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Biologia

活体哺乳动物细胞器中单膜蛋白的多色定位显微术

Published: June 30, 2018
doi:
10.3791/57690
, , , ,
1School of Biology,University of Osnabrück, 2Center of Cellular Nanoanalytics, Integrated Bioimaging Facility,University of Osnabrück, 3Department of Biology,WWU Münster

Summary

在这里, 我们提出了一个协议的多颜色本地化的单膜蛋白在细胞器的活体细胞。要附加显影, 使用自贴标蛋白。蛋白质, 位于同一细胞器的不同的细胞膜车厢, 可以本地化的精度为 18 nm。

Abstract

了解细胞 subcompartments 中蛋白质的定位是理解其特定功能的关键。在这里, 我们提出了一种超分辨率技术, 允许通过生成这些蛋白质的定位和跟踪图来确定蛋白质的 microcompartments。此外, 通过多色定位显微术, 可同时获得不同 subcompartments 蛋白质的定位和跟踪特征。该技术是特定的活细胞, 是基于重复成像的单一移动膜蛋白。感兴趣的蛋白质与特定的, 所谓的自我标记标签基因融合。这些标记是以共价键方式与基体反应的酶。共轭这些基板是荧光染料。酶标记的蛋白质与荧光标记的基质的反应导致标记的蛋白质。在这里, 四甲基罗丹明 () 和硅罗丹明 (先生) 被用作附着在酶基板上的荧光染料。通过在 pM 到 nM 范围内的基质浓度, 实现了分化学计量标记, 从而产生明显的信号。这些信号是本地化的 ~ 15–27纳米精度。该技术允许单一分子的多色成像, 即颜色的数量受可用的膜渗透染料和自标记酶的汇辑的限制。我们通过确定质量控制酶 (Pten) 诱导激酶 1 (PINK1) 在不同线粒体隔间的加工过程中对其他膜蛋白的定位, 证明了该技术的可行性。然而, 由于低标号度降低了同时标记两个相邻蛋白质的概率, 因此, 对单个分子的不同标记的单个蛋白质之间的真实物理相互作用的测试受到限制。虽然该技术对膜室中的成像蛋白很强, 但在大多数情况下, 不适宜确定高流动性可溶性蛋白的定位。

Introduction

本协议的目标是提供一种成像方法来定位和跟踪活细胞内的单膜蛋白。我们称这种方法跟踪和定位显微镜 (TALM)1,2。像随机光学重建显微镜 (风暴)3和荧光 Photoactivation 定位显微镜 ((F) 棕榈)4,5, TALM 是一个单一的分子为基础的荧光定位技术。然而, 这是不同的方式, 膜蛋白的流动性与重复成像的同一标记分子在不同的位置显示的 microcompartment, 可用于移动蛋白。换句话说, 蛋白质的可能的定位由细胞器的建筑学和由蛋白质1的流动性设置。该方法是互补的其他各种超分辨率技术6,7,8 , 因为它揭示了定位和弹道图的图像移动蛋白。标签是基于使用的基因工程融合蛋白本身是非荧光的。这些融合蛋白是自我标记的酶, 反应共价键与基体共轭染料。该程序具有标记度可由添加基板的数量控制的优点。此外, 它允许改变荧光的颜色, 取决于所选的共轭染料。几个自我标记酵素标记是可利用的9。使用自标记酶标签的另一个好处是, 共轭染料通常比荧光蛋白更稳定和明亮1 , 因此, 单个蛋白质可以记录更长更精确, 直到它们被漂白。这允许记录移动蛋白的轨迹和扩散系数的提取10,11。

在这里, 我们展示了 TALM 线粒体膜蛋白的可行性, 但它也可以应用于其他细胞内和外膜蛋白, 包括不同的细胞类型12,13。我们表明, 多色 TALM 进一步允许在不同 subcompartments 中的蛋白质同时区别于现有超分辨率荧光显微技术14,15, 16. TALM 与活细胞成像17兼容。所选 rhodamines 四甲基罗丹明和硅 Rhodamien (SiR) 的光物理, 特别是它们的亮度和稳定性, 允许在提供定位 (和轨迹) 图的多个帧上记录单个膜蛋白。然而, 由于运动模糊度太高, 且每帧采集的光子过低, 无法进行适当的定位, TALM 对高扩散系数可溶性蛋白的定位有限。此外, TALM 需要较少的励磁功率比例如风暴或被刺激的尾气损耗 (STED) 显微镜6,7, 减少光毒性作用。这是重要的, 因为光毒性重音经常影响 organellar 形态学18和因而机动性分析19。总之, 我们提出多色 TALM 在活细胞作为一种技术, 填补了本地化显微镜方法风暴/STED/(F) 棕榈和技术, 分析蛋白质的流动性, 如荧光恢复后漂白 (酶)20 ,21, 荧光相关光谱学 (FCS)22, 和荧光交叉相关光谱学 (FCCS)11,23。

Protocol

以下议定书遵循地方机构研究道德委员会的准则。 1. 方法 细胞培养 培养细胞, 例如 HeLa 细胞 (人宫颈癌), 在一个 T25 细胞培养瓶包含5毫升生长培养基在37°c 和 5% CO2。注: 用于成像, 将单元格拆分到准备好的盖玻片 (参见步骤1.3 和 1.4) 并保持成像介质。 细胞转染注: 使用细胞线稳定地表达标记的蛋白质, ?…

Representative Results

多色成像和定位分析可以帮助确定蛋白质的亚 organellar 定位。我们早先证明了这一点与胞浆磷酸酶和张力同系物, PINK1, 有不同的亚线粒体位置由于其处理的线粒体蛋白酶17。PINK1 是保证线粒体功能34,35的重要因素。为确定 PINK1 在不同线粒体隔间的定位过程 (图 4A), 进行了多色超分辨显微镜?…

Discussion

本文提出了一种移动膜蛋白双色单分子定位技术。根据该协议, 膜蛋白被融合到自我标记的蛋白质, 与罗丹明染料的反应, 和先生共轭他们各自的基质。罗丹明染料是明亮和 photostable, 从而允许重复成像1。为了取得成功, 必须牢记几个条件和关键主题。

首先, 选择适当的过滤器和分配器, 以便将其信号与船长分开是很重要的。为了减少细胞外的背景, 盖玻片的 pr…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者要感谢 Osnabrück 大学生物物理小组和雅各 Piehler 不断的支持, Wladislaw 科尔的技术援助和材料的制备, 以及 CellNanOs 板提供显微镜供使用。该项目由 SFB 944 资助。

Materials

(2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid, 1 M) (HEPES) Biochrom #1104E
DC1: Dichroid beam splitter  Chroma 640 dcxr NC506031
DC2: Polychroic Mirror, beamsplitter Chroma zt405/488/561/640rpc discontinued
Dulbecco´s Phosphate-Buffered Saline (PBS) 1x (w/o Ca & Mg) Sigma-Aldrich & Co. #RNBF8311
Earle´s MEM without phenol red, without L-Glutamine and without NaHCO3 containing 1% FBS, 0.1% HEPES, 0.1% NEAA, 0.1% Alanyl-L-Glutamine and 34.78% sodium hydrogen carbonate (NaHCO3 0.75g/l) Imaging medium
Earle´s minimum essential medium (MEM) with phenol red, containing 1% Fetal Bovine Serum Superior (FBS), 0.1% HEPES (2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid, 1 M), and 0.1% non-essential amino acids (NEAA) Growth medium
EF: Emission filter quadbandpass AHF analysentechnik F72-866 Brightline HC 446 nm/523 nm/600 nm/677 nm
EMCCD camera Andor Andor iXON 897 EMCCD camera
Emission filter QuadView filter cubes, orange AHF analysentechnik F39-637 bandpass 582 – 619 nm
Emission filter QuadView filter cubes, red Chroma bandpass 655 – 725 nm (HQ 690/70)
FBS (Fetal bovine serum) superior Biochrom S0615
Fluorescent beads: TetraSpeck™ Microspheres, 0.1 µm, fluorescent blue/green/orange/dark red Thermo Fisher Scientific T7279 fluorescent microspheres
Glutamine Biochrom #0951C
HeLa cells  DSMZ ACC-57 Cervical carcinoma cells from patient Henrietta Lacks
Hela cells CI::paGFP, stable Muster et al., PLOSOne 2010
Hela cells CV g::Halo7-Tag, stable Appelhans et al., NanoLett 2012
Hela cells Tom20::Halo7-Tag, stable Appelhans et al., NanoLett 2012
Image splitter Photometrics Dual-View QV2 image splitter emission
Imaging processing software  ImageJ2 / Fiji freeware
Immersion Oil – ImmersolTM 518 F (ne = 1.518, ve = 45) Carl Zeiss Jena GmbH 444960-0000-000
Inverted epifluorescence microscope Olympus IX-71/73/83
Laser 561 nm, 200 mW CrystaLaser CL-561-200 561 nm emission
Laser 642 nm, 140 mW Omicron Luxx-642-140 642 nm emission
MATLAB MathWorks version R2013a
MEM with Earle's Balanced Salt Solution 2.2 g/L NaHCO3, stable glutamine w/o PR Biochrom FG-0385
MEM with Earle's Balanced Salt Solution with 2.2 g/L NaHCO3, stable glutamine, Phenolred Biochrom FG-0325
MitoTracker® Deep Red FM Thermo Fisher Scientific M22426 dye
MitoTracker® Green FM Thermo Fisher Scientific M7514 dye
Multi-mode-optical polarization maintaining monomode fiber Pointsource/Qioptiq KineFLEX
NHS-PEG-MAL, Rapp Polymer  Rapp Polymere GmbH Tübingen coverslip coating
non-essential amino acids (NEAA) Biochrom #0802E
PEG 800 (Polyethylene glycol) 10 % Carl Roth GmbH Art No. 0263.1 coverslip coating
Penicillin/Streptomycin Biochrom #0122E
Plasmid for PINK1-Halo7-Tag expression Beinlich et al., ACS Chemical Biology 2015
Poly-L-lysine (1.2 mg/ml) Sigma-Aldrich & Co. Cat. No.P9155 coverslip coating
RGD Peptide (Ac-CGRGDS-COOH) Coring System Diagnostix GmbH, Gernsheim coverslip coating / Intergrin receptor motif
Silicon Rhodamine linked to HaloTag®-Ligand (SiRHTL)  personal gift from Kai Johnson dye
Software analysis plugin self-written C. P. Richter, Biophysik Osnabrück SLIMFAST 16g
Tetramethylrhodamine / SNAP-Cell® TMR-Star linked to SNAP-Ligand (TMRstar)  New England Biolab® S9105S dye
Tetramethylrhodamine linked to HaloTag®-Ligand (TMRHTL)  Promega G8251 dye
TIRF condensor Olympus Cell^TIRF MITICO System TIRF condensor
TIRF microscope controlling software Olympus cellSens 1.12
TIRF objective Olympus 150x oil objective (N.A. 1.45; Olympus UAPO)
Trypsin/EDTA 10x Biochrom #0266
Water H2O 99,5 % Rotipuran® Low organic  Carl Roth GmbH Art. No. HN57.1
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Tags

Keywords: Multi-color Localization MicroscopySingle Membrane ProteinsOrganellesLive Mammalian CellsProtein DynamicsSpatiotemporal OrganizationMitochondrial Membrane ProteinsTransfectionSample PreparationMicroscope SetupFluorescent BeadsObjective LensImmersion OilTransmission LightLaserFluorescence ChannelsImage SplitterTIRF MicroscopyTransformation MatrixDual-color ImagesCalibrationUnit ConversionLocalization ManagerPoint Spread Function (PSF)
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Appelhans, T., Beinlich, F. R., Richter, C. P., Kurre, R., Busch, K. B. Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells. J. Vis. Exp. (136), e57690, doi:10.3791/57690 (2018).
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