Novel ecto-tagged integrins reveal their trafficking in live cells

Clotilde Huet-Calderwood,David A Calderwood,Felix Rivera-Molina,Derek Toomre,Daniel V Iwamoto,Emil B Kromann

doi:10.1038/s41467-017-00646-w

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.

Highlights

Integrins are abundant heterodimeric cell-surface adhesion receptors essential in multicellular organisms
Several factors restrict the number of suitable sites for placement of a large tag such as GFP, Halo or SNAP: (i) the large aforementioned nested N-terminal domains, (ii) the presence of an N-terminal signal peptide that gets cleaved off during polypeptide maturation, (iii) the need to retain the ability to pair with integrin α subunits to bind ligand, and (iv) the need to accommodate the large-scale conformational rearrangements thought to occur during integrin activation
We establish that the insertion of GFP, the pH-sensitive GFP variant pHluorin, or the enzymatic tag Halo at this site does not disrupt β1 integrin function

Summary

Introduction

Integrins are abundant heterodimeric cell-surface adhesion receptors essential in multicellular organisms. Many molecular adapters involved in membrane trafficking have been found to regulate integrin surface levels and to affect integrinmediated activities, with some adapters shown to directly bind integrin subunits[4, 5, 10, 14, 15]. Biochemical assays such as cell-surface biotinylation or integrin labeling with ligand or a. Β1 integrin KO Cyto-GFP β1 Ecto-Halo β1 Ecto-pH9 β1 Ecto-pH4 β1 Ecto-GFP9 β1 Ecto-GFP4 β1 No-tag β1

Methods

Results

Conclusion