Name: imaging the intracellular trafficking of app with photoactivatable gfp
Uploaded: 2015-10-17T14:00:00.000+00:00
Duration: 7 min 55 s
Description: Watch this Scientific Journal Video about Imaging the Intracellular Trafficking of APP with Photoactivatable GFP at JoVE.com

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>

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I., 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=Retrieval+of+the+Alzheimer's+amyloid+precursor+protein+from+the+endosome+to+the+TGN+is+S655+phosphorylation+state-dependent+and+retromer-mediated.">Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated.</a> Mol Neurodegener. 5 (1), 40 (2010).</li><li>Sullivan, C. P., 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+disruption+promotes+amyloidogenic+APP+processing.">Retromer disruption promotes amyloidogenic APP processing.</a> Neurobiol. Dis. 43 (2), 338-3345 (2011).</li><li>Fjorback, A. 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><tbody><tr><td>35 mm glass bottom culture dishes&amp;nbsp;</td><td>Matek</td><td>P-35G-1.5-20-C</td><td></td></tr><tr><td>Dulbecco&#039;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&#039;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&amp;nbsp;</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&amp;ndash;2 digital 2-channel</td><td>PeCon GmbH</td><td></td><td></td></tr><tr><td>Zeiss LSM-510 META laser- scanning microscope&amp;nbsp;</td><td>Carl Zeiss</td><td></td><td>with laser diode, argon laser, and HeNe1 laser</td></tr><tr><td>Zeiss 63X 1.4 numerical aperture oil immersion lens</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>L685, 458&amp;nbsp;</td><td>EMD Millipore</td><td>565771</td><td>dissolved in DMSO</td></tr><tr><td>cholorquine&amp;nbsp;</td><td>Sigma</td><td>C6628&amp;nbsp;</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&amp;nbsp;</td><td>Sigma</td><td>472301</td><td></td></tr><tr><td>Imaris&amp;nbsp;</td><td>Bitplane</td><td></td><td>with colocalization package&amp;nbsp;</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

11.8K 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

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

在原代培养海马神经元的密集核心囊泡的实时成像

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

科研

生物学

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

神经科学

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

荧光寿命成像显微镜观察细胞内圈套的贩运

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

免疫与感染

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

在培养的小鼠神经元中可视化轴突生长锥塌陷和早期淀粉样β效应

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

一种用于监测 ampa 受体贩运的高含量检测方法

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

<meta charset="utf8"></head><body>通过活细胞成像可视化原代海马神经元中的蛋白质

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

<meta charset="utf8"></head><body>作者聚焦：了解病理蛋白对神经退行性疾病轴突转运的影响

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.

成像APP的细胞内运输与光活化GFP

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此视频中的章节

在原代培养海马神经元的密集核心囊泡的实时成像

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

荧光寿命成像显微镜观察细胞内圈套的贩运

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

在培养的小鼠神经元中可视化轴突生长锥塌陷和早期淀粉样β效应

一种用于监测 ampa 受体贩运的高含量检测方法

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

使用活细胞成像测量病理蛋白对原代海马神经元轴突运输的影响

使用活细胞成像测量病理蛋白对原代海马神经元轴突运输的影响

使用活细胞成像测量病理蛋白对原代海马神经元轴突运输的影响