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 beta 1 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 beta 1 integrin knockout fibroblasts. Importantly, beta 1 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 beta 1 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.