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Neurociência

具有邻近标记蛋白质组学的神经元溶酶体相互作用组表征

Published: June 23, 2022
doi:
10.3791/64132
, ,
1Department of Chemistry,The George Washington University

Summary

这里描述了神经元溶酶体邻近标记蛋白质组学协议，以表征人类诱导的多能干细胞衍生神经元中的动态溶酶体微环境。溶酶体膜蛋白和与溶酶体相互作用（稳定或瞬时）的蛋白质可以在该方法中准确定量，在活体人类神经元中具有出色的细胞内空间分辨率。

Abstract

溶酶体经常与各种生物分子交流，以实现降解和其他不同的细胞功能。溶酶体对人类大脑功能至关重要，因为神经元是有丝分裂后，严重依赖自噬-溶酶体途径来维持细胞稳态。尽管在理解各种溶酶体功能方面取得了进展，但捕获溶酶体和其他细胞成分之间的高度动态通讯在技术上具有挑战性，特别是在高通量方式中。在这里，为最近发表的人诱导多能干细胞（hiPSC）衍生神经元中的内源性（敲入）溶酶体邻近标记蛋白质组学方法提供了详细的协议。

溶酶体膜蛋白和10-20nm半径内溶酶体周围的蛋白质都可以在活的人类神经元中可靠地鉴定和准确定量。详细描述了协议的每个步骤，即hiPSC神经元培养，邻近标记，神经元收获，荧光显微镜，生物素化蛋白质富集，蛋白质消化，LC-MS分析和数据分析。总之，这种独特的内源性溶酶体邻近标记蛋白质组学方法为研究活体神经元中的高动态溶酶体活性提供了一种高通量和强大的分析工具。

Introduction

溶酶体是分解代谢细胞器，通过溶酶体自噬途径1降解大分子。除了降解，溶酶体还参与多种细胞功能，如信号转导、营养传感和分泌2，3，4。溶酶体功能的扰动与溶酶体贮积症、癌症、衰老和神经变性有关3，5，6，7。对于有丝分裂后和高度极化的神经元，溶酶体在神经元细胞稳态、神经递质释放和沿轴突的长距离运输中起着关键作用8，9，10，11。然而，研究人类神经元中的溶酶体一直是一项具有挑战性的任务。诱导多能干细胞（iPSC）衍生神经元技术的最新进展使得能够培养以前无法获得的活体人类神经元，弥合了动物模型和人类患者研究人脑之间的差距12，13。特别是先进的i3神经元技术在多西环素诱导的启动子作用下，将神经原蛋白-2转录因子稳定地整合到iPSC基因组中，驱动iPSC在2周内分化为纯皮质神经元14，15。

由于高动态溶酶体活性，捕获溶酶体与其他细胞成分的相互作用在技术上具有挑战性，特别是以高通量方式。邻近标记技术非常适合研究这些动态相互作用，因为它能够以出色的空间特异性捕获稳定和瞬时/弱蛋白质相互作用16，17。工程化过氧化物酶或生物素连接酶可以在遗传上融合到诱饵蛋白上。激活后，产生高反应性生物素自由基以共价标记相邻蛋白质，然后可以通过液相色谱-质谱（LC-MS）平台富集链霉亲和素包被的珠子进行下游自下而上的蛋白质组学17，18，19，20，21。

最近开发了一种内源性溶酶体邻近标记蛋白质组学方法，以捕获i3神经元22中的动态溶酶体微环境。工程抗坏血酸过氧化物酶（APEX2）在iPSCs溶酶体相关膜蛋白1（LAMP1）的C末端被敲入，然后可以分化为皮质神经元。LAMP1是一种丰富的溶酶体膜蛋白和经典溶酶体标志物23。LAMP1也在晚期内体中表达，内体成熟为溶酶体;这些晚期内体溶酶体和非降解溶酶体在本方案中都被称为溶酶体。这种内源性LAMP1-APEX探针在生理水平上表达，可以减少LAMP1错误定位和过表达伪影。可以在活的人类神经元中以出色的空间分辨率鉴定和定量数百种溶酶体膜蛋白和溶酶体相互作用体。

在这里，描述了人类iPSC衍生神经元中溶酶体邻近标记蛋白质组学的详细方案，并进一步改进了最近发表的方法22。整个工作流程如图 1 所示。该方案包括hiPSC衍生的神经元培养、神经元中的邻近标记激活、通过荧光显微镜验证APEX活性、确定最佳链霉亲和素珠与输入蛋白的比例、生物素化蛋白的富集、磁珠蛋白消化、肽脱盐和定量、LC-MS分析和蛋白质组学数据分析。还讨论了故障排除指南和实验优化，以改进邻近标记质量控制和性能。

Protocol

所有程序均由乔治华盛顿大学生物安全和伦理委员会批准。 表1提供了该方案中使用的培养基和缓冲液的组成。此处使用的商业产品信息在 材料表中提供。 1. 人iPSC衍生神经元培养 人 iPSC 培养和 LAMP1-APEX 探针整合（7 天）将基质胶储备溶液在4°C的冰桶中解冻过夜，将500μL溶液等分到冷无菌管中，并将等分试样储存在-80°C。 ?…

Representative Results

这项溶酶体邻近标记蛋白质组学研究是在人iPSC衍生的神经元中进行的，以在活神经元中 原位 捕获动态溶酶体微环境。hiPSCs和hiPSC衍生神经元在不同时间点的细胞形态如图 2A所示。人 iPSC 在 E8 培养基中的菌落中生长。通过将 iPSC 接种到含多西环素的神经元诱导培养基中来启动分化。在 3 天分化期间，神经突延伸每天变得更加明显。在神经元培养基中切换到PLO涂层板后?…

Discussion

使用该LAMP1-APEX探针，溶酶体膜上和附近的蛋白质被生物素化和富集。鉴于典型的溶酶体直径为100-1，200nm，该方法可提供出色的细胞内分辨率，标记半径为10-20nm。LAMP1是一种丰富的溶酶体膜蛋白，也是溶酶体的经典标志物，可作为内源表达水平溶酶体APEX标记的优良诱饵蛋白。然而，当使用LAMP1靶向溶酶体时也存在局限性，因为LAMP1也存在于晚期内体和非降解溶酶体中35。大多数溶?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项研究得到了NIH资助（R01NS121608）的支持。A.M.F. 承认 ARCS-Metro 华盛顿分会奖学金和波旁 F. 斯克里布纳捐赠奖学金。我们感谢美国国家神经疾病和中风研究所（NINDS）的Michael Ward实验室提供的分子生物学支持和i3神经元技术开发。

Materials

10% (w/v) Saponin solution Acros Organics 419231000 Flourescent Microscopy
Accutase Life Technologies A1110501 cell detachment solution, Cell Culture
B27 Supplement Fisher Scientific 17504044 Cell Culture, Cortical Neuron Medium
BDNF PeproTech 450-02 Cell Culture, Cortical Neuron Medium
Boric acid Sigma-Aldrich B6768 Cell Culture, Borate Buffer
Bovine Serum Albumin Millipore Sigma A8806 To make standard solutions to measure total protein concentrations
Brainphys neuronal medium STEMCELL Technologies 5790 Cell Culture, Cortical Neuron Medium
CD45R (B220) Antibody Alexa Fluor 561 Thermo Fisher Scientific 505-0452-82 Flourescent Microscopy
Chroman1 ROCK inhibitor Tocris 716310 Cell Culture
cOmplete mini Protease Inhibitor Roche 4693123001 cocktail inhibitor in Lysis Buffer
DC Protein Assay Kit II Bio-Rad 5000112 To determine total protein concentrations of cell lysate
Dimethyl sulfoxide (DMSO) Sigma-Aldrich D8418 Proximity-labeling Reaction
DMEM/F12 medium Thermo Fisher Scientific 11320082 Cell Culture, Dish Coating
DMEM/F12 medium with HEPES Thermo Fisher Scientific 11330057 Cell Culture, Induction Medium
Donkey serum Sigma-Aldrich D9663 Flourescent Microscopy
Doxycycline hyclate, ≥98% (HPLC) Sigma-Aldrich D9891-1G Cell Culture, Induction Medium
Essential 8 Medium Thermo Fisher Scientific A1517001 Cell Culture
Essential 8 Supplement (50x) Thermo Fisher Scientific A1517101 Cell Culture
Extraction plate vacuum manifold kit Waters WAT097944 For Peptide desalting
Formic Acid (FA) Fisher Scientific A11750 For LC-MS analysis
GDNF PeproTech 450-10 Cell Culture, Cortical Neuron Medium
Hoechst dye Thermo Fisher Scientific 62239 Flourescent Microscopy
HPLC grade methanol Fisher Scientific A452 For Peptide desalting
HPLC grade water Fisher Scientific W5 For Peptide desalting
Human induced pluripotent stem cells Corriell Institute GM25256 Cell Culture
Hydrogen peroxide, ACS, 29-32% w/w aq. soln., stab. Thermo Fisher Scientific AA33323AD Proximity-labeling Reaction
Iodoacetamide (IAA) Millipore Sigma I6125 For Protein Digestion
Laminin Fisher Scientific 23017015 Cell Culture, Cortical Neuron Medium
LC-MS grade Acetonitrile Fisher Scientific A955 For LC-MS analysis
LC-MS grade water Fisher Scientific W64 For LC-MS analysis
L-glutamine Fisher Scientific 25-030-081 Cell Culture, Induction Medium
Matrigel Thermo Fisher Scientific 08-774-552 basement membrane matrix, Cell Culture, Dish Coating
Mouse anti-human LAMP1 monoclonal antibody Developmental Studies Hybridoma Bank h4a3 Flourescent Microscopy
N-2 Supplement (100x) Fisher Scientific 17-502-048 Cell Culture, Induction Medium
Nitrocellulose Membrane, Precut, 0.45 µm, 7 x 8.5 cm Bio-Rad 1620145 To conduct dot blot assay for bead titration
Non-essential amino acids (NEAA) Fisher Scientific 11-140-050 Cell Culture, Induction Medium
NT-3 PeproTech 450-03 Cell Culture, Cortical Neuron Medium
Oasis HLB 96-well solid phase extraction plate Waters 186000309 For Peptide desalting
Odyssey Blocking Buffer (TBS) LI-COR Biosciences 927-50000 To conduct dot blot assay for bead titration
Paraformaldehyde Electron Microscopy Sciences 15710 Flourescent Microscopy
Phenol Biotin (1,000x stock) Adipogen 41994-02-9 Proximity-labeling Reaction
Phosphate-buffered saline (PBS) without calcium or magnesium Gibco 10010049 Cell Culture, Proximity-labeling Reaction, Flourescent Microscopy
Pierce Quantitative Colorimetric Peptide Assay Thermo Fisher 23275 Peptide Concentration Assay
Poly-L-Ornithine (PLO) Millipore Sigma P3655 Cell Culture, Dish Coating
Sodium Ascorbate Sigma-Aldrich A4034 Proximity-Labeling Quench Buffer, Lysis Buffer
Sodium azide Sigma-Aldrich S8032 Proximity-Labeling Quench Buffer, Lysis Buffer, Flourescent Microscopy
Sodium chloride Thermo Fisher Scientific S271500 Cell Culture, Borate Buffer
Sodium dodecyl sulfate (SDS) Thermo Fisher Scientific BP1311220 Lysis Buffer, Dot blot assay buffer, Beads wash buffer
Sodium hydroxide Sigma-Aldrich 415413 Cell Culture, Borate Buffer
Sodium tetraborate Sigma-Aldrich 221732 Cell Culture, Borate Buffer
SpeedVac concentrator vacuum concentrator
Streptavidin Magnetic Sepharose Beads Cytiva (formal GE) 28-9857-99 Enrich biotinylated proteins
Streptavidin, Alexa Fluor 680 Conjugate Thermo Fisher Scientific S32358 To conduct dot blot assay for bead titration
Thermomixer temperature-controlled mixer
Trifluoacetic acid (TFA) Millipore Sigma 302031 For Peptide desalting
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) Millipore Sigma C4706 For Protein Digestion
Tris-HCl Thermo Fisher Scientific BP152500 Lysis Buffer, Dot blot assay buffer, Beads wash buffer
Triton-X Thermo Fisher Scientific BP151500 Beads wash buffer
TROLOX Sigma-Aldrich 648471 Proximity-Labeling Quench Buffer, Lysis Buffer
Trypsin/Lys-C Mix, Mass Spec Grade Promega V5073 For Protein Digestion
TWEEN 20 Millipore Sigma P1379 Dot blot assay buffer
Urea Thermo Fisher Scientific BP169500 Beads wash and On-Beads Digestion Buffer
Vitronectin STEMCELL Technologies 7180 Cell Culture, Dish Coating
Y-27632 ROCK inhibitor Selleck S1049 Cell Culture
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Tags

Proximity Labeling ProteomicsLysosomal MicroenvironmentIPSC-derived NeuronsLysosomal DysfunctionBrain DiseasesBiotin PhenolHydrogen PeroxideAcetone PrecipitationProtein PelletDCA Assay
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Frankenfield, A., Ni, J., Hao, L. Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics. J. Vis. Exp. (184), e64132, doi:10.3791/64132 (2022).
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