A new approach to analysis of intracellular proteins and subcellular localization using cellprofiler and imageJ in combination
A new approach to analysis of intracellular proteins and subcellular localization using cellprofiler and imageJ in combination
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DOI：
10.1016/j.ymeth.2021.04.019
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发表时间：
2022-05-28
期刊：
影响因子：
4.8
通讯作者：
Okano, Hideyuki
中科院分区：
文献类型：
--
作者：
Hattori, Akito;Ohta, Etsuro;Okano, Hideyuki
细胞成像分析开源软件CellProfiler的分析流水线在各种分析应用中起着至关重要的作用。在这项研究中，我们分析了神经母细胞瘤和单核细胞系，以及疾病特异性诱导的多能干细胞(IPSC)来源的神经元，以检验联合使用CellProfiler和ImageJ是否可以区分细胞内的蛋白质。这表明，通过在CellProfiler中创建分析管道，可以清楚地检测到神经母细胞瘤系中分散的Rab7和转铁蛋白斑点。然后，我们构建了测量距离细胞核中心的距离的管道，以便研究Rab7或转铁蛋白在细胞内的定位。结合使用CellProfiler和ImageJ，我们证实我们的管道在定量和客观地分析Rab蛋白和转铁蛋白等蛋白质的膜转运方面都是适用的。此外，当应用于吞噬功能的定量测量时，我们的管道清楚地检测到吞噬了生物物品的单核细胞系。最后，我们开发了新的管道，使用来自一名携带I2020T LRRK2突变(Park8)的家族性帕金森病(PD)患者的IPSCs来分析疾病表型。这些方法能够成功地在Park8患者IPSC来源的神经元中检测到Rab5点和Rab7点。有趣的是，在长期培养中，我们发现单个Park8患者IPSC来源的神经元中Rab7斑点的数量低于对照IPSC来源的神经元。另一方面，在体外培养14天时，Park8患者IPSC来源的神经元中Rab5点的数量低于同基因的iPSC来源的神经元，但Rab7点的数量不低于同基因的iPSC来源的神经元。此外，Park8患者IPSC来源的神经元的Rab5点相对于同基因的IPSC来源的神经元显示出不同的定位模式。这些结果表明，这一新的分析工具可以作为细胞内蛋白质定量的辅助方法。
Analytical pipeline, which is used for various analysis application, of CellProfiler, an open-source software for cell imaging analysis, is very important. In the present study, to examine whether intracellular proteins can be discriminated using a combination of CellProfiler and ImageJ, we analyzed neuroblastoma and monocytic cell lines, and disease-specific induced pluripotent stem cell (iPSC)-derived neurons. This revealed that scattered puncta of Rab7 and transferrin in neuroblastoma lines were clearly detectable by created analytical pipelines in CellProfiler. We then constructed pipelines for measuring the distance from the center of the nucleus to allow investigation of the intracellular localization of Rab7 or transferrin. Using CellProfiler and ImageJ in combination, we confirmed that our pipelines were applicable both quantitatively and objectively to analysis of membrane trafficking of proteins such as Rab proteins and transferrin. In addition, when applied to quantitative measurement of phagocytosis, our pipelines clearly detected monocytic cell lines that had engulfed bioparticles. Finally, we developed new pipelines for analysis of disease phenotype using iPSCs from a patient with familial Parkinson's disease (PD), harboring the I2020T LRRK2 mutation (PARK8). These were able to successfully detect Rab5 puncta and Rab7 puncta in PARK8 patient iPSC-derived neurons. Interestingly, in long-term culture, we found that the numbers of Rab7 puncta in a single PARK8 patient iPSC-derived neurons were lower than that of control iPSC-derived neurons. On the other hands, at 14 days in vitro, the numbers of Rab5 puncta in PARK8 patient iPSC-derived neurons were lower than those of isogenic iPSC-derived neurons, but not Rab7 puncta. Furthermore, Rab5 puncta of PARK8 patient iPSC-derived neurons exhibited distinct localization pattern relative to isogenic iPSC-derived neurons. These present results suggest that this new analytical tool can be used as a supporting method for quantification of intracellular protein.