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>

<ol>
	<li>Barbosa, I. C. R., Shikata, H., Zourelidou, M., Heilmann, M., Heilmann, I., Schwechheimer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+composition+and+a+polybasic+motif+determine+D6+PROTEIN+KINASE+polar+association+with+the+plasma+membrane+and+tropic+responses.">Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.</a> Development. 143 (24), 4687-4700 (2016).</li><li>Kato, M., Aoyama, 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=The+Ca2+-binding+protein+PCaP2+located+on+the+plasma+membrane+is+involved+in+root+hair+development+as+a+possible+signal+transducer.">The Ca2+-binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer.</a> Plant J. 74 (4), 690-700 (2013).</li><li>Kubitscheck, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence Microscopy: From Principles to Biological Applications. , (2013).</li><li>Janecki, A. J., Janecki, M., Akhter, S., Donowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+plasma+membrane+expression+of+a+fusion+protein+of+Na/H+exchanger+NHE3+and+green+fluorescence+protein+(GFP)+in+living+PS120+fibroblasts.">Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts.</a> J Histochem Cytochem. 48 (11), 1479-1492 (2000).</li><li>Peitzsch, R. M., McLaughlin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+acylated+peptides+and+fatty+acids+to+phospholipid+vesicles:+pertinence+to+myristoylated+proteins.">Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins.</a> Biochemistry. 32 (39), 10436-10443 (1993).</li><li>Vosolsob&#283;, S., Petr&#225;&#353;ek, J., Schwarzerov&#225;, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+plasticity+of+plasma+membrane+interaction+in+DREPP+family+proteins.">Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.</a> Biochim Biophys Acta - Biomembr. 1859 (5), 686-697 (2017).</li><li>Bolte, S., Talbot, C., Boutte, Y., Catrice, O., Read, N. D., Satiat-Jeunemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM-dyes+as+experimental+probes+for+dissecting+vesicle+trafficking+in+living+plant+cells.">FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells.</a> J Microsc. 214 (2), 159-173 (2004).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>R Development Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> R: A language and environment for statistical computing. , (2008).</li><li>Logan, D. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+phosphatidylinositol+4-phosphate+dynamics+in+living+plant+cells.">Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells.</a> Plant J. 57 (2), 356-372 (2009).</li><li>La&#328;kov&#225;, 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=Determination+of+Dynamics+of+Plant+Plasma+Membrane+Proteins+with+Fluorescence+Recovery+and+Raster+Image+Correlation+Spectroscopy.">Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.</a> Microsc Microanal. 22 (2), 290-299 (2016).</li></ol>

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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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 border="0" cellpadding="0" cellspacing="0" style="width:944px;" width="944">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">FM 4-64</td>
			<td style="width:135px;">ThermoFisher Scientific</td>
			<td style="width:384px;">T13320</td>
			<td style="width:184px;">Plasma membrane dye</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Dimethyl sulfoxide</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:384px;">D4540 Sigma</td>
			<td style="width:184px;">Dye solvent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Ordinary equipment (microscopic slides, pipettes, tips, tubes)</td>
			<td style="width:135px;"></td>
			<td></td>
			<td style="width:184px;">Equipment for cell labelling and microscopy</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Confocal laser scanning microscope</td>
			<td style="width:135px;"></td>
			<td style="width:384px;"></td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Ordinary computer</td>
			<td style="width:135px;"></td>
			<td style="width:384px;"></td>
			<td style="width:184px;"></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

Universidad de Cartagena UDEC

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

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 ...

科研

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

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. ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

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, ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...