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Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking.

Xia P, Liu X, Wu B, Zhang S, Song X, Yao PY, Lippincott-Schwartz J, Yao X - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

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.

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EB1 molecules at the microtubule plus ends from the leading edge and cell body of migrating cells exhibit distinct distribution patterns. (A) Precise localization of single EB1-PACF molecules in the cell bodies of live MCF7 cells. Arrows indicate the plus ends of microtubules. Scale bar: 250 nm. (B) Fluorescence intensity distribution for PALM and TIRFM imaging of a rectangular region of 1 μm × 200 nm in the front of a plus end in the cell body was calculated and plotted. The fluorescence intensity was normalized to the highest value in the region to facilitate the comparison. (C) Precise localization of EB1-PACF proteins in the leading edge of live MCF7 cells. Arrows indicate the plus ends. Scale bar: 250 nm. (D) Fluorescence intensity distribution for PALM and TIRFM imaging of a rectangular region of 1 μm × 200 nm in the front of a plus end in the leading edge was calculated and plotted. The fluorescence intensity was normalized to the highest value in the region to facilitate the comparison. (E) The percentage of plus ends classified as type A and type B are plotted for the leading edge and cell body. Data were collected from six individual experiments. The total sample size (number of plus ends classified) is indicated in the parentheses on the abscissa. (F) A working model of plus-end tracking in a migrating cell was proposed and presented. PACF-based imaging demonstrates that EB1 molecules exhibit a context-dependent distribution pattern at the leading edge, cell body, and trailing edge of migrating cells. The distribution pattern of EB1 molecules reports the characteristics of microtubule plus-end dynamics in the aforementioned regions.
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Figure 3: EB1 molecules at the microtubule plus ends from the leading edge and cell body of migrating cells exhibit distinct distribution patterns. (A) Precise localization of single EB1-PACF molecules in the cell bodies of live MCF7 cells. Arrows indicate the plus ends of microtubules. Scale bar: 250 nm. (B) Fluorescence intensity distribution for PALM and TIRFM imaging of a rectangular region of 1 μm × 200 nm in the front of a plus end in the cell body was calculated and plotted. The fluorescence intensity was normalized to the highest value in the region to facilitate the comparison. (C) Precise localization of EB1-PACF proteins in the leading edge of live MCF7 cells. Arrows indicate the plus ends. Scale bar: 250 nm. (D) Fluorescence intensity distribution for PALM and TIRFM imaging of a rectangular region of 1 μm × 200 nm in the front of a plus end in the leading edge was calculated and plotted. The fluorescence intensity was normalized to the highest value in the region to facilitate the comparison. (E) The percentage of plus ends classified as type A and type B are plotted for the leading edge and cell body. Data were collected from six individual experiments. The total sample size (number of plus ends classified) is indicated in the parentheses on the abscissa. (F) A working model of plus-end tracking in a migrating cell was proposed and presented. PACF-based imaging demonstrates that EB1 molecules exhibit a context-dependent distribution pattern at the leading edge, cell body, and trailing edge of migrating cells. The distribution pattern of EB1 molecules reports the characteristics of microtubule plus-end dynamics in the aforementioned regions.

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).


Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking.

Xia P, Liu X, Wu B, Zhang S, Song X, Yao PY, Lippincott-Schwartz J, Yao X - Mol. Biol. Cell (2014)

EB1 molecules at the microtubule plus ends from the leading edge and cell body of migrating cells exhibit distinct distribution patterns. (A) Precise localization of single EB1-PACF molecules in the cell bodies of live MCF7 cells. Arrows indicate the plus ends of microtubules. Scale bar: 250 nm. (B) Fluorescence intensity distribution for PALM and TIRFM imaging of a rectangular region of 1 μm × 200 nm in the front of a plus end in the cell body was calculated and plotted. The fluorescence intensity was normalized to the highest value in the region to facilitate the comparison. (C) Precise localization of EB1-PACF proteins in the leading edge of live MCF7 cells. Arrows indicate the plus ends. Scale bar: 250 nm. (D) Fluorescence intensity distribution for PALM and TIRFM imaging of a rectangular region of 1 μm × 200 nm in the front of a plus end in the leading edge was calculated and plotted. The fluorescence intensity was normalized to the highest value in the region to facilitate the comparison. (E) The percentage of plus ends classified as type A and type B are plotted for the leading edge and cell body. Data were collected from six individual experiments. The total sample size (number of plus ends classified) is indicated in the parentheses on the abscissa. (F) A working model of plus-end tracking in a migrating cell was proposed and presented. PACF-based imaging demonstrates that EB1 molecules exhibit a context-dependent distribution pattern at the leading edge, cell body, and trailing edge of migrating cells. The distribution pattern of EB1 molecules reports the characteristics of microtubule plus-end dynamics in the aforementioned regions.
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Figure 3: EB1 molecules at the microtubule plus ends from the leading edge and cell body of migrating cells exhibit distinct distribution patterns. (A) Precise localization of single EB1-PACF molecules in the cell bodies of live MCF7 cells. Arrows indicate the plus ends of microtubules. Scale bar: 250 nm. (B) Fluorescence intensity distribution for PALM and TIRFM imaging of a rectangular region of 1 μm × 200 nm in the front of a plus end in the cell body was calculated and plotted. The fluorescence intensity was normalized to the highest value in the region to facilitate the comparison. (C) Precise localization of EB1-PACF proteins in the leading edge of live MCF7 cells. Arrows indicate the plus ends. Scale bar: 250 nm. (D) Fluorescence intensity distribution for PALM and TIRFM imaging of a rectangular region of 1 μm × 200 nm in the front of a plus end in the leading edge was calculated and plotted. The fluorescence intensity was normalized to the highest value in the region to facilitate the comparison. (E) The percentage of plus ends classified as type A and type B are plotted for the leading edge and cell body. Data were collected from six individual experiments. The total sample size (number of plus ends classified) is indicated in the parentheses on the abscissa. (F) A working model of plus-end tracking in a migrating cell was proposed and presented. PACF-based imaging demonstrates that EB1 molecules exhibit a context-dependent distribution pattern at the leading edge, cell body, and trailing edge of migrating cells. The distribution pattern of EB1 molecules reports the characteristics of microtubule plus-end dynamics in the aforementioned regions.
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).

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.

View Article: PubMed Central - PubMed

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