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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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Superresolution imaging with EB1-PACF in live cells visualizes spatial dynamics of EB1 dimer. (A) Live PALM and TIRFM images of MCF7 cells transiently transfected to express EB1-PACF dimerized proteins. Frames indicate the regions of microtubule plus ends at the leading edge and in the cell body. Scale bar: 5 μm. (B) Time-lapse PALM and TIRFM images of the EB1-PACF dimerized proteins on a microtubule plus end in the region of the cell body indicated in the frame in A. Images of every time point were collected with 100 cycles of photoactivation and excitation. The exposure time is 15 ms per cycle. Paired images collected from PALM were merged with those from TIRFM to compare the clarity of molecules visualized. Scale bar: 500 nm. (C) Time-lapse PALM and TIRFM images of the EB1-PACF dimerized proteins on a microtubule plus end in the region of the leading edge indicated in the frame in A. Images of every time point were collected with 100 cycles of photoactivation and excitation. The exposure time is 15 ms per cycle. Paired images collected from PALM and TIRFM were merged to compare the clarity of molecules visualized by PALM. Scale bar: 500 nm.
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Figure 2: Superresolution imaging with EB1-PACF in live cells visualizes spatial dynamics of EB1 dimer. (A) Live PALM and TIRFM images of MCF7 cells transiently transfected to express EB1-PACF dimerized proteins. Frames indicate the regions of microtubule plus ends at the leading edge and in the cell body. Scale bar: 5 μm. (B) Time-lapse PALM and TIRFM images of the EB1-PACF dimerized proteins on a microtubule plus end in the region of the cell body indicated in the frame in A. Images of every time point were collected with 100 cycles of photoactivation and excitation. The exposure time is 15 ms per cycle. Paired images collected from PALM were merged with those from TIRFM to compare the clarity of molecules visualized. Scale bar: 500 nm. (C) Time-lapse PALM and TIRFM images of the EB1-PACF dimerized proteins on a microtubule plus end in the region of the leading edge indicated in the frame in A. Images of every time point were collected with 100 cycles of photoactivation and excitation. The exposure time is 15 ms per cycle. Paired images collected from PALM and TIRFM were merged to compare the clarity of molecules visualized by PALM. Scale bar: 500 nm.

Mentions: For probing microtubule plus-end dynamics at the nanometer scale in live cells, every single image was generated from 100 frames of 15 ms per exposure (Figure 1, F and G). The efforts to reduce frames and exposure time to obtain every single image resulted in a 10-nm loss in localization precision (Supplemental Figure 4B). However, this brief loss enabled us to collect superresolution images in 1.5 s and successfully trace the dynamic microtubule plus ends in live cells. Our results also demonstrated the advantage of EB1-PACF compared with EB1-PAGFP; the former has decreased background without perturbing the signal at the microtubule plus ends (Figure 1, F and G). Furthermore, we found that the EB1-PACF molecules at the leading edges of migrating cells exhibit a slow motion compared with those tracking the microtubule plus ends in the cell body (Figure 2, A–C, and Supplemental Figure 4C). We speculate that the growth rate of microtubule plus ends is reduced in the leading edge of migrating MCF7 cells, as these plus ends may dock into the cell cortex. This hypothesis was further confirmed by conventional live-cell imaging of MCF7 cells expressing EB1-eGFP (enhanced green fluorescent protein; Supplemental Figure 5).


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)

Superresolution imaging with EB1-PACF in live cells visualizes spatial dynamics of EB1 dimer. (A) Live PALM and TIRFM images of MCF7 cells transiently transfected to express EB1-PACF dimerized proteins. Frames indicate the regions of microtubule plus ends at the leading edge and in the cell body. Scale bar: 5 μm. (B) Time-lapse PALM and TIRFM images of the EB1-PACF dimerized proteins on a microtubule plus end in the region of the cell body indicated in the frame in A. Images of every time point were collected with 100 cycles of photoactivation and excitation. The exposure time is 15 ms per cycle. Paired images collected from PALM were merged with those from TIRFM to compare the clarity of molecules visualized. Scale bar: 500 nm. (C) Time-lapse PALM and TIRFM images of the EB1-PACF dimerized proteins on a microtubule plus end in the region of the leading edge indicated in the frame in A. Images of every time point were collected with 100 cycles of photoactivation and excitation. The exposure time is 15 ms per cycle. Paired images collected from PALM and TIRFM were merged to compare the clarity of molecules visualized by PALM. Scale bar: 500 nm.
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Related In: Results  -  Collection

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Figure 2: Superresolution imaging with EB1-PACF in live cells visualizes spatial dynamics of EB1 dimer. (A) Live PALM and TIRFM images of MCF7 cells transiently transfected to express EB1-PACF dimerized proteins. Frames indicate the regions of microtubule plus ends at the leading edge and in the cell body. Scale bar: 5 μm. (B) Time-lapse PALM and TIRFM images of the EB1-PACF dimerized proteins on a microtubule plus end in the region of the cell body indicated in the frame in A. Images of every time point were collected with 100 cycles of photoactivation and excitation. The exposure time is 15 ms per cycle. Paired images collected from PALM were merged with those from TIRFM to compare the clarity of molecules visualized. Scale bar: 500 nm. (C) Time-lapse PALM and TIRFM images of the EB1-PACF dimerized proteins on a microtubule plus end in the region of the leading edge indicated in the frame in A. Images of every time point were collected with 100 cycles of photoactivation and excitation. The exposure time is 15 ms per cycle. Paired images collected from PALM and TIRFM were merged to compare the clarity of molecules visualized by PALM. Scale bar: 500 nm.
Mentions: For probing microtubule plus-end dynamics at the nanometer scale in live cells, every single image was generated from 100 frames of 15 ms per exposure (Figure 1, F and G). The efforts to reduce frames and exposure time to obtain every single image resulted in a 10-nm loss in localization precision (Supplemental Figure 4B). However, this brief loss enabled us to collect superresolution images in 1.5 s and successfully trace the dynamic microtubule plus ends in live cells. Our results also demonstrated the advantage of EB1-PACF compared with EB1-PAGFP; the former has decreased background without perturbing the signal at the microtubule plus ends (Figure 1, F and G). Furthermore, we found that the EB1-PACF molecules at the leading edges of migrating cells exhibit a slow motion compared with those tracking the microtubule plus ends in the cell body (Figure 2, A–C, and Supplemental Figure 4C). We speculate that the growth rate of microtubule plus ends is reduced in the leading edge of migrating MCF7 cells, as these plus ends may dock into the cell cortex. This hypothesis was further confirmed by conventional live-cell imaging of MCF7 cells expressing EB1-eGFP (enhanced green fluorescent protein; Supplemental Figure 5).

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
Related in: MedlinePlus