监测存根1介导的吞噬
Summary
该协议提供了触发和监测活细胞中Stub1介导的吞噬的说明。
Abstract
哺乳动物细胞可以通过Stub1介导的噬菌体翻转过氧化物酶体。该途径可能允许细胞控制过氧化物酶体的数量和质量。在此过程中，热休克蛋白70和泛素E3连接酶Stub1易位到过氧化物酶体上以翻转以启动pexophation。Stub1连接酶活性允许泛素和其他自噬相关模块在靶向过氧化物酶体上的积累。提高过氧化物酶体腔内的活性氧 （ROS） 水平可以激活 Stub1 介导的噬菌。因此，可以使用染料辅助ROS生成来触发和监测该途径。本文概述了使用两类染料（荧光蛋白和合成荧光团）在哺乳动物细胞培养物中启动噬寡糖的程序。这些基于染料辅助ROS生成的方案不仅可以用于靶向全球细胞群中的所有过氧化物酶体，还可以允许操纵单个细胞内的单个过氧化物酶体。我们还描述了如何使用活细胞显微镜跟踪Stub1介导的噬寡噬。
Introduction
过氧化物酶体是存在于大多数真核细胞中的单膜结合细胞器。过氧化物酶体是进行超长链脂肪酸的β-氧化、嘌呤分解代谢以及醚磷脂和胆汁酸合成所必需的代谢区室1。过氧化物酶体衍生的乙酰辅酶A通过调节代谢中的中枢信号传导来控制脂质稳态2。因此，过氧化物酶体功能受损隐含在各种疾病中也就不足为奇了，包括神经退行性疾病、衰老、癌症、肥胖和糖尿病3，4，5。维持过氧化物酶体操作的一个基本过程是噬菌。噬菌是一种分解代谢过程，用于通过自噬选择性周转过氧化物酶体。细胞使用pexophagy来帮助控制过氧化物酶体的数量和质量，从而确保适当的过氧化物酶体功能。最近的一项研究表明，由PEX1过氧化物酶体生物发生因子1和6突变引起的过氧化物酶体丢失是由不受控制的pexophagy6引起的。值得注意的是，65% 的过氧化物酶体生物发生障碍 （PBD） 患者存在过氧化物酶体 AAA ATP 酶复合物缺陷，该复合物由哺乳动物细胞中的 PEX1、PEX6 和 PEX26 组成7。
许多方法可用于启动和研究噬菌。在酵母中，当提供的营养物质从过氧化物酶体依赖性碳源切换到过氧化物酶体依赖性碳源（以降低细胞过氧化物酶体数量）时，就会触发噬菌8。例如，甲醇生长的毕赤酵母细胞从甲醇培养基转移到葡萄糖培养基和乙醇培养基分别诱导微噬和巨噬8，9，10。微食螯合通过重塑液泡形成杯状液泡封存膜和称为微食特异性膜装置（MIPA）的盖状结构来聚集过氧化物酶体以进行降解。在巨噬中，单个过氧化物酶体被称为噬小体的双膜结构吞噬，然后与液泡融合以进行降解8，9，10。噬菌受体的磷酸化，例如酿酒酵母中的Atg36p和毕赤酵母中的Atg30p，对于受体募集核心自噬机制和促进过氧化物酶体靶向自噬体8，11至关重要。
在哺乳动物细胞中，泛素化可诱导食脂。在胞质侧用泛素标记过氧化物酶体膜蛋白PMP34或PEX3诱导噬寡糖12。PEX3的过表达诱导过氧化物酶体泛素化和溶酶体消除过氧化物酶体13。此外，PEX5与C末端EGFP的融合会损害单泛素化PEX5的输出并导致噬菌14。另一方面，食肉也可以由H2O2 治疗触发。过氧化物酶体产生活性氧（ROS）;具体来说，催化超长链脂肪酸（>22碳）β-氧化初始步骤的过氧化物酶体酶Acox1不仅产生乙酰辅酶A，还产生过氧化物酶体ROS。为了响应H2O2 处理下ROS水平升高，哺乳动物细胞激活噬菌以降低ROS产生并缓解压力。据报道，H2O2 治疗驱动共济失调-毛细血管扩张突变 （ATM） 募集到过氧化物酶体。然后ATM磷酸化PEX5，通过噬菌15促进过氧化物酶体周转。
由于过氧化物酶体是产生ROS的中心，因此它们也容易受到ROS损伤。ROS引发的过氧化物酶体损伤迫使细胞激活食磷脂，以启动过氧化物酶体质量控制途径（通过自噬去除受损的过氧化物酶体）。在这里，我们概述了一种按需触发ROS诱发的过氧化物酶体损伤的方法。该协议利用细胞器16，17，18，19，20内的光激活ROS产生（图1）。染料标记的过氧化物酶体被照亮，导致过氧化物酶体腔内产生ROS，从而特异性触发过氧化物酶体损伤。使用该协议，显示ROS应激过氧化物酶体通过泛素依赖性降解途径去除。ROS应激的过氧化物酶体招募泛素E3连接酶Stub1，以允许它们吞噬到自噬体中，以便通过噬菌16进行个体去除。可以使用该协议通过延时显微镜比较同一细胞内受伤和健康过氧化物酶体的命运。该方法还可用于全局破坏培养皿上的所有过氧化物酶体（在所有细胞中），从而允许对噬菌途径进行生化分析。
Protocol
Representative Results
Discussion
该协议详细介绍了如何通过用光提高过氧化物酶体ROS水平来触发细胞培养物中Stub1介导的噬菌。由于该方案依赖于染料辅助ROS生成，因此需要确保在目标细胞内充分表达diKillerRed-VKSKL或染料标记的SLP配体染色。鉴于不同的细胞类型或不同遗传背景的细胞可能含有性质略有不同的过氧化物酶体，可能需要调整确切的照明条件以确保诱导pexophagy。筛查Stub1介导的噬细胞活化的一种可靠措施是监测EGFP-Ub是…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了台湾国家科学技术委员会的MOST 111-2311-B-001-019-MY3研究资助。
Materials
| 35 mm culture dish with a 20 mm diameter glass microwell | MatTek | P35G-1.5-20-C | 20 mm glass bottomed |
| 3-amino-1,2,4-triazole (3-AT) | Sigma Aldrich | A8056 | |
| bovine serum | ThermoFisher Scientific | 16170060 | |
| Cell culture incubator | Nuaire | NU-4750 | |
| diKillerRed-PTS1 | Academia Sinica | made by appending the KillerRed tandem dimer with PTS1(VKSKL) | |
| Dulbecco's Modified Eagle Medium (DMEM) | ThermoFisher Scientific | 11965092 | |
| Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) | ThermoFisher Scientific | 11330032 | |
| EGFP-C1 | Clontech | pEGFP-C1 | The backbone of EGFP-C1 was used for cloning EGFP-Stub1, EGFP-Hsp70, EGFP-p62 |
| EGFP-Hsp70 | Academia Sinica | Hsp70 gene (HSPA1A) PCR amplified from HeLa cDNA and cloned into EGFP-C1 | |
| EGFP-LC3B | Addgene | 11546 | |
| EGFP-p62 | Academia Sinica | generated by inserting the human SQSTM1 gene (through PCR amplification of the HeLa cell cDNA) into EGFP-C1 | |
| EGFP-Stub1 | Academia Sinica | generated by inserting the mouse Stub1 gene (through PCR amplification of the total mouse kidney cDNA) into EGFP-C1 | |
| EGFP-Ub | Addgene | 11928 | |
| fetal bovine serum | ThermoFisher Scientific | 10437028 | |
| HaloTag TMR ligand | Promega | G8252 | |
| HaloTag-PTS1 | Academia Sinica | PTS1 appended and cloned into EGFP-C1 backbone | |
| HEPES | ThermoFisher Scientific | 15630080 | |
| Inverted Confocal Microscope | Olympus | FV3000RS | 405 nm Ex, 488 nm Ex, 561 nm Ex, microscope with a TOKAI HIT chamber incubator and the UNIV2-D35 dish attachment |
| Janelia Fluor 646 HaloTag Ligand | Promega | GA1120 | |
| LED | VitaStar | PAR64 | 80 W, 555-570 nm |
| lipofectamine 2000 | ThermoFisher Scientific | 11668 | transfection reagent |
| NIH3T3 cell | ATCC | CRL-1658 | adherent |
| Opti-MEM | ThermoFisher Scientific | 319850 | reduced serum media |
| penicillin/streptomycin | ThermoFisher Scientific | 15140 | |
| PMP34-TagBFP | Academia Sinica | PMP34 PCR amplified from HeLa cDNA and cloned intoTagBFP-C (Evrogen FP171) | |
| roGFP2-PTS1 | Academia Sinica | generated by appending eroGFP (taken from Addgene plasmid 20131) with the amino acid sequence VKSKL, and cloned into the EGFP-C1 | |
| SHSY5Y cell | ATCC | CRL-2266 | adherent |
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