利用外显整联蛋白监测整联蛋白胞吐作用和胞吞作用。
Use of Ecto-Tagged Integrins to Monitor Integrin Exocytosis and Endocytosis.
机构信息
Departments of Pharmacology, Yale University School of Medicine, Yale University, New Haven, CT, USA.
Departments of Cell Biology, Yale University School of Medicine, Yale University, New Haven, CT, USA.
出版信息
Methods Mol Biol. 2023;2608:17-38. doi: 10.1007/978-1-0716-2887-4_2.
Controlled exocytosis and endocytosis of integrin adhesion receptors is required for normal cell adhesion, migration, and signaling. In this chapter, we describe the design of functional β1 integrins carrying extracellular fluorescent or chemically traceable tags (ecto-tag) and methods for their use to image β1 integrin trafficking in cells. We provide approaches to generate cells in which endogenous β1 integrins are replaced by ecto-tagged integrins containing a pH-sensitive fluorophore pHluorin or a HaloTag and describe strategies using photobleaching, selective extracellular/intracellular labeling, and chase, quenching, and blocking to reveal β1 integrin exocytosis, endocytosis, and recycling by live total internal reflection fluorescence (TIRF) microscopy.
整合素黏附受体的受控胞吐作用和胞吞作用对于正常的细胞黏附、迁移和信号转导是必需的。在本章中，我们描述了携带细胞外荧光或化学可追踪标签（ecto-tag）的功能性β1 整合素的设计及其在细胞内成像β1 整合素运输中的用途。我们提供了生成细胞的方法，在这些细胞中，内源性β1 整合素被带有 pH 敏感荧光团 pHluorin 或 HaloTag 的 ecto-tagged 整合素所取代，并描述了使用光漂白、选择性细胞外/细胞内标记以及追踪、淬灭和阻断来揭示通过活全内反射荧光（TIRF）显微镜观察β1 整合素胞吐作用、胞吞作用和再循环的策略。
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