Name: monitoring stub1-mediated pexophagy
Uploaded: 2023-05-12T12:35:00
Description: Watch this Scientific Journal Video about Monitoring Stub1-Mediated Pexophagy at JoVE.com

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。

1. 制备在过氧化物酶体腔中表达diKillerRed或自标记蛋白（SLP）的细胞
<ol><li>将所需细胞接种在玻璃底细胞培养皿上。对于此处的实验，在 840 μL 培养基（补充有 10% 胎牛血清和 1% 青霉素/链霉素的 DMEM/F-12）中接种 2 x 105 人 SHSY5Y 细胞，或在 840 μL 培养基（补充有 10% 牛血清和 1% 青霉素/链霉素的 DMEM）中接种 2 x 105 个人 SHSY5Y 细胞，在直径为 20 mm 的玻璃微孔的 35 mm 培养?...

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bit-by-bit+autophagic+removal+of+parkin-labelled+mitochondria.">Bit-by-bit autophagic removal of parkin-labelled mitochondria.</a> Nature Communications. 4, 2428 (2013).</li><li>Los, G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HaloTag:+A+novel+protein+labeling+technology+for+cell+imaging+and+protein+analysis.">HaloTag: A novel protein labeling technology for cell imaging and protein analysis.</a> ACS Chemical Biology. 3 (6), 373-382 (2008).</li><li>Lismont, C., Walton, P. 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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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm culture dish with a 20 mm diameter glass microwell&#160;</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C</td>
			<td>20 mm glass bottomed</td>
		</tr>
		<tr>
			<td>3-amino-1,2,4-triazole (3-AT)</td>
			<td>Sigma Aldrich</td>
			<td>A8056</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>16170060</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Nuaire</td>
			<td>NU-4750</td>
			<td></td>
		</tr>
		<tr>
			<td>diKillerRed-PTS1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>made by appending the KillerRed tandem dimer with PTS1(VKSKL)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12)</td>
			<td>ThermoFisher Scientific</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>EGFP-C1</td>
			<td>Clontech</td>
			<td>pEGFP-C1</td>
			<td>The backbone of EGFP-C1 was used for cloning EGFP-Stub1, EGFP-Hsp70, EGFP-p62</td>
		</tr>
		<tr>
			<td>EGFP-Hsp70</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>Hsp70 gene (HSPA1A) PCR amplified from HeLa cDNA and cloned into EGFP-C1</td>
		</tr>
		<tr>
			<td>EGFP-LC3B</td>
			<td>Addgene</td>
			<td>11546</td>
			<td></td>
		</tr>
		<tr>
			<td>EGFP-p62</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>generated by inserting the human SQSTM1 gene (through PCR amplification of the HeLa cell cDNA) into EGFP-C1</td>
		</tr>
		<tr>
			<td>EGFP-Stub1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>generated by inserting the mouse Stub1 gene (through PCR amplification of the total mouse kidney cDNA) into EGFP-C1</td>
		</tr>
		<tr>
			<td>EGFP-Ub</td>
			<td>Addgene</td>
			<td>11928</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>HaloTag TMR ligand&#160;</td>
			<td>Promega</td>
			<td>G8252</td>
			<td></td>
		</tr>
		<tr>
			<td>HaloTag-PTS1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>PTS1 appended and cloned into EGFP-C1 backbone</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Confocal Microscope&#160;</td>
			<td>Olympus</td>
			<td>FV3000RS</td>
			<td>405 nm Ex, 488 nm Ex, 561 nm Ex,&#160; microscope with a TOKAI HIT chamber incubator and the UNIV2-D35 dish attachment</td>
		</tr>
		<tr>
			<td>Janelia Fluor 646 HaloTag Ligand</td>
			<td>Promega</td>
			<td>GA1120</td>
			<td></td>
		</tr>
		<tr>
			<td>LED</td>
			<td>VitaStar</td>
			<td>PAR64</td>
			<td>80 W, 555-570 nm</td>
		</tr>
		<tr>
			<td>lipofectamine 2000&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>11668</td>
			<td>transfection reagent</td>
		</tr>
		<tr>
			<td>NIH3T3 cell</td>
			<td>ATCC</td>
			<td>CRL-1658</td>
			<td>adherent</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>ThermoFisher Scientific</td>
			<td>319850</td>
			<td>reduced serum media</td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>PMP34-TagBFP</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>PMP34 PCR amplified from HeLa cDNA and cloned intoTagBFP-C (Evrogen FP171)</td>
		</tr>
		<tr>
			<td>roGFP2-PTS1</td>
			<td>Academia Sinica</td>
			<td></td>
			<td>generated by appending eroGFP (taken from Addgene plasmid 20131) with the amino acid sequence VKSKL, and cloned into the EGFP-C1</td>
		</tr>
		<tr>
			<td>SHSY5Y cell</td>
			<td>ATCC</td>
			<td>CRL-2266</td>
			<td>adherent</td>
		</tr>
	</tbody>
</table>

monitoring stub1-mediated pexophagy

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。

此处显示的Stub1介导的噬菌诱导方案利用了过氧化物酶体腔内的染料辅助ROS生成。此操作需要最小的光强度。因此，含有荧光蛋白或染料的过氧化物酶体可以使用标准激光扫描共聚焦显微镜照射。聚焦照明导致单个过氧化物酶体内瞬时和局部ROS产生，如荧光报告基因roGFP2-VKSKL所示（图9）。我们没有在roGFP2-VKSKL测量中对diKillerRed-VKSKL进行成像，以避免在成像过程中产生额外的RO...

Watch this Scientific Journal Video about Monitoring Stub1-Mediated Pexophagy at JoVE.com

Monitoring Stub1-Mediated Pexophagy

该协议提供了触发和监测活细胞中Stub1介导的吞噬的说明。

监测存根1介导的吞噬

Mammalian cells can turn over peroxisomes through Stub1-mediated pexophagy. The pathway potentially permits cellular control of the quantity and quality ...

过氧化物酶体进行β氧化，容易引起ROS损伤。这种方法可以研究细胞如何处理ROS应激的过氧化物酶体。该协议不仅用于全局靶向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。染料辅助ROS生成也可用于靶向过氧化物酶体外的细胞器，以探测细胞如何处理细胞器损伤。首先，在玻璃底35毫米培养皿上接种两次10至第五个人SHSY5Y细胞或六次10至第四只小鼠NIH H3T3细胞，在玻璃底35毫米培养皿上接种2次至50毫米，并带有20毫米直径的玻璃微孔。在 5% 的二氧化碳下在 37 摄氏度下培养细胞 24 小时。接下来，使用转染试剂或SHSY5Y细胞转染用所需质粒转染细胞。在 55 μL 无血清 DMEM/F-12 中稀释质粒，在 55 μL 无血清 DMEM/F-12 中稀释 1 μL 转染试剂。将稀释的DNA与稀释的试剂相结合。轻轻混合并在室温下孵育 30 分钟。将复合物添加到先前接种有100微升含有不含抗生素的SHSY5Y细胞的培养基的20毫米微孔中。轻轻地来回摇晃盘子。如前所述进行NIH 3T3细胞转染，但用还原血清培养基代替无血清DMEM/F-12作为质粒稀释和转染试剂。转染两小时后，取出含转染试剂的培养基，加入一毫升新鲜培养基。将NIH 3T3或SHSY5Y细胞在37摄氏度和5%二氧化碳下孵育24小时。对于光活化ROS生产，在转染后24小时取出培养基，并将10微升200纳摩尔HaloTag TMR配体或200纳摩尔Janelia Fluor 646 HaloTag配体加入20毫米玻璃微孔中。将培养皿转移到 37 摄氏度和 5% 二氧化碳培养箱中，放置一小时以染色细胞。一小时后，彻底除去染色培养基并加入一毫升新鲜培养基。将含有所需细胞的玻璃底培养皿放在配备有载物台顶部培养箱的激光扫描共聚焦显微镜上。在软件中，单击工具窗口，选择目镜，然后单击DIA按钮打开透射光。通过显微镜目镜观察培养皿，并通过转动聚焦旋钮使细胞聚焦。选择 LSM，然后单击染料检测器选择按钮。在弹出窗口中双击所需的染料，选择适当的激光和滤光片设置来对细胞进行成像。单击Live面板中的Live4x以启动快速激光扫描，以开始筛选细胞的荧光信号。使用操纵杆控制器移动载物台，找到表达培养基的diKillerRed VKSKL或SLP-VKSKL细胞，以在过氧化物酶体上施加ROS应力。获取所需细胞的图像。单击“工具窗口”，然后选择“LSM 刺激”。然后单击椭圆按钮，在“实时图像”窗口中使用鼠标绘制直径为15微米的ROI，其中包含将施加ROS应力的所需过氧化物酶体。要施加ROS应力，对具有diKillerRed VKSKL或TMR标记的SLP配体的细胞使用561纳米，对用Janelia Fluor 646标记的SLP配体染色的细胞使用640纳米。选择用于施加 ROS 应力的激光波长。输入所需的激光百分比和持续时间。对于过氧化物酶体中的ROS产生，请单击“获取”，然后单击“刺激”按钮，以启动561或640纳米光通过ROI扫描，每次扫描30秒。这种刺激对应于561和640纳米的大约5%激光功率设置。激光照射30秒后，ROI内的过氧化物酶体会失去其diKillerRed VKSKL，TMR或Janelia Fluor 646信号。这种信号损失允许区分细胞内的发光和非发光过氧化物酶体。为了监测噬菌活细胞，通过使用帧之间的 10 分钟间隔进行延时成像，跟踪 EGFP 标记的 Stub1、Hsp70、泛素、p62 或 LC3B 易位到发光或 ROS 应激过氧化物酶体上。聚焦照明导致单个过氧化物酶体内瞬时和局部ROS产生，如荧光报告基因roGFP2-VKSKL所示。diKillerRed VKSKL图像是在进行所有roGFP2-VKSKL测量后拍摄的，以指示受损的过氧化物酶体的位置。数据还表明，未发光的过氧化物酶体不受影响，表明该方法的精确性。该方法用于监测Stub1介导的噬菌。在此过程中，Hsp70，Stub1，泛素化蛋白，自噬适配器p62和LC3B随后出现在ROS应激的过氧化物酶体中以驱动pexophagy。使用相同的策略，可以同时破坏培养皿上的所有过氧化物酶体，以对Stub1介导的pexophagy进行生化表征。在LED照明之前，EGFP-泛素荧光在细胞质中是均匀的，并且不与过氧化物酶体共定位。经过九小时的LED照明后，EGFP-泛素信号积累到共聚焦培养皿中生长的细胞中的所有过氧化物酶体上。使用该协议，我们发现ROS应激的过氧化物酶体通过泛素依赖性降解途径被去除。ROS应激的过氧化物酶体招募泛素E3连接酶Stub1以允许它们通过pexophatic去除。

Research

JoVE Journal

Biology

Monitoring Actin Disassembly with Time-lapse Microscopy

拆装时间推移显微镜监测肌动蛋白

科研

生物学

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

加固，Eyetracking，生理监测的功能成像

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Monitoring Plant Hormones During Stress Responses

植物激素在应激反应监测

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Monitoring Acupuncture Effects on Human Brain by fMRI

通过功能磁共振成像监测针灸对人脑的影响

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Bioassays for Monitoring Insecticide Resistance

生物测定对杀虫剂的抗药性监测

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...