我们感谢我们的团队成员提供技术援助，特别是管委会Blache，以及M Irla和乙Imhof，对他们的支持和富有成效的讨论。通缩和分娩的数字再现自然方法提供。支持这个项目是由国家科学研究中心（INSERM）和马赛大学，体制补助和特定地区研究所国家杜癌症，法新社国立德拉Recherche（普罗旺斯 - 阿尔卑斯 - 科特迪瓦-d&#39;Azur补助的ANR-08-PCVI 0034-02，国家情报局2010汪曾祺1214 01）基金会争取，LA RECHERCHEMédicale（队报labéliséeFRM-2009）。 VR是支持从法甲国立CONTRE LE癌症的奖学金。

在这段视频中，我们提出了一个完整的单粒子追踪实验，利用量子点定位到一个特定的膜受体。这项实验的主要目标包括在鉴别不同类型的测量活细胞的细胞膜内的分子扩散行为。事实上，在膜产生的分子运动，通常可以从布朗扩散偏离线性指示或局限在nanodomains 26日至29日，例如。我们的目标，同时为许多技术上是可行的受体后，提供了在一个活细胞的细胞膜内发生的动力学产生的各种快照。这是最终有望使细胞表面的受体信号的调节机制破译。 1。细胞培养<ol><li>准备细胞样品:使用附着COS-7细胞内源性表达的表皮生长因子受体（EGFR）30。何时与活细胞的工作，不用抗生素，确保有没有潜子检出污染，使用适当的无菌技术，在任何时候都在准备。 </li><li>生长在细胞完全培养基（DMEM培养液，10％小牛血清，1％谷氨酰胺，肝素钠1％和1％丙酮酸钠，见下面的特定试剂的表），在37°C，7％的CO 2，照顾，让他们分合流，之前他们在Lab-Tek的传播呈指数增长。 </li><li>实验前的一天，传播5000细胞/以及在8室（实验室TEK）孵育过夜。计数细胞，保证了恒定的细胞密度，因此每个细胞中的量子点的重现率。 </li></ol> 2。细胞标记量子点准备与一个特定的涂层。量子点是半导体荧光纳米粒子组成。这些纳米粒子呈现出高息，因为它们是非常明亮，耐光古典荧光探针31,32，这使得单分子成像成就一个适当的信号噪声比（SNR）。 <ol><li>实验前，产生杂交瘤细胞株（108单克隆抗体，ATCC HB - 9764）针对表皮生长因子受体的Fab片段与木瓜蛋白酶消化，如前面所述21。 </li><li>共轭厂与生物素，为制造商的说明（EZ链接磺酸基NHS-LC-生物素包; Thermo Scientific的， 图1A）。 </li><li>使用功能在605纳米（最佳发射波长的辉煌，从细胞自体荧光检测和分离）与链霉亲和素（Invitrogen公司）和发光的量子点。 </li><li>准备在完全培养基的标签解决方案（见特定试剂的表），以饱和的streptavidins目前量子点周围，以及防止聚集和非特异性结合到细胞和盖玻片。 </li><li>准备两个中间的解决方案:量子点 - 亲和另一个与生物素厂之一，在20纳米之一。 </li><li>混合等量的这两个解决方案:在1:1的比例混合厂的量子点，获​​得最后的工作浓度为10纳米晶圆厂:量子点复杂。使用摩尔比，有利于形成一个量子点和一个生物素化的Fab片段组成的单官能络合物。这有利于单价标签，由于受体交联33,34限制工件的意见。 </li><li>在25°C孵育15分钟，每分钟1200转振动筛，以防止聚集。混合，然后准备使用活细胞。 </li><li>在37℃孵育细胞100μL混合5分钟，7％的CO 2。 </li><li>用细胞非萤光成像介质（HBSS中的缓冲区，1％肝素钠，看到表的特定试剂）预温37°C。 </li><li>仔细去除多余的标签，以防止联合国必要通过绑定破坏信噪比，重点量子点，通过广泛与成像介质，在室温下通常为5倍延迟至少5分钟前，最后一次洗涤洗涤每口井。 </li></ol> 3。光学装置视频显微镜设置四个主要部分组成: <ol><li>倒置显微镜与一个特定的荧光立方体（FF01-457/50激励，FF495-Di02分色，FF01-617/73排放过滤器，Semrock）和高数值孔径（1.3或1.49）油浸泡100X目标。 </li><li>一个100瓦汞灯（通过光纤耦合到显微镜，避免干扰与体温）。 </li><li> 512 x 512像素高灵敏度EMCCD相机达到了足够的信噪比。 </li><li>孵化器保持在37°C，在实验过程中的生物样品。 </li></ol> 4。获得<ol><li>选择孤立和传播良好的细胞。这利于延长不错，平坦的伪足，最适合寻找分子膜的平面运动，。 </li><li>评估细胞的生理状态，其外观都在透射光荧光:紧张水疱交通，坏死或凋亡的迹象，低自体荧光，平均强大的量子点标记（通常高达1000每单元量子点的情况下，请参阅图1b）。 </li><li>选择一个高的标签密度，同时监测尽可能多的受体是至关重要的。这实际上是有限的生理（表面的表达，抗原无障碍，横向运动）和算法参数（非重叠的点扩散函数（PSF），即使考虑到由于议案的帐户模糊，允许适当的检测和重新连接）。 </li><li>对于每一个细胞，首次获得明场图像（优先使用，DIC，如果有的话），可以进一步让检查细胞方面和空间限制的lamellipodia。 </li><li>保存该图像，视频栈（如例如cell1.tif及cell1.stk）使用相同的名称，在名为DIC的一个子文件夹，因为，这些公约，该算法可以自动找回来的图像匹配每个堆栈。 </li><li>收购每单元1至3部影片，连续，通常在36毫秒率，以最快的速度在全画幅，这台相机实现。但是人们可以获取更高的频率，可达1毫秒率，使用专用的CCD具有更高的灵敏度和/或更少的像素。帧传输技术，提供帧之间的延迟可以忽略不计。 </li><li>电子乘法增强应始终设置为最大（仅低于饱和，如果有关的话），可以达到单个分子的敏感性，有足够的信噪比，至少超过20分贝（高效的峰值检测1），通常约为25-30分贝。 </li><li>通常情况下获得300帧，每部影片，罪CE轨迹〜100帧平均多长的闪烁事件的限制，重建了。视频频率和长度可以适应一个给定的措施，例如可能需要更长的痕迹。 </li></ol> 5。 MTT法分析<ol><li>目录中包含的视频文件与Matlab或倍频评价一个给定的数据集选择的路径。 </li><li>要启动的完全自动化的分析1,35，键入精明，或MTT23i在Matlab命令detect_reconnex23。这个方案表示"2.3版本的用户界面，"可供下载，与以前的2.2版本。它首先显示的图形界面，列出所有使用的参数， 如图所示。 2。 </li></ol> 6。代表结果 MTT法自动分析每个视频录像机，实现目标的检测和估计的痕迹，进一步补充如坐月子检测vestigations。这最终使细胞图像（ 图1C＆3）映射的痕迹。 MTT法说明核心MTT法进行了每一帧，调用3（ 图3）主要任务: <ol><li>作为一个双维高斯峰:存在存在或没有一个目标（量子点），在滑动分区域先后围绕每个像素的中心，其中两个假设进行了比较，无论是信号检测与PSF模型化，或唯一的噪音，投保足够低的误报的一个门槛，用虚假检测不到百分之一帧。这将导致检测概率图。被认为是一个假定的目标，每个地方的最高投保，其概率是根据所需的误报警（PFA）的概率，比最低值高。此限制设置得足够低，有效地辨别信号噪音花费较长时间，避免在（10-6 PFA默认情况下，以保证在512×512像素的图像误差不到一）虚假检测，同时仍然允许足够高的检测概率，达到了理论预测最佳1。需要注意的是，使用一个分区域的要求意味着，图像边界（3个像素，为7×7像素的默认窗口）无法评估。 </li><li> 估计，对有关参数，如亚像素位置和信号强度，每个检测到的目标。对于每一个检测到的目标，最小二乘高斯 - 牛顿适合未来执行估计检测高斯的位置，宽度和高度。这提供了显着的染料的亚像素位置（10至20纳米精度典型的SNR值和静止或缓慢扩散的染料，增加染料在0.1微米的2 / s的扩散到约100 nm）。 </li><li> 重联的新目标已建成超过前一帧的痕迹。以前的痕迹，再配上一套新的目标。为此，为了给每个目标跟踪，如果可能的话，使用所有可用的统计资料，从检测步骤获得，不仅地位，但也强度，宽度，闪烁和相关的统计。因此，目标不只是分配到最近的痕迹:在过路的痕迹，强度，速度，宽度和闪烁的情况下将被视为。这也提供了统计最优的重联得分。此策略避免了可能的时候，向近邻偏置重联。 </li></ol>检测峰可以拒绝，如果他们的估计或重新连接失败后验。一个特殊的测试处理检测新的高峰，这将启动新的痕迹。本试验采用更严格的PFA（10-7），自重新高峰跟踪验证Ø可以解释为事实上<html> f的相关性（这个标准定义不适用新的高峰）。 轨迹分析未来可能短暂监禁评估当地扩散24-29负相关的功能。应用门槛，允许定义限制或不发作。通过遍历所有的痕迹，我们可以将膜动力学，在短暂的禁闭/放慢事件。这可以交替使用二进制或本隔离指数的离散值代表。 默认情况下，MTT法自动执行这些任务，保存在一个文本文件中的8个峰参数:车架号，i和j的位置，信号强度，半径，偏移和闪烁每个视频帧（组7行）和微量元素（列）， 。这些输出参数，可以重新使用Matlab或倍频fread_data_spt脚本，为进一步分析即走线或信号强度，作为体现在<html> "http://www.jove.com/files/ftp_upload/3599/MTT_example.txt"&gt; MTT_example脚本附录。 进一步的分析，导致映射的每个单元的痕迹（ 图1C），并提供相关参数（如峰值强度，信噪比或局部扩散值）直方图分布。对于每个文件，平均每个参数的标准偏差都保存在一个文本文件，图像的直方图。对数分布，如R 2平方位移，导致以几何平均数。从以上的MSD曲线的第一个五点的线性拟合计算的扩散系数D。这些值涉及细胞反应或影响膜组织的制药/酶处理的例子动力学实验提供了一个概述。由于MTT法是一个开放的源代码，这方面可以很容易适应任何专门的调查。 599fig1.jpg"ALT ="图1"/&gt; 图1。膜受体的动态监测采用MTT。（一）膜元件，如表皮生长因子受体，与生物素化的Fab片段（约正确的，每个分子尺度的示意图）耦合量子点标记。 （二）典型的荧光图像获得从现场的COS-7细胞，用36毫秒的曝光时间，描绘有限衍射峰对应的个别标记的受体。 （三）MTT法显示输出受体重组后的轨迹，对细胞的明图像覆盖。 <img src="/files/ftp_upload/3599/3599fig2.jpg" alt="图2" /> 图2。 MTT法输入参数。运行MTT23i打开图形用户界面，列出所有的输入参数，名称和默认值，在我们以前出版1。在算法，空间和时间参数（搜索窗口，峰值半径，最大扩散<html>闪烁）是无量纲的标准单位，像素和帧。校准可用于事后转换输出结果。默认值，相应的一个梯级512BFT 100X倍率，像素大小:156海里/ PXL和帧延迟:36毫秒/帧。 调查人员应优化几个关键参数，如预期的最大扩散系数（"变化最大"）和最大闪烁失踪（"T OFF"）。这两个时间和空间的限制，几乎是唯一的，需要重新考虑，对于一个给定的实验条件下，健壮的默认值被设置为其他的。例如，误报的数量直接设置，以确保每万像素不到一个错误，因此比每一个框架，这在大多数情况下是令人满意的。所有参数都保存在一个文本文件输出文件夹中，让用户核实后的设置被用于分析。 s/ftp_upload/3599/3599fig3.jpg"ALT ="图3"/&gt; 图3。 MTT法的主要步骤。从实验的荧光图像栈开始，峰顺序，并自动检测，估计高斯拟合和多帧重新连接（第一的经营范围，上部流程图）。假定禁闭的痕迹可以进一步加以分析，例如，最终导致了相关描述（第二经营范围，下部）的动态地图。 <img src="/files/ftp_upload/3599/3599fig4.jpg" alt="图4" /> 图4。标签价不会影响MTT法评估可能的偏差，通过人工的多价标签介绍，MTT法进行跟踪标签使用2个不同的方案，以生成和分析的轨迹图的内源性表皮生长因子受体。 （A）受体与生物素化的Fab和量子dots605的 - 亲标签，在协议中所述。在这种情况下，量子点和streptavidins多价可能会导致在一个单一的染料耦合几个受体。 （二）受体标记直接耦合到一个有机染料，Atto647N晶圆厂。在这种情况下，一个晶圆厂，因此一个受体，可以再加上一个以上的染料。 （三）平均平方位移（MSD）的曲线，计算出每个单元的所有痕迹，标记与量子点或阿托染料（左边和右边的图形，分别）。超过五个第一的MSD（红色虚线）点的线性拟合计算扩散系数。每个标签导致类似的扩散值（图中央）的计划。 qdot:量子dots605（N = 5个细胞），阿托:Atto647N（N = 7细胞），NS:不显着（学生t检验P值&gt; 0.05）。

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没有利益冲突的声明。

旁边的细胞和显微镜的各个方面，在单粒子跟踪，分析代表了相当一部分的工作。这解决了算法用于执行三大任务:检测，评估和重新连接了每帧的山峰。但随之而来的这项工作方面驻留在拟订算法本身，这可能需要适应任何新的专门调查，基本上最后，额外的步骤（如破译运动模式，相互作用或化学计量学）。 然而，一旦该算法充分开发，运行，它是简单的，特别是因为我?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 数量 </td></tr><tr><td> COS-7细胞系</td><td> ATCC </td><td> CRL-1651 </td><td> 5000细胞/孔</td></tr><tr><td>无钙的HBSS中2 + </td><td> GIBCO公司</td><td> 14175 </td><td> 1毫升</td></tr><tr><td>胰蛋白酶EDTA的0.05％ </td><td> GIBCO公司</td><td> 25300 </td><td> 1毫升</td></tr><tr><td> 8以及实验室TEK </td><td> NUNC </td><td> 155441 </td><td> 1 </td></tr><tr><td> QDot-605链霉</td><td> Invitrogen公司</td><td> Q10101MP </td><td> 20毫米</td></tr><tr><td colspan="4"> 生物素厂 （为晶圆厂合成，见参考文献21） </td></tr><tr><td>从单克隆抗体的Fab 108 </td><td> ATCC </td><td> HB-9764 </td><td> 200微克</td></tr><tr><td> NHS-生物素<html></td><td> Thermo Scientific的</td><td> 21435 </td><td> 18.5微克</td></tr><tr><td colspan="4"> 完全培养基 </td></tr><tr><td> DMEM培养液</td><td> GIBCO公司</td><td> 41965 </td><td> 500毫升</td></tr><tr><td>胎牛血清</td><td>西格玛</td><td> F7524 </td><td> 50毫升</td></tr><tr><td> L-谷氨酰胺</td><td> GIBCO公司</td><td> 25030 </td><td> 5毫升</td></tr><tr><td>肝素钠</td><td> GIBCO公司</td><td> 15630 </td><td> 5毫升</td></tr><tr><td>丙酮酸钠</td><td> GIBCO公司</td><td> 11360 </td><td> 5毫升</td></tr><tr><td colspan="4"> 成像介质 </td></tr><tr><td>与钙的HBSS中2 + </td><td> GIBCO公司</td><td> 14025 </td><td> 25毫升</td></tr><tr><td>肝素钠</td><td> GIBCO公司</td><td> 15630 </td><td> 250μL </td></tr></tbody></table>1"&gt; <tbody><tr><td> 设备 </td><td> 公司 </td><td> 参考 </td></tr><tr><td>倒置显微镜</td><td>尼康</td><td>日食TE2000U </td></tr><tr><td>日光灯</td><td>尼康</td><td> intensilightÇ-HGFIE </td></tr><tr><td> 1.3适用100X目标</td><td>尼康</td><td>计划氟1.30 </td></tr><tr><td> 1.49不适用100X目标</td><td>尼康</td><td>亚洲生产力组织的TIRF 1.49 </td></tr><tr><td>相机</td><td>罗珀科学</td><td>级联512 B的</td></tr><tr><td>恒温箱</td><td>生活影像服务</td><td>包装盒</td></tr></tbody></table>附录:例如MTT的补充分析脚本 ，功能 MTT_example（FILE_NAME） ％％％Basic示例显示如何恢复MTT法的输出结果 ％％％绘制每个TRACe和建立直方图 荧光强度的％％ nargin &lt;1％，如果没有提供FILE_NAME？ 文件= DIR（&#39;* STK&#39;）; 如果的isEmpty（文件），DISP（&#39;没有数据&#39;在当前目录）， 返回，结束 FILE_NAME =文件（1）名称;％默认:第一STK文件 DISP（[&#39;使用&#39;FILE_NAME&#39;默认&#39;]） 结束 file_param = [FILE_NAME"_tab_param.dat&#39;];％的输出文件 ％负载数据 CD％或（"output22）（"output23），根据版本使用 声明:版本2.2只产生7个参数， ％一个额外的参数，噪声，2.3版本中添加了 ％在一个表中一次读取所有的参数， ％tab_param = fread_all_param（file_param）; ％TAb_i = tab_param（2:8结束，:); tab_j = ... ％在不同的表读取所有参数（除frame_number） ％[tab_i，tab_j，tab_alpha，tab_radius，tab_offset，tab_blk，tab_noise] = fread_all_data_spt（file_param）; tab_i = fread_data_spt（file_param，3）;％，指数是3，因为跟踪号码和车架号码，非知识性，被丢弃！ tab_j = fread_data_spt（file_param，4）; tab_alpha = fread_data_spt（file_param，5）; tab_blk = fread_data_spt（file_param，8）; ％％循环过的痕迹 n_traces =大小（tab_i，1）; ％表是由N_frames colums N_traces线 ITRC = 1:N_traces no_blk_index = tab_blk（ITRC，:)&gt; 0％，非只有闪烁的步骤 情节（tab_i（ITRC No_blk_index）的，tab_j（ITRC No_blk_index）的） ＆NBSP; xlabel（&#39;I（像素）&#39;），ylabel（&#39;J（像素）"） 标题（[微量＃&#39;num2str（ITRC）]） DISP（&#39;请取得任何未来跟踪的关键"），暂停 结束 ％FLUO直方图的 n_datapoints = SUM（tab_blk（:)&gt; 0）;％，无闪烁的步骤 * SQRT（N_datapoints）的）历史（tab_alpha（tab_blk&gt; 0），2％使用2sqrt（N）的垃圾箱 xlabel（&#39;强度（非盟）&#39;），ylabel（&#39;发生&#39;） 标题（"粒子的荧光强度的直方图&#39;）

mapping molecular diffusion in the plasma membrane by multiple-target tracing (mtt)

我们的目标是获得细胞膜发生在不同的生物功能的分子过程的全面描述。我们的目标是在描述复杂的组织和细胞膜的动态，在单分子水平，开发的分析工具，专门在高密度单粒子追踪 （SPT）: 多目标跟踪 （MTT）1。电视显微镜，单分子提供毫秒级和纳米级分辨率1-11，允许膜组织12-14详细表示，通过精确映射，如细 ​​胞受体的定位，流动性，禁闭或相互作用的描述。 我们重新审视六方会谈，实验和算法。实验方面包括:优化设置和细胞标记，以达到尽可能高的标签密度特别强调，为了提供一个分子动力学动态快照就这样发生在膜。算法的问题有关的每一步都用于重建的轨迹:峰值检测，估计和重新从15,16图像分析处理，通过特定的工具。实施后，检测通缩允许抢救峰最初隐藏相邻，峰强。值得注意的是，提高检测直接影响重联，减少内轨迹的差距。已评估使用各种标签密度和噪声值，这通常代表两个主要局限在高时空分辨率的并行测量的蒙特卡罗模拟表演。 17纳米的精度得到单分子，无论是连续使用开/关photoswitching或非线性光学，可以提供详尽的意见。这是19,20风暴18日 ，棕榈，RESOLFT 21 STED 22,23，WHI等17 nanoscopy方法的基础上CH可能常常需要固定的样品成像。中心任务是有限的单分子所产生的衍射峰的检测和估计。因此，提供足够的假设，如恒定的定位精度，而不是处理布朗运动，MTT法是直截了当地适用于纳米级的分析。此外，MTT法可以从根本上被使用在任何规模:不仅为分子，同时也为细胞或动物，例如。因此，MTT法是一个功能强大的跟踪算法，发现在细胞和分子尺度的应用。

Watch this Scientific Journal Video about Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT at JoVE.com

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

多目标跟踪是一个自制的算法跟踪单独标记分子在活细胞的细胞膜。有效地检测，估计和跟踪在高密度的分子随着时间的推移，提供一个用户友好，全面的工具来研究纳米膜动力学。

测绘在细胞膜分子扩散，多目标跟踪（MTT）

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

mapping-molecular-diffusion-plasma-membrane-multiple-target-tracing

Research

JoVE Journal

Biology

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

17.2K 次观看.  Parc scientifique de Luminy. 多目标跟踪是一个自制的算法跟踪单独标记分子在活细胞的细胞膜。有效地检测，估计和跟踪在高密度的分子随着时间的推移，提供一个用户友好，全面的工具来研究纳米膜动力学。

视频: Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

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

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

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 ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

视黄醇运输的血浆视黄醇结合蛋白的膜受体的实时分析

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

绿色荧光蛋白标记的质膜蛋白质的扩散系数的简易测量采用K空间图像相关光谱

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Imaging Plasma Membrane Deformations With pTIRFM

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

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

成像质膜变形随着pTIRFM

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

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

药物与DNA相互作用的细胞全基因组映射与COSMIC（小分子交联，染色质隔离）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

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 ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.