成像APP的细胞内运输与光活化GFP
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 35-mm glass bottom culture dishes | Matek | P-35G-1.5-20-C | |
| Dulbecco's Phosphate Buffered Saline | Life Technologies | 14190-144 | |
| Hanks Balanced Salt Solution | Life Technologies | 14025-092 | |
| Lipofectamine 2000 | Life Technologies | 11668019 | |
| Dulbecco's Modified Eagle Medium | Life Technologies | 11995-092 | |
| Penicilin/Streptomycin | Life Technologies | 15140-122 | |
| dibutyrl cyclic AMP | Sigma | D0627 | |
| Heat in activated Fetal Bovine Serum | Life Technologies | 10082147 | |
| Heated Microscopy stage insert P | PeCon GmbH | ||
| Tempcontrol 37–2 digital 2-channel | PeCon GmbH | ||
| Zeiss LSM-510 META laser- scanning microscope | Carl Zeiss | with laser diode, argon laser, and HeNe1 laser | |
| Zeiss 63× 1.4 numerical aperture oil immersion lens | Carl Zeiss | ||
| L685, 458 | EMD Millipore | 565771 | dissolved in DMSO |
| cholorquine | Sigma | C6628 | dissolved in water |
| Nocodazole | Sigma | M1404 | dissolved in DMSO |
| Dimethyl sulphoxide | Sigma | 472301 | |
| Imaris | Bitplane | with colocalization package |
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