Although the basic process of receptor-mediated endocytosis (RME) is well established, certain specific aspects, like the endosomal redox state, remain less characterized. Previous studies used chemically labeled ligands or antibodies with a FRET (fluorescence resonance energy transfer) probe to gauge the redox activity of the endocytic pathway with a limitation being their inability to track the apo receptor. New tools that allow direct labeling of a cell surface receptor with synthetic probes would aid in the study of its endocytic pathway and function. Herein, we use a peptide ligase, butelase 1, to label the human transferrin receptor 1 (TfR1) in established human cell lines with a designer disulfide FRET probe. This strategy enables us to obtain real-time live cell imaging of redox states in TfR1-mediated endocytosis, attesting a reducing environment in the endosomal compartments and the dynamics of TfR1 trafficking. A better understanding of endocytosis of different cell surface receptors has implications in designing strategies that hijack this natural process for intracellular drug delivery.

中文翻译：

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用二硫化物FRET探针标记转铁蛋白受体，以测定内膜隔室中的氧化还原状态。

尽管受体介导的内吞作用（RME）的基本过程已得到广泛确立，但某些特定方面（如内体氧化还原状态）的特征仍然较少。先前的研究使用化学标记的配体或具有FRET（荧光共振能量转移）探针的抗体来测量内吞途径的氧化还原活性，但由于无法追踪载脂蛋白受体而受到限制。允许使用合成探针直接标记细胞表面受体的新工具将有助于研究其内吞途径和功能。在这里，我们使用肽连接酶，butelase 1，用设计的二硫键FRET探针在已建立的人细胞系中标记人转铁蛋白受体1（TfR1）。这种策略使我们能够获得TfR1介导的内吞作用中氧化还原状态的实时活细胞成像，证明内体区室的还原环境和TfR1转运的动力学。更好地理解不同细胞表面受体的胞吞作用对设计劫持细胞内药物传递这一自然过程的策略具有影响。