中设置适当的强度阈值,并使用过量的胞外 (胞内) 信号。指定将强度阈值应用于x 坐标下的字段 (大于)的区域。</li>
		<li>再次运行图形配置文件数据宏(步骤 4.4) 与复选框使用筛选输入数据？选定;确保已成功删除所有异常配置文件。否则, 请改进筛选参数 (步骤 4.5.1)。</li>
	</ol></li>
	<li>运行创建模型宏(短切 F3), 根据校准数据创建等离子体膜和胞浆荧光分布模型。请按照 "调用的宏" 对话框窗口中的参数进行设置。<ol>
<li>通过选择 "对输入数据使用筛选" 复选框, 指定是否应筛选输入数据 (步骤 4.5)。</li>
		<li>指定源数据类比, 如步骤4.4.2 中所列。</li>
		<li>指定在微米中 "开始"、"结束" 和 "步长" 值的定义预测模型的间隔。 注意: 所有配置文件测量应长于指定的间隔。</li>
		<li>指定在适当的领域中使用的染色体的荧光激发和发射极值的波长。设置 GFP 和 FM 4-64 组合的默认值。 注: "gfp" 表示用于蛋白质标记的生色 (gfp、CFP、YFP等), "FM4" 表示用于等离子膜染色的生色 (通常为 FM 4-64)。</li>
		<li>指定 "输出文件前缀" 字段。前缀将用于将生成的模型作为 RData 文件保存到源数据目录中。</li>
		<li>选择复选框图形模型？如果需要图形输出。如果选择了pdf 输出复选框, 则该情节将被保存为 PNG, 也可以作为 pdf 文件。</li>
		<li>单击"确定"。在显示执行的 R 代码的对话框窗口中单击"确定", 在下一个对话框窗口中指定输入或输出路径, 并确认分析。 注: 在新的图像窗口中将显示外周相关蛋白荧光的模拟胞浆和膜复合物的情节, 而 R 分析输出将在文本窗口中列出。</li>
	</ol></li>
	<li>处理表达感兴趣蛋白质的细胞的图像。<ol>
<li>导入图像类比, 如步骤4.1 所做。</li>
		<li>在步骤4.2 中设置分析类比。平滑和线条宽度应相同。</li>
		<li>在步骤4.3 中测量配置文件类比。</li>
		<li>通过绘制 (步骤 4.4) 检查配置文件的准确性, 并根据需要设置筛选 (步骤 4.5)。</li>
	</ol></li>
	<li>运行计算分布宏(短切 F4), 用于计算血浆膜与细胞质之间的蛋白质分配。按照 "调用的宏" 对话框窗口中的参数设置进行。<ol>
<li>类比上一步 (步骤 4.4), 指定数据源、筛选和文件名前缀。</li>
		<li>指定是否从斐济 "外围" 宏的最近实例中的最后一个模型计算中加载蛋白质分布计算模型(选择复选框最近的结果), 或者从RData 文件(选中"从文件"复选框)。</li>
		<li>如果需要结果筛选, 请保持复选框移除结果的剩余可变性大于激活状态。指定可由单个剖面测量的信号分解无法解释的最大允许剩余变异性的阈值。例如: 值为0.200 表示所有测量的不解释性变异性高于20% 的荧光强度的总变异性将被丢弃。</li>
		<li>选择示例分组因素, 可用于创建方框 (步骤 4.4)。</li>
		<li>激活复选框pdf 输出, 以将方框情节保存为 pdf 文件。</li>
		<li>单击"确定", 指定输入或输出路径, 并在显示执行的 R 代码的对话框窗口中单击"确定" 确认分析。 注: 在新的图像窗口中显示了膜与细胞质之间的外周相关蛋白分割的盒状图, 并将在文本窗口中列出 R 分析输出。</li>
		<li>对于外部处理, 请将结果窗口中显示的结果保存到文件 |另存为...在 "窗口" 菜单中。</li>
	</ol></li>
</ol>

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C., Domergue, O., Teyssendier de la Serve, B., Rossignol, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+family+of+plasma+membrane+polypeptides+differentially+regulated+during+plant+development.">A new family of plasma membrane polypeptides differentially regulated during plant development.</a> Biochem Mol Biol Int. 43 (5), 1051-1062 (1997).</li><li>Ide, Y., Nagasaki, N., Tomioka, R., Suito, M., Kamiya, T., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+properties+of+a+novel,+hydrophilic+cation-binding+protein+associated+with+the+plasma+membrane.">Molecular properties of a novel, hydrophilic cation-binding protein associated with the plasma membrane.</a> J Exp Bot. 58 (5), 1173-1183 (2007).</li><li>Nagasaki-Takeuchi, N., Miyano, M., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plasma+membrane-associated+protein+of+Arabidopsis+thaliana+AtPCaP1+binds+copper+ions+and+changes+its+higher+order+structure.">A plasma membrane-associated protein of Arabidopsis thaliana AtPCaP1 binds copper ions and changes its higher order structure.</a> J Biochem. 144 (4), 487-497 (2008).</li><li>Li, 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=MDP25,+A+Novel+Calcium+Regulatory+Protein,+Mediates+Hypocotyl+Cell+Elongation+by+Destabilizing+Cortical+Microtubules+in+Arabidopsis.">MDP25, A Novel Calcium Regulatory Protein, Mediates Hypocotyl Cell Elongation by Destabilizing Cortical Microtubules in Arabidopsis.</a> Plant Cell. 23 (12), 4411-4427 (2011).</li><li>Qin, T., Liu, X., Li, J., Sun, J., Song, L., Mao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+microtubule-destabilizing+protein+25+functions+in+pollen+tube+growth+by+severing+actin+filaments.">Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube growth by severing actin filaments.</a> Plant Cell. 26 (1), 325-339 (2014).</li><li>Nagata, T., Nemoto, Y., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+BY-2+Cell+Line+as+the+&amp;#34;HeLa&amp;#34;+Cell+in+the+Cell+Biology+of+Higher+Plants.">Tobacco BY-2 Cell Line as the &amp;#34;HeLa&amp;#34; Cell in the Cell Biology of Higher Plants.</a> Int Rev Cytol. 132, 1-30 (1992).</li><li>Hellens, R. P., Anne Edwards, E., Leyland, N. R., Bean, S., Mullineaux, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pGreen:+A+versatile+and+flexible+binary+Ti+vector+for+Agrobacterium-mediated+plant+transformation.">pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation.</a> Plant Mol Biol. 42 (6), 819-832 (2000).</li><li>Vermeer, J. E. M., Thole, J. M., Goedhart, J., Nielsen, E., Munnik, T., Gadella, T. W. 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作者没有什么可透露的。

本文所描述的方法比起基于测量荧光强度5的其他方法, 更准确地估计了外围相关蛋白质的等离子膜分割。该方法的主要改进是考虑了等离子体膜和胞质信号的光衍射和叠加。虽然这些方法的结果与一个简单的方法的结果相关联的基础上的荧光强度在膜位置与平均细胞质信号 (如前6), 这一新方法的主要好处是通过信号分解来确定残留变异性 (无法解释的), 从而可?...

An erratum was issued for: <a href="https://www.jove.com/video/57837/determination-plasma-membrane-partitioning-for-peripherally">Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins</a>. The Affiliations section was updated.
One of the affiliations was updated from:
Department of Experimental Plant Biology, University of Science, Charles University
to:
Department of Experimental Plant Biology, Faculty of Science, Charles University

An erratum was issued for: <a href="https://www.jove.com/video/57837/determination-plasma-membrane-partitioning-for-peripherally">Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins</a>. The Affiliations section was updated.

Erratum: Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

外周相关的血浆膜蛋白是细胞信号通路的关键组成部分。它们的一个基本作用是它们的瞬态等离子体膜的关联和离解, 这对于细胞膜和细胞质之间的信号传导是重要的。外周相关的等离子膜蛋白可以通过脂质锚 (n-myristoylation、S 酰化或 prenylation) 或脂结合域 (与磷酸肌醇磷酸盐、磷脂酸相互作用) 附着在等离子膜上。等)。
这些蛋白质的等离子体膜结合特性可以在体内进行检查,例如, 当荧光标记的蛋白质被一个部位定向突变的关键氨基酸所修饰, 或者当它被各种抑制剂处理, 影响脂质信号。外周膜蛋白的分布主要有定性评价, 特别是在蛋白质再分布明显的情况下。所提出的方法对蛋白质再分配仅部分的情况是最佳的, 定量评价是必要的。一种常用的方法, 当等离子膜联想估计从共焦激光扫描显微图像作为荧光强度的比率在等离子膜和细胞质1,2, 是简单的, 但不准确。等离子体膜的荧光强度反映了等离子体膜和胞浆信号的叠加, 这是由于特殊荧光显微技术和光学元件所使用的光衍射特性的3。因此, 细胞质信号也包括在膜区。因此, FM 4-64 染色模式不能用作膜信号选择的掩码4。此外, 在 FM 4-64 染色最大的位置对膜信号的简单测量总是系统高估了外围相关的等离子体膜蛋白的真实等离子体膜信号, 因为膜和细胞质化合物。荧光标记的外周相关蛋白的最大观测信号也不与血浆膜标记物 (即FM 4-64 苯乙烯染料) 的最大值共同定位, 而是移向细胞质。另一个限制是基于这样一个事实: FM 4-64 发射峰与绿色荧光蛋白 (如 GFP) 的发射峰相比较宽, 因为光衍射3的波长依赖性。
在这里描述的方法中, 被标记的蛋白质信号由两个经验函数拟合, 分别描述了等离子体膜和细胞质信号的假设分布。该信号分解适用于垂直于源图像中的等离子体膜的线性荧光剖面, 它们是荧光标记蛋白表达的规则、双通道共焦切片。用 FM 4-64 染料标记的细胞。
用于拟合的第一个函数描述了细胞边缘上细胞质信号的衍射。它是从以前获得的荧光剖面得到的测量在细胞表达的细胞质蛋白标记相同的生色与血浆膜外周相关蛋白的兴趣。第二个函数描述一个等离子膜信号的衍射是从4-64 的荧光。这一信号首先被一个高斯函数所逼近, 正被用于一个点源的光衍射的近似建模。其次, 这个模型, 有效的红色 FM 4-64 发射, 是数学上转换为与生色的发射波长相关的形式, 用于标记的外围相关的蛋白质感兴趣的等离子膜。两种功能均按最大强度和平均值分别由 FM 4-64 信号和细胞质蛋白信号的最高值的10% 来规范化。通过这种信号分解 (非线性最小二乘法拟合方法), 可以方便、准确地估计等离子体膜的比值和所检测蛋白质的胞浆含量。由于细胞质体积浓度与等离子体膜表面浓度的比较, 计算分区系数的实际物理维度在千分尺范围内。它定义了从血浆膜到细胞质的距离, 在这种范围内, 相同数量的蛋白质被本地化, 如在相邻区域的等离子膜。此值等效于以前5引入的分区系数K2 。该方法是非常快速的, 只需要单一共焦部分获得使用常规共焦激光扫描显微镜, 它不是计算要求。分析内核已在便携式 R 包中实现, 并编写了一个附加的 ImageJ 宏, 以提供图形用户界面, 以便从直观的对话框中运行分析。该方法的软件和更详细的描述 (以前发布的6) 可以在 http://kfrserver.natur.cuni.cz/lide/vosolsob/Peripheral/中找到。
该方法适用于分离细胞、原生质体和组织, 其中单个细胞的血浆膜明显区分, 表达了经检查的外周相关蛋白的荧光标记结构。必须使用与 FM 4-64 染色兼容的生色。FM 4-64 发出红色荧光;因此, 检测到的蛋白质可以被标记的荧光蛋白与蓝色, 绿色, 或黄色的排放 (如GFP, CFP, YFP)。推荐生物材料的稳定转化, 因为它能够减少人工和更可重现的蛋白质分布观察。检验后的蛋白质具有相对均匀的胞质分布是必要的。蛋白质在内质网或另一种胞内膜室中的定位可以产生人工结果。
此外, 同样的生物材料表达细胞质标记必须用于比较。细胞可以由一个自由的 chomophore (与用于外周蛋白标记,例如, 游离 GFP) 或被标记的蛋白质感兴趣的被取消的膜结合能力改变。膜结合能力可以取消, 例如, 修剪膜结合领域或通过现场定向诱变的关键氨基酸残渣 (例如, myristoylation, s-酰化, 或 prenylation 的地点等)。
对于共焦扫描显微镜, 细胞必须标记的膜标记, 如 FM 4-64 染料。如果 FM 4-64 染色不适合所研究的材料 (由于干扰自发荧光, 差染料渗透等), 等离子膜可以标记, 例如, 由整体等离子膜蛋白标记为适当的 chomophore (mCherry, RFP等)。在细胞内膜隔室 (endomembranes) 中, 标记物的定位至关重要。
如果使用固定的样本和抗体, 可以使用可修复的模拟 FM 4-64FX 或用抗体对合适的靶标贴上等离子膜。在这种情况下, 重要的是要非常仔细地评估结果, 因为固定程序可以导致选择性地丢失蛋白质从细胞质和血浆膜。

<table><tbody><tr><td>FM 4-64</td><td>ThermoFisher Scientific</td><td>T13320</td><td>Plasma membrane dye</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D4540 Sigma</td><td>Dye solvent</td></tr><tr><td>Ordinary equipment (microscopic slides, pipettes, tips, tubes)</td><td></td><td></td><td>Equipment for cell labelling and microscopy</td></tr><tr><td>Confocal laser scanning microscope</td><td></td><td></td><td></td></tr><tr><td>Ordinary computer</td><td></td><td></td><td></td></tr></tbody></table>

determination of plasma membrane partitioning for peripherally-associated proteins

这种方法提供了一个快速的方法, 以确定任何荧光标记的外周相关蛋白的血浆膜分配使用的荧光强度分布在整个等离子膜。所测量的荧光剖面由一个垂直于细胞周围的直线上的膜和细胞质荧光分布模型拟合而成。该模型是建立在参考细胞的荧光强度值, 表达荧光标记标记的细胞质和 FM 4-64 标记的等离子膜。该方法可应用于各种细胞类型和生物体;然而, 只有非相邻细胞的等离子膜才能被评估。这种快速显微技术适用于实验, 在这种情况下, 需要对等离子体膜相关标记进行细微和动态的变化, 并需量化,例如, 在分析蛋白质突变体、抑制剂治疗、和信号转导观察。该方法是在多平台 R 包中实现的, 该软件包与作为用户友好接口的 ImageJ 宏结合在一起。

DREPP10是一种植物特异的外周相关的等离子膜蛋白, 与等离子膜通过 n-myristoylation 和静电相互作用与磷酸肌醇磷酸盐11, 12. DREPP 被描述为植物细胞中钙信号机械的一个组成部分, 也与细胞骨架13,14相互作用。在提出的实验中, 野生型烟草 DREPP2 及其非 myristoylated 突变体 (Gly...

Watch this Scientific Journal Video about Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins at JoVE.com

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

在这里, 我们提出了一个协议, 以进行定量分析的血浆膜协会的水平荧光标记的外围相关蛋白。该方法是以等离子体膜荧光标记细胞中所观察到的细胞膜和胞质成分的计算分解为基础的。

外周相关蛋白血浆膜分型的测定

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

determination-plasma-membrane-partitioning-for-peripherally

Research

JoVE Journal

Biology

8.3K 次观看.  Faculty of Science, Charles University. 在这里, 我们提出了一个协议, 以进行定量分析的血浆膜协会的水平荧光标记的外围相关蛋白。该方法是以等离子体膜荧光标记细胞中所观察到的细胞膜和胞质成分的计算分解为基础的。

视频: Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

科研

生物化学

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

Here we present protocols for detergent-free homogenization of cultured mammalian cells based on nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. This method is ideal for monitoring the partitioning of peripheral membrane proteins between soluble and membrane fractions.

氮气气蚀和差速离心允许监测外周血膜蛋白在细胞中的分布

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

点变异荧光相关光谱用于分析活细胞质膜上的分子扩散

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

生物学

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip:蛋白质 - 磷酸肌醇相互作用的无标记研究

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

生物工程

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

脂筏的测定分区在活细胞中的荧光标记探针的荧光相关光谱（FCS）

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

免疫与感染

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

外周相关蛋白血浆膜分型的测定

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