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Nat Commun. 2017 Sep 18;8(1):570. doi: 10.1038/s41467-017-00646-w.

Novel ecto-tagged integrins reveal their trafficking in live cells.

Author information

1
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut, 06520, USA.
2
Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut, 06520, USA.
3
Department of Biomedical Engineering, Yale University, 333 Cedar Street, New Haven, Connecticut, 06520, USA.
4
Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut, 06520, USA. derek.toomre@yale.edu.
5
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut, 06520, USA. david.calderwood@yale.edu.
6
Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut, 06520, USA. david.calderwood@yale.edu.

Abstract

Integrins are abundant heterodimeric cell-surface adhesion receptors essential in multicellular organisms. Integrin function is dynamically modulated by endo-exocytic trafficking, however, major mysteries remain about where, when, and how this occurs in living cells. To address this, here we report the generation of functional recombinant β1 integrins with traceable tags inserted in an extracellular loop. We demonstrate that these 'ecto-tagged' integrins are cell-surface expressed, localize to adhesions, exhibit normal integrin activation, and restore adhesion in β1 integrin knockout fibroblasts. Importantly, β1 integrins containing an extracellular pH-sensitive pHluorin tag allow direct visualization of integrin exocytosis in live cells and revealed targeted delivery of integrin vesicles to focal adhesions. Further, using β1 integrins containing a HaloTag in combination with membrane-permeant and -impermeant Halo dyes allows imaging of integrin endocytosis and recycling. Thus, ecto-tagged integrins provide novel powerful tools to characterize integrin function and trafficking.Integrins are cell-surface adhesion receptors that are modulated by endo-exocytic trafficking, but existing tools to study this process can interfere with function. Here the authors develop β1 integrins carrying traceable tags in the extracellular domain; a pH-sensitive pHlourin tag or a HaloTag to facilitate dye attachment.

PMID:
28924207
PMCID:
PMC5603536
DOI:
10.1038/s41467-017-00646-w
[Indexed for MEDLINE]
Free PMC Article

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