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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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Characterization of photoactivatable complementary green fluorescent proteins. (A) Protein A and B were conjugated with nPACF or cPACF, respectively. On interaction of protein A with protein B, nPACF and cPACF were programmed to form a functional fluorescent protein under photoactivation. The photoactivation of PACF proteins was achieved with a 405-nm laser and excited with a 475-nm laser. The emission spectrum peaks at 517 nm. (B) Schematic illustration of engineered constructs containing EB1 dimer fused to nPACF and cPACF proteins. (C) HeLa cells transiently transfected to express EB1-PACF proteins were fixed and stained; this was followed by imaging under a Zeiss LSM 710 laser confocal microscope. After PA under UV irradiation, the EB1-PACF signal intensity increased (in frame), while the signal intensity of α-tubulin-rhodamine decreased (by photobleaching). Scale bars: 20 μm. (D) Statistical analyses of fluorescence intensity changes of EB1-PACF and α-tubulin marked by rhodamine in C before and after PA. (E) Comparative analyses of PALM and sum of TIRFM images of fixed MCF7 cells transiently transfected to express EB1-PACF proteins. Scale bar (top panels): 5 μm. Bottom panels are magnified views of insets mentioned above. Plus-end localization of EB1-PACF dimerized proteins was readily apparent and clearly defined under superresolution imaging. Scale bars: 1 μm. (F) Comparative analyses of live PALM and sum of TIRFM images of MCF7 cells expressing EB1-PACF. Scale bars: 5 μm (top panels); 1 μm (bottom panels). (G) Comparative analyses of live PALM and sum of TIRFM images of MCF7 cells expressing EB1-PAGFP. Scale bars: 5 μm (top panels); 1 μm (bottom panels).
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Figure 1: Characterization of photoactivatable complementary green fluorescent proteins. (A) Protein A and B were conjugated with nPACF or cPACF, respectively. On interaction of protein A with protein B, nPACF and cPACF were programmed to form a functional fluorescent protein under photoactivation. The photoactivation of PACF proteins was achieved with a 405-nm laser and excited with a 475-nm laser. The emission spectrum peaks at 517 nm. (B) Schematic illustration of engineered constructs containing EB1 dimer fused to nPACF and cPACF proteins. (C) HeLa cells transiently transfected to express EB1-PACF proteins were fixed and stained; this was followed by imaging under a Zeiss LSM 710 laser confocal microscope. After PA under UV irradiation, the EB1-PACF signal intensity increased (in frame), while the signal intensity of α-tubulin-rhodamine decreased (by photobleaching). Scale bars: 20 μm. (D) Statistical analyses of fluorescence intensity changes of EB1-PACF and α-tubulin marked by rhodamine in C before and after PA. (E) Comparative analyses of PALM and sum of TIRFM images of fixed MCF7 cells transiently transfected to express EB1-PACF proteins. Scale bar (top panels): 5 μm. Bottom panels are magnified views of insets mentioned above. Plus-end localization of EB1-PACF dimerized proteins was readily apparent and clearly defined under superresolution imaging. Scale bars: 1 μm. (F) Comparative analyses of live PALM and sum of TIRFM images of MCF7 cells expressing EB1-PACF. Scale bars: 5 μm (top panels); 1 μm (bottom panels). (G) Comparative analyses of live PALM and sum of TIRFM images of MCF7 cells expressing EB1-PAGFP. Scale bars: 5 μm (top panels); 1 μm (bottom panels).

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).


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)

Characterization of photoactivatable complementary green fluorescent proteins. (A) Protein A and B were conjugated with nPACF or cPACF, respectively. On interaction of protein A with protein B, nPACF and cPACF were programmed to form a functional fluorescent protein under photoactivation. The photoactivation of PACF proteins was achieved with a 405-nm laser and excited with a 475-nm laser. The emission spectrum peaks at 517 nm. (B) Schematic illustration of engineered constructs containing EB1 dimer fused to nPACF and cPACF proteins. (C) HeLa cells transiently transfected to express EB1-PACF proteins were fixed and stained; this was followed by imaging under a Zeiss LSM 710 laser confocal microscope. After PA under UV irradiation, the EB1-PACF signal intensity increased (in frame), while the signal intensity of α-tubulin-rhodamine decreased (by photobleaching). Scale bars: 20 μm. (D) Statistical analyses of fluorescence intensity changes of EB1-PACF and α-tubulin marked by rhodamine in C before and after PA. (E) Comparative analyses of PALM and sum of TIRFM images of fixed MCF7 cells transiently transfected to express EB1-PACF proteins. Scale bar (top panels): 5 μm. Bottom panels are magnified views of insets mentioned above. Plus-end localization of EB1-PACF dimerized proteins was readily apparent and clearly defined under superresolution imaging. Scale bars: 1 μm. (F) Comparative analyses of live PALM and sum of TIRFM images of MCF7 cells expressing EB1-PACF. Scale bars: 5 μm (top panels); 1 μm (bottom panels). (G) Comparative analyses of live PALM and sum of TIRFM images of MCF7 cells expressing EB1-PAGFP. Scale bars: 5 μm (top panels); 1 μm (bottom panels).
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Related In: Results  -  Collection

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Figure 1: Characterization of photoactivatable complementary green fluorescent proteins. (A) Protein A and B were conjugated with nPACF or cPACF, respectively. On interaction of protein A with protein B, nPACF and cPACF were programmed to form a functional fluorescent protein under photoactivation. The photoactivation of PACF proteins was achieved with a 405-nm laser and excited with a 475-nm laser. The emission spectrum peaks at 517 nm. (B) Schematic illustration of engineered constructs containing EB1 dimer fused to nPACF and cPACF proteins. (C) HeLa cells transiently transfected to express EB1-PACF proteins were fixed and stained; this was followed by imaging under a Zeiss LSM 710 laser confocal microscope. After PA under UV irradiation, the EB1-PACF signal intensity increased (in frame), while the signal intensity of α-tubulin-rhodamine decreased (by photobleaching). Scale bars: 20 μm. (D) Statistical analyses of fluorescence intensity changes of EB1-PACF and α-tubulin marked by rhodamine in C before and after PA. (E) Comparative analyses of PALM and sum of TIRFM images of fixed MCF7 cells transiently transfected to express EB1-PACF proteins. Scale bar (top panels): 5 μm. Bottom panels are magnified views of insets mentioned above. Plus-end localization of EB1-PACF dimerized proteins was readily apparent and clearly defined under superresolution imaging. Scale bars: 1 μm. (F) Comparative analyses of live PALM and sum of TIRFM images of MCF7 cells expressing EB1-PACF. Scale bars: 5 μm (top panels); 1 μm (bottom panels). (G) Comparative analyses of live PALM and sum of TIRFM images of MCF7 cells expressing EB1-PAGFP. Scale bars: 5 μm (top panels); 1 μm (bottom panels).
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).

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