Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale.

Jing-Ming Dong, Felicia Pei-Ling Tay, Hannah Lee-Foon Swa, Jayantha Gunaratne, Thomas Leung, Brian Burke, Ed Manser

Science signaling 2016 Jun 14

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Focal adhesions are protein complexes that link metazoan cells to the extracellular matrix through the integrin family of transmembrane proteins. Integrins recruit many proteins to these complexes, referred to as the "adhesome." We used proximity-dependent biotinylation (BioID) in U2OS osteosarcoma cells to label proteins within 15 to 25 nm of paxillin, a cytoplasmic focal adhesion protein, and kindlin-2, which directly binds β integrins. Using mass spectrometry analysis of the biotinylated proteins, we identified 27 known adhesome proteins and 8 previously unknown components close to paxillin. However, only seven of these proteins interacted directly with paxillin, one of which was the adaptor protein Kank2. The proteins in proximity to β integrin included 15 of the adhesion proteins identified in the paxillin BioID data set. BioID also correctly established kindlin-2 as a cell-cell junction protein. By focusing on this smaller data set, new partners for kindlin-2 were found, namely, the endocytosis-promoting proteins liprin β1 and EFR3A, but, contrary to previous reports, not the filamin-binding protein migfilin. A model adhesome based on both data sets suggests that focal adhesions contain fewer components than previously suspected and that paxillin lies away from the plasma membrane. These data not only illustrate the power of using BioID and stable isotope-labeled mass spectrometry to define macromolecular complexes but also enable the correct identification of therapeutic targets within the adhesome. Copyright © 2016, American Association for the Advancement of Science.

Citation

Jing-Ming Dong, Felicia Pei-Ling Tay, Hannah Lee-Foon Swa, Jayantha Gunaratne, Thomas Leung, Brian Burke, Ed Manser. Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale. Science signaling. 2016 Jun 14;9(432):rs4