Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking.
Bottom Line: Using PACF, we obtained precise localization of dynamic microtubule plus-end hub protein EB1 dimers and their distinct distributions at the leading edges and in the cell bodies of migrating cells.Surprisingly, our analyses revealed critical role of a previously uncharacterized EB1 linker region in tracking microtubule plus ends in live cells.Thus PACF provides a unique approach to delineating spatial dynamics of homo- or heterodimerized proteins at the nanometer scale and establishes a platform to report the precise regulation of protein interactions in space and time in live cells.
Affiliation: Anhui Key Laboratory for Cellular Dynamics & Chemical Biology and the Center for Integrated Imaging, Hefei National Laboratory for Physical Sciences at the Nanoscale and University of Science and Technology of China, Hefei, Anhui 230026, China.Show MeSH
Mentions: To this end, N-terminus/C-terminus (n/c) PACF was designed to fuse with different proteins to generate fluorescent protein (Figure 1A and Supplemental Figure 1). The interaction of target proteins provides the opportunity for PACF protein capture and maturation. After maturation, the green PACF proteins can be photoactivated with a 405-nm laser. To test the photoactivation property of PACF, we fused n/cPACF to EB1 to detect homodimerized EB1 proteins (Figure 1B). The expression of EB1-PACF in cells was confirmed by Western blot analyses of the right size (Supplemental Figure 2A). PACF can be activated with a 405-nm laser in cold methanol–fixed HeLa cells (Figure 1, C and D). As shown in Supplemental Figure 2, B–D, native gel analyses also confirm a functional activation of PACF and EB1-PACF by UV light, suggesting that PACF indeed possesses the property of being photoactivatable after its complementation. Photobleaching properties of EB1-PACF and EB1-PAGFP were also examined, and the parameters of PACF are comparable with those of PAGFP (Supplemental Figure 3, A and B).
Affiliation: Anhui Key Laboratory for Cellular Dynamics & Chemical Biology and the Center for Integrated Imaging, Hefei National Laboratory for Physical Sciences at the Nanoscale and University of Science and Technology of China, Hefei, Anhui 230026, China.