Quick and Mild Isolation of Intact Lysosomes Using Magnetic-Plasmonic Hybrid Nanoparticles
Quick and Mild Isolation of Intact Lysosomes Using Magnetic-Plasmonic Hybrid Nanoparticles
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DOI：
10.1021/acsnano.1c08474
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发表时间：
2022-01-03
期刊：
影响因子：
17.1
通讯作者：
Maenosono, Shinya
中科院分区：
文献类型：
--
作者：
Le, The Son;Takahashi, Mari;Maenosono, Shinya
快速有效地分离完整的溶酶体对于通过蛋白质组学分析研究其功能和代谢产物是必要的。我们开发了一种快速和稳健的基于纳米颗粒的磁性分离方法，其中磁性-等离子体杂化纳米颗粒（MPNPs）与氨基葡聚糖（aDxt）缀合，通过内吞途径靶向溶酶体的内腔。对于溶酶体的良好定向的磁性分离，重要的是追踪内吞作用途径中aDxt缀合的MPNP（aDxt-MPNP）的细胞内运输。因此，我们通过使用阈值Manders共定位系数（R-t）研究aDxt-MPNP和细胞器的免疫染色标记蛋白的等离子体散射的时间依赖性共定位来分析aDxt-MPNP的细胞内转运过程。早期和晚期内体和溶酶体的R-t的时间变化的详细分析允许我们推导出细胞中aDxt-MPNP的转运动力学。在确认aDxt-MPNP在溶酶体中充分积累所需的孵育时间后，将溶酶体尽可能完整地磁性分离。通过改变从匀浆到完全分离溶酶体的时间（t（delay））和温度（T），用聚丙烯酰胺凝胶电泳和氨基酸分析研究了t（delay）和T对溶酶体蛋白质组成的影响。我们发现，溶酶体的完整性可能会很快受损，为了分离尽可能完整的高纯度溶酶体，t（延迟）= 30 min和T = 4 ℃是最佳设置。
Rapid and efficient isolation of intact lysosomes is necessary to study their functions and metabolites by proteomic analysis. We developed a swift and robust nanoparticle-based magnetic separation method in which magnetic-plasmonic hybrid nanoparticles (MPNPs) conjugated with amino dextran (aDxt) were targeted to the lumen of lysosomes via the endocytosis pathway. For well-directed magnetic separation of the lysosomes, it is important to trace the intracellular trafficking of the aDxt-conjugated MPNPs (aDxt-MPNPs) in the endocytosis pathway. Therefore, we analyzed the intracellular transport process of the aDxt-MPNPs by investigating the time-dependent colocalization of plasmonic scattering of aDxt-MPNPs and immunostained marker proteins of organelles using the threshold Manders' colocalization coefficient (R-t). Detailed analysis of time variations of R-t for early and late endosomes and lysosomes allowed us to derive the transport kinetics of aDxt-MPNPs in a cell. After confirming the incubation time required for sufficient accumulation of aDxt-MPNPs in lysosomes, the lysosomes were magnetically isolated as intact as possible. By varying the elapsed time from homogenization to complete isolation of lysosomes (t(delay)) and temperature (T), the influences of t(delay) and T on the protein composition of the lysosomes were investigated by polyacrylamide gel electrophoresis and amino acid analysis. We found that the intactness of lysosomes could become impaired quite quickly, and to isolate lysosomes as intact as possible with high purity, t(delay) = 30 min and T = 4 degrees C were optimal settings.