两种剥离方法，用于分离小鼠视网膜中的光感受器细胞区室以进行蛋白质分析
Summary
该协议提出了两种技术来分离小鼠杆光感受器的亚细胞区室以进行蛋白质分析。第一种方法利用活视网膜和纤维素滤纸来分离杆外段，而第二种方法采用冻干视网膜和胶带剥离杆内段和外段层。
Abstract
视杆光感受器是具有不同区室的高度极化感觉神经元。小鼠杆长（~80μm）和薄（~2μm），横向堆积在视网膜的最外层，即光感受器层，导致类似的亚细胞区室对齐。传统上，冷冻的平板视网膜的切向切片已被用于研究不同杆室内蛋白质的运动和定位。然而，杆状小鼠视网膜的高曲率使得切向切片具有挑战性。在对区室间蛋白质转运研究的推动下，我们开发了两种剥离方法，可以可靠地分离出杆外段（ROS）和其他亚细胞区室用于蛋白质印迹。我们相对快速和简单的技术提供富集和亚细胞特异性级分，以定量测量正常视杆中重要光感受器蛋白的分布和再分布。此外，这些分离技术也可以很容易地用于分离和定量研究健康和退化视网膜中其他细胞层的蛋白质组成。
Introduction
棒状光感受器细胞紧密地聚集在神经视网膜的最外层，是昏暗光视觉的一个组成部分。为了充当忠实的光子计数器，杆利用基于G蛋白的信号通路（称为光转导）来产生对单光子捕获的快速，放大和可重复的响应。这种对光的响应最终会触发质膜电流的变化，并随后向视觉系统的其余部分发出信号1。顾名思义，每个杆细胞都具有独特的杆状形状，并表现出高度极化的细胞形态，由外段（OS），内段（IS），细胞体（CB）和突触末端（ST）组成。每个亚细胞区室具有特定的蛋白质机制（膜结合和可溶性）、生物分子特征和蛋白质复合物，这些蛋白质复合物起着至关重要的作用，如视觉光转导、一般内务管理和蛋白质合成以及突触传递2，3。
30多年前，首次观察到亚细胞蛋白的光依赖性相互运动，特别是转导素（远离OS）和停滞素（朝向OS），4，5，6，7。早期，这种观察到的现象受到怀疑，部分原因是免疫组织化学对表位掩蔽的脆弱性8。在2000年代初，通过使用严格而艰苦的物理切片技术证实了刺激依赖性蛋白质易位9。对冷冻的平装啮齿动物视网膜进行连续切向切片，然后进行免疫印迹，结果显示，转导素9，10，arrestin11，12和recoverin13都响应于光的亚细胞再分布。据信，这些关键信号蛋白的光驱动易位不仅调节光转导级联的灵敏度9，14，15，而且还可能对光损伤具有神经保护作用16，17，18。由于视杆细胞中的光驱动蛋白转运似乎对视杆细胞生物学和生理学非常重要，因此允许分离不同亚细胞区室以确定蛋白质分布的技术是有价值的研究工具。
目前，有几种旨在分离杆亚细胞区室的方法。然而，这些方法可能很长且难以复制，或者需要相当数量的视网膜分离物。例如，通过密度梯度离心19制备棒外段（ROS）通常用于将ROS与视网膜匀浆分离。这种方法广泛用于蛋白质印迹，但该过程非常耗时，并且至少需要8-12个小鼠视网膜20。另一方面，冷冻小鼠和大鼠视网膜的连续切向切片已成功用于分离OS，IS，CB和ST9，11，13。然而，这种方法在技术上具有挑战性，因为在切向切片之前，必须完全压平小而高度弯曲的小鼠视网膜以对齐视网膜层。由于有大量的小鼠模型和转基因小鼠概括视觉系统的疾病，因此创建一种可靠，快速且容易地分离单个杆室的技术有望揭示每个专用隔室中发生的生理过程以及健康和疾病中视觉过程背后的机制。
为了促进这些研究，我们描述了两种剥离方法，它们比目前的方案更容易分离杆亚细胞区室。第一种剥离方法改编自一种暴露荧光标记的双极细胞以进行膜片钳记录的技术21，它采用纤维素滤纸从活的，分离的小鼠视网膜中依次去除ROS（图1）。第二种方法改编自从chick22和frog23视网膜中分离出三个主要视网膜细胞层的程序，利用胶带从冻干视网膜中去除ROS和杆内段（RIS）（图2）。这两个程序都可以在1小时内完成，并且非常人性化。我们通过利用来自C57BL / 6J小鼠的黑暗适应和光暴露的视网膜来证明光诱导的杆转导素（GNAT1）和逮捕素（ARR1）的易位，从而验证了这两种分离方案对蛋白质印迹的有效性。此外，使用胶带剥离方法，我们提供了额外的证据，证明我们的技术可用于检查和解决通过免疫细胞化学（ICC）和蛋白质印迹获得的蛋白质定位数据之间的不一致。具体而言，我们的技术表明：1）蛋白激酶C-α（PKCα）亚型不仅存在于双极细胞中，还存在于小鼠ROS和RIS中，尽管浓度低24，25和2）视紫红质激酶（GRK1）主要存在于分离的OS样品中。这些数据证明了我们的两种剥离技术在分离和定量特定视网膜蛋白和视网膜蛋白方面的有效性。
Protocol
所有实验均根据南加州大学（USC）研究动物护理委员会的当地机构指南进行。 1. 活细胞视网膜剥离法 准备艾姆斯的缓冲液、去皮纸和解剖皿 使用钝尖虹膜剪刀（或等效的剪刀型），将纤维素滤纸（413级）切割成长方形，尺寸约为5 mm x 2.5 mm。储存插条以备将来使用。 通过混合一瓶Ames’培养基，2.38克HEPES和0.877克NaCl来制备1升Ames的HEPES缓冲液。…
Representative Results
本策略的开发是为了提供相对快速和简单的方法来分离和分析特定杆亚细胞区室中的蛋白质，以进行蛋白质印迹分析。我们应用了两种顺序剥离技术（图1 和 图2），然后进行免疫印迹，以证明这些方法可以可靠地用于检测黑暗和光适应动物中杆转导素（GNAT1）和停滞素（ARR1）的已知分布。为了验证我们的两种方案在精确分离杆亚细胞区室而不污染其他…
Discussion
许多视网膜疾病会影响视杆细胞，导致视杆细胞死亡，并最终导致完全视力丧失37。多年来，人类视网膜变性的遗传和机制起源的很大一部分已经在许多小鼠模型中成功概括。在这种情况下，能够轻松和选择性地将单个杆亚细胞区室与小小鼠视网膜分开，这将大大增强我们对视网膜疾病的局部生化和分子基础的理解。此外，神经视网膜的分层性质，结合杆光感受器独特的高度极?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH Grant EY12155，EY027193和EY027387对JC的支持。我们感谢Spyridon Michalakis博士（美国帕萨迪纳加州理工学院）和Natalie Chen博士（美国洛杉矶南加州大学）校对手稿。我们还要感谢Seth Ruffins博士（南加州大学，洛杉矶，美国）和Janos Peti-Peterdi博士（南加州大学，洛杉矶，美国）提供必要的设备来收集作者提供的镜头。材料来自：Kasey Rose等人，分离小鼠视网膜中的感光细胞区室进行蛋白质分析，分子神经变性，发表[2017]，[施普林格自然]。
Materials
| 100 mL laboratory or media bottle equipped with a tubing cap adapter | N/A | N/A | |
| 100% O2 tank | N/A | N/A | |
| 1000mL Bottle Top Filter, PES Filter Material, 0.22 μm | Genesee Scientific | 25-235 | |
| 4X SDS Sample Buffer | Millipore Sigma | 70607-3 | |
| 50 mL Falcon tube | Fisher Scientific | 14-432-22 | |
| 95% O2 and 5% CO2 tank | N/A | N/A | |
| Ames’ Medium with L-glutamine, without bicarbonate | Sigma-Aldrich | A1420 | |
| CaCl2 (99%, dihydrate) | Sigma | C-3881 | |
| Drierite (Anhydrous calcium sulfate, >98% CaSO4, >2% CoCl2) | WA Hammond Drierite Co LTD | 21005 | |
| Falcon Easy-Grip Petri Dish (polystyrene, 35 x 10 mm) | Falcon-Corning | 08-757-100A | |
| Falcon Easy-Grip Tissue Culture Dish (60 x 15 mm) | Falcon-Corning | 08-772F | |
| Feather Scalpel (No. 10, 40 mm) | VWR | 100499-578 | |
| Feather Scalpel (No. 11, 40 mm) | VWT | 100499-580 | |
| KCl (99%) | Sigma | P-4504 | |
| Kimble Kontes pellet pestle | Sigma | z359971 | |
| Labconco Fast-Freeze Flasks | Labconco | N/A | |
| LN2 (liquid nitrogen) + Dewar flask or similar vacuum flask | N/A | N/A | |
| MgCl2 | Sigma | M-9272 | |
| Milli-Q/de-ionized water | EMD Millipore | N/A | |
| Na2HPO4 (powder) | J.T. Baker | 4062-01 | |
| NaCl (crystal) | EMD Millipore | Sx0420-3 | |
| NaHCO3 | Amresco | 0865 | |
| OmniPur EDTA | EMD | 4005 | |
| OmniPur HEPES, Free Acid | EMD | 5320 | |
| Parafilm M | Sigma-Aldrich | P7793 | |
| Reynolds Wrap Aluminum Foil | Reynolds Brands | N/A | |
| Scotch Magic Tape (12.7 mm x 32.9 m) | Scotch-3M | N/A | |
| Sodium deoxycholate | Sigma-Aldrich | D67501 | |
| Spectrafuge mini centrifuge | Labnet International, Inc | C1301 | |
| Tissue incubation chamber (purchased or custom made) | N/A | N/A | |
| Tris-HCl | J.T.Baker | 4103-02 | |
| Triton X-100 | Signma-Aldrich | T8787 | |
| VirTis Benchtop 2K Lyophilizer or equivalent machine | SP Scientific | N/A | |
| VWR Grade 413 Filer Paper (diameter 5.5 cm, pore size 5 μm) | VWR | 28310-015 | |
| Whatman Grade 1 Qualitative Filter Paper (diameter 9 cm, pore size 11 μm) | Whatman/GE Healthcare | 1001-090 | |
| Wide bore transfer pipet, Global Scientific | VWR | 76285-362 |
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Cite This Article
Rose, K., Lokappa, S., Chen, J. Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis. J. Vis. Exp. (178), e62977, doi:10.3791/62977 (2021).