利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化
Summary
该协议概述了一种定量分析从原人类小岛样本的β细胞中专门形成米托巴细胞蛋白复合体的方法。因此,该技术允许从有限的生物材料分析米托巴,这在珍贵的人类胰腺β细胞样本中至关重要。
Abstract
Mitophagy 是一种重要的线粒体质量控制途径,对于胰腺胰岛β细胞生物能量促进葡萄糖刺激的胰岛素释放至关重要。评估米托巴经具有挑战性,通常需要基因报告员或多种补充技术,这些辅助技术不易用于组织样本,如原发性人类胰腺胰岛。在这里,我们演示了一种强大的方法来可视化和量化主要人类胰腺胰岛中关键内源性米托巴细胞复合物的形成。利用灵敏的接近结扎检测技术来检测米托巴风调节器NRDP1和USP8的相互作用,我们能够具体量化原位基本米托巴结物的形成。通过结合这种方法来对抗转录因子PDX1的染色,我们可以量化米托巴结物复合物,以及可能损害米托巴的因子,特别是在β细胞内。我们描述的方法克服了其他蛋白质-蛋白质相互作用研究(如免疫沉淀 (IP) 或质谱仪)所需的大量细胞提取物的需求,并且通常不适合珍贵的人类胰岛样本,通常不为这些方法提供足够数量的产品。此外,该方法消除了对流分选技术的需求,从异质小岛种群中纯化β细胞,用于下游蛋白质应用。因此,我们描述了一种有价值的方案,用于可视化米托巴细胞,高度兼容,用于异质和受限细胞群。
Introduction
胰腺β细胞产生维持正常葡萄糖平衡所需的胰岛素,其失败导致所有形式的糖尿病的发展。Β细胞保持强大的线粒体能力,以产生将葡萄糖代谢与胰岛素释放耦合所需的能量。最近,很明显,维持功能线粒体质量对于最佳β细胞功能1,2,3至关重要。为了维持功能线粒体质量,β细胞依靠质量控制机制来去除功能失调、受损或老化的线粒体4。我们和其他人以前已经证明,β细胞依靠一种特殊形式的线粒体周转,称为线粒体自噬(或线粒体),以保持线粒体质量控制在啮齿动物和人类胰岛1, 2,5.然而,不幸的是,在人类胰腺β细胞中,没有简单的方法来检测米托巴细胞或内分泌的米托巴细胞成分。
我们最近已经表明,β细胞中米托巴的上游调节依赖于形成一种蛋白质复合物,包括E3利气CLEC16A和NRDP1和二苯基酸酶USP81。NRDP1和USP8已经独立显示,通过行动对关键的米托巴沙第6,7影响米托巴吉。NRDP1的目标是PARKIN的泛化和降解关闭米托巴希6,和USP8专门二苯基丁基丁K6链接的PARKIN,以促进其易位线粒体7。接近结扎测定(PLA)技术是蛋白质相互作用生物学领域的一个最新进展,允许在单个细胞中原位实现内源性蛋白质相互作用的可视化,不受稀缺样品材料的限制。这种方法对人类小岛/β细胞生物学特别诱人,因为样品可用性的稀疏性,加上需要了解异质细胞类型中生理相关的蛋白质复合物。
利用PLA方法,我们能够观察原发性人类胰腺β细胞和神经元细胞系中的关键内源性米托巴细胞复合物,并演示糖尿病环境对米托巴病途径1的影响。总之,该协议的首要目标是分析缺乏丰富物质的组织中的特定米托巴状蛋白复合物,或者无法进行常规蛋白质相互作用研究。
Protocol
使用已识别的捐赠者人类胰腺胰岛是通过机构审查委员会(IRB)豁免,并符合密歇根大学IRB政策。人类胰腺胰岛由NIH/NIDDK赞助的综合胰岛分布方案(IIDP)提供。 1. 人类小岛样品制备 单细胞分离 培养人类胰岛样品(4000~6000胰岛当量/10 mL介质),在胰腺胰岛介质(PIM(S))介质中至少1天,辅以1 mM谷氨酰胺(PIM(G))、100单位/mL抗霉菌抗生素、1mM丙酸钠和10%胎儿牛血清(FBS)?…
Representative Results
我们在MIN6胰腺β细胞系和SH-SY5Y神经母细胞瘤细胞系SH-SY5Y进行了初步实验,以优化和确认抗体的特异性和可视化的蛋白质相互作用。MIN6细胞以30,000个细胞/mL镀在盖玻片上,并留下粘附48小时,SH-SY5Y细胞以15,000个细胞/mL镀在盖玻片上,然后留在附着24小时。然后,从步骤 1.2.3 开始,遵循了上述 PLA 协议。为了确保PLA信号的特异性,我们首先在(i)没有原抗体(图2A,<st…
Discussion
在这里,我们描述了一种简单而有效的方法,用于在感兴趣的组织/细胞中使用NRDP1:USP8 PLA,以量化上游米托巴细胞复合物的形成。我们之前通过多种方法确认在胰腺β细胞中形成CLEC16A-NRDP1-USP8米托巴细胞复合物,包括共免疫沉淀实验、无细胞相互作用研究、体外和基于细胞泛化测定,并演示了这个复杂驱动调节米托巴哈通通1,15。我们在这里报告和描述的PLA研究…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢JDRF(CDA-2016-189和SRA-2018-539)、国家糖尿病、消化和肾脏疾病研究所、国家卫生研究院(R01-DK-108921)、布雷姆家族和安东尼家族的资助。JDRF职业发展奖由丹麦糖尿病学院获得部分支持,该学院由诺和诺德基金会支持。
Materials
| 0.25% trypsin-EDTA 1X | Life Technologies | 25200-056 | |
| Antibiotic-Antimycotic | Life Technologies | 15240-062 | |
| Block solution | Homemade | Use 1X PBS, add 10 % donkey serum, and 0.3% Triton X-100 detergent. | |
| Buffer A | Homemade | To make 1L: Mix 8.8g NaCl, 1.2g Tris base, 500ul Tween-20, with 750mL ddH20. pH to 7.5 with HCl, and fill to 1L. Filter solution and store at 4C. Bring to RT before experimental use | |
| Buffer B | Homemade | To make 1L: Mix 5.84g NaCl, 4.24g Tris base, 26g Tris-HCl with 500mL ddH20. pH to 7.5, and fill to 1L. Filter solution and store a 4C. Bring to RT before experimental use | |
| Cy5-conjugated AffiniPure donkey anti-goat | Jackson Labs | 705-175-147 | |
| Detection Reagents Red | Sigma- Aldrich | DU092008-100RXN | Kit containing: ligation solution stock (5X), ligase, amplification solution stock (5X) and polymerase. |
| DuoLink PLA probe anti-mouse MINUS | Sigma- Aldrich | DU092004-100RXN | |
| DuoLink PLA probe anti-rabbit PLUS | Sigma- Aldrich | DU092002-100RXN | |
| Fetal bovine serum | |||
| Goat polyclonal anti-PDX1 (clone A17) | Santa Cruz | SC-14664 | RRID: AB_2162373 |
| HEPES (1M) | Life Technologies | 15630-080 | |
| MIN6 pancreatic cell line | Gift from D. Stoffers | Mouse insulinoma cell line, utilized for cell-based assays. | |
| Mouse monoclonal anti-USP8 antibody (clone US872) | Sigma- Aldrich | SAB200527 | |
| Pap-pen | Research Products International | 195505 | |
| Parafilm | Use to seal antibody and probe solutions on your cells to prevent evaporation when using small solution volumes. | ||
| PBT (phosphate buffered saline with triton) | Homemade | To make 50mL: 43.5mLddH2O, 5mL 10X PBS, 0.5mL 10X BSA(100mg/mL solution), 1mL 10% triton X-100 solution in ddH20) | |
| Penicillin-Streptomycin (100X) | Life Technologies | 15140-122 | |
| Phosphate buffered saline, 10X | Fisher Scientific | BP399-20 | |
| PIM(ABS) Human AB serum | Prodo Labs | PIM-ABS001GMP | |
| PIM(G) (glutamine) | Prodo Labs | PIM-G001GMP | |
| PIM(S) media | Prodo Labs | PIM-S001GMP | |
| PR619 | Apex Bio | A812 | |
| Prolong Gold antifade reagent with DAPI | Life Technologies (Molecular Probes) | P36935 | |
| Rabbit polyclonal anti-FLRF/RNF41 (Nrdp1) | Bethyl Laboratories | A300-049A | RRID: AB_2181251 |
| SH-SY5Y cells | Gift from L. Satin | Human neuroblastoma cell line, utilized for cell-based assays. | |
| Sodium Pyruvate (100X) | Life Technologies | 11360-070 | |
| Triton X-100 | Fisher Scientific | BP151-100 | |
| Tween-20 | Fisher Scientific | BP337-100 | |
| Water for RNA work (DEPC water) | Fisher Scientific | BP361-1L |
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Citar este artigo
Pearson, G., Soleimanpour, S. A. Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay. J. Vis. Exp. (147), e59398, doi:10.3791/59398 (2019).