This work was funded by a grant from the Canadian Institute for Health Research MOP 82890 to SHP. The authors wish to thank G.H. Patterson and J. Lippincott Schwartz for the paGFP construct. SN56 cells were a gift of Dr. Jane Rylett.

1.电池电镀和转染<ol><li>在细胞培养罩，钢板〜30万至五十万元SN56细胞（简Rylett博士惠赠）在35毫米的玻璃底焦的菜，并与预染的媒体覆盖（贝科改良Eagle培养基，DMEM，辅以10％FBS）。 </li><li>在一个5％CO 2培养箱增殖过夜孵育细胞，在37℃。 注：细胞应为约50％的转染前-70％汇合。 </li><li>在细胞培养罩，转染细胞用质粒表达的荧光蛋白标记的TGN标记（半乳糖基转移酶耦合到青色荧光蛋白，GALT-CFP），荧光蛋白标签隔室的标志（这里，相关溶酶体膜蛋白1，LAMP1，具有标记MRFP），和感兴趣的具有标记paGFP（βAPP-paGFP的蛋白）。 （这些构建之前已描述30） </li><li>孵育细胞与转染试剂和质粒DNA˚F或24小时，在37℃下在5％CO 2培养箱。使用的DNA的〜2微克的比率为5微升转染试剂（例如脂质体2000），以最佳平衡的细胞毒性和转染效率。实际浓度可能需要为不同的质粒进行优化。在这里描述的实验中，使用βAPP-paGFP的1微克/共聚焦盘，LAMP1-MRFP 0.3微克/共聚焦盘，和GALT-CFP的0.2微克/共焦菜。 注意：使用质粒的量将取决于转染效率。 </li><li>神经元细胞系：步骤1.4之后，在细胞培养罩，除去预转染培养基和分化SN56细胞在2ml的DMEM补充有0.1％青霉素/链霉素用1mM二丁环AMP（dbcAMP）24小时。 </li></ol> 2.显微镜阶段准备<ol><li>预温热的磷酸盐缓冲盐水（PBS）和Hank氏平衡盐溶液（HBSS）至37℃。意马前热身显微镜阶段吴至37℃。 </li><li>用PBS洗涤细胞两次，以除去分化培养基和2毫升HBSS中替换，在37℃。在显微镜阶段将细胞加热到37℃。 注：细胞是活的，在HBSS中约1.5-2小时。 </li><li>允许5-10分钟为共焦板温度达到平衡与显微镜阶段。 </li></ol> 3.光激活的ROI在高尔基<ol><li>用显微镜目镜，发现转染GALT-CFP，LAMP1-MRFP，和βAPP-paGFP。 <ol><li>使用的油镜镜头，高倍率（ 即 63X或100X）。对于荧光视觉形态，使用设置能够可视化FITC（适用于CFP和GFP荧光）和罗丹明（对于MRFP荧光）的过滤器。 </li></ol></li><li>共焦切片为每个通道设置为1微米。取一个低分辨率图像。 </li><li>裁剪图像所感兴趣的细胞。 </li><li>利用调整旋钮，手动设置焦平面到小区的中间。这通常是其中核是最大的小区的一部分。 </li><li>在TGN（GALT-CFP荧光）内画利益（投资回报）​​3-5圆形区域。 <ol><li>为了吸引投资回报（ 即蔡司LSM程序），选择“编辑ROI”命令控制台按钮。 </li><li>选择圆形ROI按钮。 </li><li>单击并拖动超过GALT-CFP电池的正面区域。 注意：光漂白包许多显微镜具有这种能力。 </li></ol></li><li>采取与重叠的ROI的细胞的图像。保存ROI的副本，以供日后参考。 <ol><li>要将投资回报保存图像，选择“宏”按钮。 </li><li>按“加载”按钮，启动“CopyRoisToOverlay”（文件路径：C：/AIM/Macros/AdvancedTimeSeries/Utility.lvb）。 注：共聚焦显微镜配备有475-525纳米（CFP）带通（BP），一个500-530纳米的BP（GFP），和560纳米长通（LP）滤波器组是必需的。也需要458纳米和488纳米发射和540nm的发射氦氖激光器的氩激光。 </li></ol></li><li>设置在显微镜的漂白时间过程。 <ol><li>设置激光二极管（25毫瓦405纳米的激光），以最大功率。 注意：过度的光激活会导致光毒性或损害细胞膜。使用保护眼睛，避免视网膜损伤。 </li><li>设置了显微​​镜成像120次。 注：一个成像周期包括在投资回报中的漂白和整个小区的成像。这是漂白时间过程。 </li><li>将显微镜漂白（光激活），只有投资回报的每张图片后20-30迭代。的时间，以图像和漂白的图像会有所不同。设置图像之间的时间延迟，所以每个周期大约是30秒。 注：每个周期的成像部分大约需要15-30秒取决于图像大小。漂白每个RO余为约50毫秒。所有4个感兴趣区为20次迭代的漂白需要4秒在每个漂白/图象周期​​的结束。 </li></ol></li><li>启动漂白时间过程。 </li><li>关掉漂白15分钟（成像和漂白的30个循环）之后，但继续捕获细胞的图像。 注：时间0至15分钟是“脉冲”时期。 </li><li>继续拍摄图像的45分钟（不含漂白），跟随βAPP-paGFP的间隙。 注：时间15至45分钟是&#39;追&#39;的时期。 </li><li>使用在步骤6中描述的分析方法，共定位的兴趣（这里，βAPP-paGFP）与感兴趣的隔室（在此，LAMP1-MRFP）通过使表面到每个信道的蛋白质。 </li></ol> 4.确定下游腔<ol><li>以确定是否溶酶体（在这些实验中）是最后的隔室中，添加膜可渗透蛋白酶抑制剂以引起蛋白质积累在溶酶体。<ol><li>对于βAPP，使用0.5微米L685，458（具体的γ分泌酶抑制剂）晚上或100μM氯喹（deacidifies溶酶体）成像前30分钟。 </li></ol></li><li>查找细胞和光活化按照步骤3.1 - 3.8。 </li><li>图像的细胞另外45分钟（没有光激活）来确定该βAPP-paGFP积累在缺乏裂解。 </li><li>根据在步骤6中描述的方法进行分析的递送蛋白的溶酶体（或其它下游隔室）和随后的间隙。 </li></ol> 5.高尔基内确保光活化的精度<ol><li>热身HBSS至37℃。 </li><li>制备2ml的溶液，对于每一个共焦板要被成像的66μM诺考达唑（处理）或DMSO（对照）在HBSS中。 <ol><li>其一共聚焦板，加入2毫升37℃HBSS和从16.60毫米诺考达唑库存增加7.96微升诺考达唑的。 </li><li>作为控制如此而定，加入2毫升37℃HBSS到2ml管中，并加入7.96微升DMSO中。 </li></ol></li><li>孵育细胞，在HBSS中与诺考达唑或DMSO进行成像之前5分钟。 </li><li>发现细胞在步骤3.1。 </li><li>光激活前，准备显微镜拍照，激活期限后Z堆栈。 <ol><li>点击&#39;Z堆栈“按钮。开始扫描使用快速XY。 </li><li>调整焦平面与显微镜调整旋钮电池的顶部。按“马克先&#39;设置在堆栈中的第一个位置。 </li><li>调整焦平面与细胞的显微镜调整旋钮的底部（最靠近玻璃）。按&#39;马克最后&#39;来设置在堆栈中的最后一个位置。 </li><li>在Z堆叠面板，按“Z切片”按钮。将“间隔”为1微米。 注意：对于更高的空间分辨率堆栈较小堆栈间隔，但更多的图像将需要采取延长为栈的摄像期间。 </li></ol></li><li>漂白细胞，如在步骤3.7-3.8中描述。光激活和图像从0分钟至15分钟（30帧）。 </li><li> 30帧后停止成像。立即保存视频的图像。 注意：有些显微镜，可能会覆盖第一视频，如果它不保存启动第二成像序列之前。 </li><li>保存的视频后，立即获取Z堆栈，使用步骤5.5中的参数。 </li><li>共定位βAPP-paGFP与GALT-CFP（或其它高尔基标记物），如在步骤6中描述。 注意：CFP光漂白剂迅速且由成像期间结束可能不可见。共定位可能无法与GALT-CFP。如果共定位是必需的，使用MRFP标记哥尔特。 </li></ol> 6.囊泡过滤和共定位<ol><li>使用一个分析程序 （例如了Imaris）以使隔室的表面 （如LAMP1-MRFP）和蛋白O-˚F 利益 （例如βAPP-paGFP）。 </li><li>通过点击顶部菜单栏上的超越按钮，进入分析程序的“超越”部分。 </li><li>在最左边的面板（左起第 5按钮）的顶部选择曲面向导按钮。 </li><li>选择一个频道来过滤囊泡（ 即 βAPP-paGFP荧光，感兴趣的蛋白质）。 </li><li>通过提交大泡的直径在现场向导，然后按下一步进行背景扣除。分析程序自动选择 - 根据从最大囊泡在现场得到的阈值 - 在感兴趣的信道的区域。 <ol><li>手动调整阈值的背景，如果需要细化选择。 </li></ol></li><li>在阈值选择屏幕的底部，选中“拆分触摸对象”选项来分隔组合表面。 注意：如果许多小泡紧密分组，GETHER，分析程序意愿组下一个表面上这些对象。 <ol><li>选择10-15代表性的囊泡，并计算出平均泡直径并输入到向导。 </li></ol></li><li>通过提高或降低对“质量”（在每个点的中部的通道的强度）的阈值缩小所选种子点。 注：感兴趣的频道的表面将被创建。选定曲面的进一步细化可以在此时执行。基于在每个泡囊的像素数阈囊泡。 </li><li>面膜使用这种表面原有的渠道。要屏蔽，请从左侧面创建向导（铅笔）第4选项卡。 </li><li>选择“屏蔽所有”。与感兴趣的囊泡一个新的通道将被创建。 注：在面膜的过程中，一个选项，复制原有的渠道提供。如果原始数据需要保存，请选择此选项。 </li><li>重复小号TEPS 6.1至6.7的未经过滤的通道（ 即 LAMP1 MRFP，车厢）。 </li><li>在顶部的工具栏，选择共定位工具。 <ol><li>在顶部面板中，作为信道被共定位直方图出现，选择最近创建的屏蔽频道。 </li><li>选择所有可用的数据。有暗像素有时分析后存在。不要选择这些像素直方图，就会影响结果。 </li><li>在最右侧的面板中，选择“生成Coloc通道”。这将创建一个只包含共定位像素的新渠道。的数据将在在&#39;频道统计“按钮相同的面板。该数据可以导出为.csv文件。 </li><li>使用“材料的共定位百分比”统计。这个选项考虑到对象的像素和尺寸的强度。 </li></ol></li></ol>

<ol>
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W., 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=Retromer+binds+the+FANSHY+sorting+motif+in+SorLA+to+regulate+amyloid+precursor+protein+sorting+and+processing.">Retromer binds the FANSHY sorting motif in SorLA to regulate amyloid precursor protein sorting and processing.</a> J. Neurosci. 32 (4), 1467-1480 (2012).</li><li>Patterson, G. H., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Photoactivatable+GFP+for+Selective+Photolabeling+of+Proteins+and+Cells.">A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells.</a> Science. 297 (5588), 1873-1877 (2002).</li><li>Patterson, G. H., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+photolabeling+of+proteins+using+photoactivatable+GFP.">Selective photolabeling of proteins using photoactivatable GFP.</a> Methods. 32 (4), 445-450 (2004).</li><li>Hirschberg, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+secretory+protein+traffic+and+characterization+of+golgi+to+plasma+membrane+transport+intermediates+in+living+cells.">Kinetic analysis of secretory protein traffic and characterization of golgi to plasma membrane transport intermediates in living cells.</a> J. Cell Biol. 143 (6), 1485-1503 (1998).</li><li>Kim, P. K., Mullen, R. T., Schumann, U., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+maintenance+of+mammalian+peroxisomes+involves+a+de+novo+PEX16-dependent+pathway+from+the+ER.">The origin and maintenance of mammalian peroxisomes involves a de novo PEX16-dependent pathway from the ER.</a> J. Cell Biol. 173 (4), 521-532 (2006).</li><li>Hailey, D. W., 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=Mitochondria+supply+membranes+for+autophagosome+biogenesis+during+starvation.">Mitochondria supply membranes for autophagosome biogenesis during starvation.</a> Cell. 141 (4), 656-667 (2010).</li><li>Tam, J. H. K., Seah, C., Pasternak, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Amyloid+Precursor+Protein+is+rapidly+transported+from+the+Golgi+apparatus+to+the+lysosome+and+where+it+is+processed+into+beta-amyloid.">The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid.</a> Mol. Brain. 7 (1), 54 (2014).</li><li>Scott, D. A., Das, U., Tang, Y., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+logic+underlying+the+axonal+transport+of+cytosolic+proteins.">Mechanistic logic underlying the axonal transport of cytosolic proteins.</a> Neuron. 70 (3), 441-454 (2011).</li><li>Herl, L., 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=Mutations+in+amyloid+precursor+protein+affect+its+interactions+with+presenilin/gamma-secretase.">Mutations in amyloid precursor protein affect its interactions with presenilin/gamma-secretase.</a> Mol. Cell. Neurosci. 41 (2), 166-174 (2009).</li><li>Lorenzen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+Direct+Transport+of+Cell+Surface+APP+to+the+Lysosome+defines+a+novel+selective+pathway.">Rapid and Direct Transport of Cell Surface APP to the Lysosome defines a novel selective pathway.</a> Mol. Brain. 3 (1), 11 (2010).</li><li>Thinakaran, G., Teplow, D. B., Siman, R., Greenberg, B., Sisodia, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+the+'Swedish'+amyloid+precursor+protein+variant+in+neuro2a+(N2a)+cells.+Evidence+that+cleavage+at+the+beta-secretase";site+occurs+in+the+golgi+apparatus.">Metabolism of the 'Swedish' amyloid precursor protein variant in neuro2a (N2a) cells. Evidence that cleavage at the beta-secretase";site occurs in the golgi apparatus.</a> J. Biol. Chem. 271 (16), 9390-9397 (1996).</li><li>Thinakaran, G., Koo, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+Precursor+Protein+Trafficking,+Processing,+and+Function.">Amyloid Precursor Protein Trafficking, Processing, and Function.</a> J. Biol. Chem. 283 (44), 29615-29619 (2008).</li><li>Schneider, M., Barozzi, S., Testa, I., Faretta, M., Diaspro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Photon+Activation+and+Excitation+Properties+of+PA-GFP+in+the+720-920-nm+Region.">Two-Photon Activation and Excitation Properties of PA-GFP in the 720-920-nm Region.</a> Biophys. J. 89 (2), 1346-1352 (2005).</li><li>Sevier, C. S., Weisz, O. A., Davis, M., Machamer, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+export+of+the+vesicular+stomatitis+virus+G+protein+from+the+endoplasmic+reticulum+requires+a+signal+in+the+cytoplasmic+tail+that+includes+both+tyrosine-based+and+di-acidic.">Efficient export of the vesicular stomatitis virus G protein from the endoplasmic reticulum requires a signal in the cytoplasmic tail that includes both tyrosine-based and di-acidic.</a> 11 (1), 13-22 (2000).</li><li>Muresan, V., Varvel, N. H., Lamb, B. T., Muresan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cleavage+products+of+amyloid-beta+precursor+protein+are+sorted+to+distinct+carrier+vesicles+that+are+independently+transported+within+neurites.">The cleavage products of amyloid-beta precursor protein are sorted to distinct carrier vesicles that are independently transported within neurites.</a> J. Neurosci. 29, 3565-3578 (2009).</li></ol>

作者有没有竞争经济利益或利益冲突。

该技术描述了精确的光激活膜蛋白标记paGFP在TGN想象以后的贩卖和切割。虽然paGFP创建十年前25，这是精确的光激活跟随新生蛋白质贩运到下游车厢的第一个例子。使用的APP-paGFP以往的研究构建光活化的细胞，其被认为是高尔基体11的周边区域的核。然而，在我们的显微照片，溶酶体，早期内涵体和晚期内涵体作为显然也可以在该区域（图1和参考30）中找到。这些实验?...

阿尔茨海默氏病（AD）的特点是老年斑和神经原纤维缠结（NFTS）在脑中的存在。老年斑的主要成分是β淀粉样（Aβ）。 Aβ是由其前体衍生的;淀粉样前体蛋白（APP）1。 APP的淀粉样蛋白形成切割开始除去胞外域的来自APP的由β分泌2。剩余的99个残基的羧基末端片段（CTF）可通过γ分泌裂解以产生Aβ的3-7。虽然许多实验已经证明的细胞表面APP的裂解从细胞表面到核内体/溶酶体系统内化后，最近的一些研究已经表明，APP的细胞内运输也是在调节其处理8-11重要。 已经有一些尝试，在调控Aβ水平与γ分泌酶抑制剂和Aβ与免疫otherapies。然而，随着这些疗法最近的临床试验表明没有好处，而且，在某些情况下，造成的损害12。一个未开发策略以调节Aβ生成是改变APP的亚细胞定位和γ分泌相互作用。高尔基体，质膜和胞内体/溶酶体都被认为是可能的语言环境γ裂解APP的。从我们的实验室的研究表明，APP和所述γ分泌是溶酶体膜13的居民的蛋白质。此外，我们已发现，溶酶体γ分泌具有酸性最适pH 13。此外，内体/溶酶体系统与氯喹或NH 4的Cl碱化，已经显示减少生产的Aβ14。 γ-分泌酶的抑制或敲除或早老导致APP-CTFS堆积在溶酶体15-17。此外，扰乱APP内吞作用降低Aβ生成18-20。 尽管高尔基作为分类站为新生蛋白质，并从内体/溶酶体系统回收蛋白的重要性，APP的细胞内运输尚未详细研究21。最近的工作表明，APP可以经由与Retromer Complex功能相互作用再循环到反面高尔基网（TGN）。在Retromer Complex上的下调减少Aβ产生8,22-24。然而，APP的从高尔基体出口未得到很好的研究。 而以下细胞表面蛋白，例如转铁蛋白受体的内吞作用，很容易被标记并且随后，以下细胞内蛋白质的贩卖是更具挑战性。事实上，很少有蛋白质有他们的细胞内运输成像。荧光蛋白标记，如光活化-GFP（paGFP）的出现，提供了新的工具来研究细胞内运输。光活化-GFP是GF形式P，该是合成后几乎不可见，但发展强烈的绿色（GFP）的荧光通过413纳米的激光活化之后并且该信号是稳定的天25,26。使用paGFP构建体已经用于证明溶酶体蛋白的溶酶体内贩卖膜蛋白1（LAMP1）25，在细胞表面递送水泡性口炎病毒糖蛋白（VSVG，分泌途径的标志物）27，和过氧化物酶的周转28和自噬体29。 为了可视化的APP从TGN中 ​​活细胞分选，我们设计了质粒表达APP的最后112个氨基酸耦合到光活化（paGFP）上的C末端（称为βAPP-paGFP）30。我们还进行这些实验，全长APP取得了相似的结果;的βAPP构建在这里使用，因为它提供更明亮的图像。然后，我们光激活＆＃946; APP-paGFP只有在TGN，所划分的TGN标记半乳糖（高尔特）。虽然APP已经被标记为paGFP可视化APP的快速轴突运输和清关的核周区域，这是APP贩运的第一个示范从一个仔细界定室到另一个11,31,32。在这里，我们展示了TGN内精确的光激活和APP的出口进入下游的车厢和随后的裂解和清除从溶酶体30。高尔基内的精确光激活可广泛适用于其它蛋白质的系统。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>35-mm glass bottom culture dishes&#160;</td>
			<td>Matek</td>
			<td>P-35G-1.5-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Life Technologies</td>
			<td>11995-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>dibutyrl cyclic AMP</td>
			<td>Sigma&#160;</td>
			<td>D0627</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat in activated Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated Microscopy stage insert P</td>
			<td>PeCon GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tempcontrol 37&#8211;2 digital 2-channel</td>
			<td>PeCon GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM-510 META laser- scanning microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>with laser diode, argon laser, and HeNe1 laser</td>
		</tr>
		<tr>
			<td>Zeiss 63&#215; 1.4 numerical aperture oil immersion lens&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L685, 458&#160;</td>
			<td>EMD Millipore</td>
			<td>565771</td>
			<td>dissolved in DMSO</td>
		</tr>
		<tr>
			<td>cholorquine&#160;</td>
			<td>Sigma</td>
			<td>C6628&#160;</td>
			<td>dissolved in water</td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma</td>
			<td>M1404</td>
			<td>dissolved in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide&#160;</td>
			<td>Sigma</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris&#160;</td>
			<td>Bitplane</td>
			<td></td>
			<td>with colocalization package&#160;</td>
		</tr>
	</tbody>
</table>

imaging the intracellular trafficking of app with photoactivatable gfp

β-淀粉样蛋白（Aβ）是在阿尔茨海默氏病的患者的脑中发现的老年斑的主要成分。 Aβ是从淀粉样前体蛋白（APP）由β和γ-分泌的顺序裂解而得。尽管Aβ对AD病理学的重要性，这些分裂的亚细胞定位还不够健全。在我们的实验室工作和其他人从细胞表面内化后牵连内体/溶酶体系统中APP加工。然而，APP的细胞内运输也比较充分研究。 而细胞表面蛋白是易于进行许多标记技术，没有简单的方法从高尔基体以下的膜蛋白的贩卖。为此目的，我们创建了APP的构建体具有标记光活化GFP（paGFP），在C末端。合成后，paGFP具有低基础荧光，但它可以与413纳米的光吨被刺激Ø产生一个强大，稳定的绿色荧光。通过使用高尔基标记半乳糖基转移酶耦合到青色荧光蛋白（GALT-CFP），为目标，我们能够准确地光活化的APP在反面高尔基网。因为它贩卖标识用荧光下游隔室的初级内体（Rab5的）时，次级内体（内Rab9）和溶酶体（LAMP1）的标签隔室标志物蛋白被光激活的APP-paGFP之后可。此外，使用抑制剂以APP加工包括氯喹或γ分泌酶抑制剂L685，458，我们能够进行脉冲追踪实验，以检查APP的处理在单个细胞。 我们发现的APP的很大一部分迅速动作，溶酶体没有出现在细胞表面，然后被从溶酶体清零分泌状分裂。该技术演示paGFP的实用程序来回回以下细胞内蛋白质的贩运和加工米高尔基到下游隔室。

典型结果表明βAPP离开TGN而显得流量迅速LAMP1（图1a 和 b）。在光激活期间，囊泡可以看出出发往溶酶体（图1b）高尔基。无抑制剂的治疗，paGFP荧光将在溶酶体可见的，而有光激活在TGN。停止光活化后，βAPP-paGFP迅速从溶酶体清零（图1a比较为 1b）。治疗与诺考达唑导致APP的GALT-CFP内的累积标记的隔室，并防止贩卖溶酶体（...

Watch this Scientific Journal Video about Imaging the Intracellular Trafficking of APP with Photoactivatable GFP at JoVE.com

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

而细胞表面蛋白的传输相对容易地研究，可视化细胞内蛋白质的贩卖是困难得多。在这里，我们使用结合光活化绿色荧光蛋白的结构和表现出的方​​法来准确地遵循从高尔基体向下游车厢淀粉样前体蛋白，并按照其清除。

成像APP的细胞内运输与光活化GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

imaging-the-intracellular-trafficking-of-app-with-photoactivatable-gfp

Research

JoVE Journal

Biology

11.7K 次观看.  Western University. 而细胞表面蛋白的传输相对容易地研究，可视化细胞内蛋白质的贩卖是困难得多。在这里，我们使用结合光活化绿色荧光蛋白的结构和表现出的方​​法来准确地遵循从高尔基体向下游车厢淀粉样前体蛋白，并按照其清除。 

视频: Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

科研

生物学

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

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 Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

快速合成及其与绿色荧光蛋白为基础的记者化学激活的转录因子筛选

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

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

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

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