Novel ecto-tagged integrins reveal their trafficking in live cells

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.