Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking.
Bottom Line: We further delineated the structure-function relationship of EB1 by generating EB1-PACF dimers (EB1(wt):EB1(wt), EB1(wt):EB1(mt), and EB1(mt):EB1(mt)) and imaging their precise localizations in culture 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
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Mentions: At the single-molecule level, we found that EB1-PACF tracking the growing microtubule plus ends in the cell body exhibits a complex construction, creating a profile shaped like a curving sheet (Figure 3A). These structures are consistent with the hypothetic model of microtubule plus ends (Vitre et al., 2008). Interestingly, we found many EB1-PACF molecules localized at the plus ends of the leading edge aligned in a narrow cone-shaped row (Figure 3C), distinctly different from the molecules tracking the microtubule plus ends in the cell body. In addition, this kind of difference could not be distinctly defined under TIRF microscopy (TIRFM) analyses at conventional resolution but were readily apparent with PACF imaging (Figure 3, B and D). We then confirmed that the difference seen between EB1-PACF signals at the cell body and leading edge is not because of blurring of the artifact; we collected 100 consecutive exposure frames (150 ms/frame) and separated them into two time series (frames 1–50 and frames 51–100). As shown in Supplemental Figure 6, A and B, the characteristics of each kind of plus end can be recognized, even with half-time exposure, although the integrity of images was obviously decreased (Supplemental Figure 6, C and D). We further classified the narrow cone-shaped EB1-PACF localization to type A and the complex curving sheet to type B (Figure 3E). Statistical analyses of type A and B plus ends at the leading edge or in the cell body exhibit significant differences. In the leading edge, 67.42 ± 6.86% plus ends are type A, while only 32.58 ± 6.86% are type B. However, in the cell body, only 13.72 ± 2.17% plus ends are type A, while 86.28 ± 2.17% plus ends are type B (Figure 3E). To illustrate the localization characteristics of EB1-PACF at the microtubule plus ends, we carried out superresolution images of EB1-PACF in fixed cells double-stained with tubulin and EB1 antibodies, respectively. The superresolution images of EB1-PACF were then merged with diffraction-limited images of tubulin and EB1 immunofluorescence. Careful examination reveals that EB1-PACF signals are superimposed onto the microtubules and microtubule plus ends (Supplemental Figure 6, C and D, respectively). As shown in Supplemental Figure 6E, statistical analyses demonstrate that the type A comet of EB1 dimeric molecules distributes preferentially at the leading edge of migrating cells, while the type B comet of EB1 dimers is enriched in the cell body. These precise localization analyses indicate that EB1 dimer molecules exhibit distinct distribution patterns on the microtubule plus ends in the leading edge, cell body, and trailing edge of a migrating cell (Figure 3F).
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.