Peng Xia and Xuebiao Yao of the Hefei National Laboratory for Physical Sciences at the Nanoscale and University of Science and Technology of China, and their colleagues have managed to visualize protein interactions at nanometer spatial resolution in live cells. The study is published in Molecular Biology of the Cell (MBoC) and will also presented at the 2014 ASCB/IFCB meeting in Philadelphia on December 8 at 1:30 pm in the ASCB Learning Center.
To move around cells have to keep reorganizing their cytoskeleton, removing pieces from one end of a microtubule and add them to the front, like a railroad with a limited supply of tracks. The EB protein family helps regulate this process and can act as a scaffold for other proteins involved in pushing the microtubule chain forward.
AdvertisementThis new method uses photoactivatable complementary fluorescent proteins (PACF) to observe and quantify protein-protein interactions in live cells at the single molecule level. Yao and Xia introduced two EB proteins into cells, one with half of a photoactivatable green fluorescent protein (PAGFP), and one with the other half of PAGFP. These PAGFP pieces will only fluoresce if the EB proteins are in a complex together and photoactivated. A different wavelength of light can also be used to switch them off. The researchers could assemble super-resolution images of protein complexes by activating and then bleaching subsets of EB molecules.
The researchers opine that their technique has revealed a critical role for a previously uncharacterized EB1 linker region in tracking microtubule plus-ends in live cells. Precise localization of dynamic microtubule plus-end hub protein EB1 dimers, and their distinct distributions at the leading edge and cell body of migrating cells can be determined with the help of this technique, which can be applied to the study of other protein complexes in unprecedented detail.