| RESEARCH HIGHLIGHTS |


[RESEARCH HIGHLIGHTS]


Rupturing a cell releases tens of thousands of proteins, which can be hard to track unless you have already tagged proteins of interest.


Cell biology: PLAYING TAG WITH PROTEINS


PROXIMITY LABELING REVEALS THE KEY COMPONENTS OF A STRUCTURE THAT GIVES CELLS THEIR SENSE OF PLACE


The protein complexes cells use to attach to the local biological matrix do more than hold cells in place — they help the cell sense what tissue they are in and what cell type they should be. However, the unstable nature of these ‘focal adhesion’ protein complexes makes them difficult to study. Researchers at A*STAR have built a working model of the focal adhesion by using a molecular tagging technique to precisely identify all the proteins involved1. With an average cell containing 10,000–


15,000 proteins, it is difficult to ascertain how specific proteins come together to perform certain functions. Paxillin is a well-known focal adhesion protein, but a “bewildering array” of other proteins have been proposed to make up the adhesion complex, says Ed Manser


30 A*STAR RESEARCH


from the A*STAR Institute of Molecular and Cell Biology, who led the work.


“YOU CAN ‘TAG’ A PROTEIN SUCH AS PAXILLIN AND GRAB IT ONCE THE CELL HAS BEEN BROKEN, IN THE HOPE OF FINDING ITS CELLULAR PARTNERS.”


“The problem is that proteomic methods


are largely a smash and grab activity,” Manser explains. “You can ‘tag’ a protein such as paxillin and grab it once the cell has been broken, in the hope of finding its cellular partners.” His team takes a more careful approach: “we tag all the local proteins before we break the cell.”


team2


The ‘BioID’ tagging technique used by the , also developed at A*STAR, involves


producing a cell line in which paxillin is fused to an enzyme that can add biotin to proteins within approximately 20 nanometers: an average protein is about five nanometers wide. These newly biotinylated proteins are easily identified among the cellular rubble inside the ruptured cell. The researchers combined BioID with


stable isotope labeling to calculate the relative enrichment of proteins relative to paxillin and kindlin-2: they used this to infer their location within the focal adhesion structure. Previous studies had proposed hundreds of


proteins make up focal adhesions. The IMCB team identified just 35 proteins involved in the


ISSUE 5 | OCTOBER – DECEMBER 2016


© KATERYNA KON/SCIENCE PHOTO LIBRARY/Getty


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