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Use of Ecto-Tagged Integrins to Monitor Integrin Exocytosis and Endocytosis.

ABSTRACT: 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.

SUBMITTER: Huet-Calderwood C

PROVIDER: S-EPMC9999384 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Use of Ecto-Tagged Integrins to Monitor Integrin Exocytosis and Endocytosis.

Huet-Calderwood Clotilde C Rivera-Molina Felix F Toomre Derek D Calderwood David A DA

Methods in molecular biology (Clifton, N.J.) 20230101

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 pHluori ...[more]

PMID: 36653699

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