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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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The linker region of EB1 molecules governs the plus-end localization of functional EB1 dimers. (A) Schematic drawing of sequence alignment of the linker region of EB1 and EB3 molecules. The conserved residues are presented and highlighted with a gray background. (B) Schematic illustration and precise localization of PACF fused EB1WT/WT, EB1KR5Q/KR5Q, and EB1WT/KR5Q. Only EB1WT/WT dimer but not EB1KR5Q/KR5Q or even EB1WT/KR5Q mutant exhibits typical comet-like plus-end localization in transiently transfected HeLa cells. Scale bars: 5 μm. (C) Statistical analyses of histograms of comet numbers per square micrometer in live MCF7 cells expressing PACF fused EB1WT/WT, EB1KR5Q/KR5Q, and EB1WT/KR5Q. Our analyses show that the numbers of comets per square micrometer are decreased in cells expressing EB1KR5Q/KR5Q and EB1WT/KR5Q mutants compared with those of EB1WT/WT-expressing cells. Error bars indicate SD. *, p < 0.005; NS, no significance. (D) FRET spectra of EB1 CH linker (aa 1–190) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine, which demonstrates the importance of those residues in intramolecular interaction of EB1 molecules. (E) FRET spectra of EB1 linker (aa 131–190) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine. (F) FRET spectra of EB1 linker-EBC (aa 131–268) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine.
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Figure 4: The linker region of EB1 molecules governs the plus-end localization of functional EB1 dimers. (A) Schematic drawing of sequence alignment of the linker region of EB1 and EB3 molecules. The conserved residues are presented and highlighted with a gray background. (B) Schematic illustration and precise localization of PACF fused EB1WT/WT, EB1KR5Q/KR5Q, and EB1WT/KR5Q. Only EB1WT/WT dimer but not EB1KR5Q/KR5Q or even EB1WT/KR5Q mutant exhibits typical comet-like plus-end localization in transiently transfected HeLa cells. Scale bars: 5 μm. (C) Statistical analyses of histograms of comet numbers per square micrometer in live MCF7 cells expressing PACF fused EB1WT/WT, EB1KR5Q/KR5Q, and EB1WT/KR5Q. Our analyses show that the numbers of comets per square micrometer are decreased in cells expressing EB1KR5Q/KR5Q and EB1WT/KR5Q mutants compared with those of EB1WT/WT-expressing cells. Error bars indicate SD. *, p < 0.005; NS, no significance. (D) FRET spectra of EB1 CH linker (aa 1–190) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine, which demonstrates the importance of those residues in intramolecular interaction of EB1 molecules. (E) FRET spectra of EB1 linker (aa 131–190) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine. (F) FRET spectra of EB1 linker-EBC (aa 131–268) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine.

Mentions: To delineate the structure–function relationship of the linker region, we first performed sequence alignment of EB1 homologues and determined that several proline (P) residues and almost all the lysine (K)/arginine (R) residues were relatively conserved in this low homologue region (Figure 4A). Proline residue often serves at the turn or loop of a protein. However, mutation of proline residues to alanine or glycine did not alter the localization of EB1 to the plus end of the microtubule (unpublished data). We then examined whether the conserved and positively charged residues K/R modulate the localization of EB1. To this end, we took the advantage of EB1-PACF to design dimers with a combination of wild-type and mutant EB1 in which the five positively charged K/R residues were mutated to glutamine (Q; EB1KR5Q). In this regard, we can ascertain the respective contribution of each molecule of the engineered EB1 dimer to stable plus-end localization using EB1-PACF (Figure 4B, left panel). We first examined the protein expression of the PACF-fused EB1 proteins with Western blotting (Supplemental Figure 9). Consistent with our demonstration above, precise localization of EB1WT/WT-PACF exhibited a typical comet-like plus end–tracking character (Figure 4B, top right panel). However, expression of either EB1KR5Q/KR5Q-PACF or EB1WT/KR5Q-PACF failed to reach a stable localization to the plus ends, as evidenced by fewer comet-like structures (Figure 4, B and C, and Supplemental Figure 10). Thus the conserved K/R residues in the linker region are essential for the functional EB1 dimers, and their plus-end tracking property is impaired, even when the linker region of one molecule of the EB1 dimer is altered.


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)

The linker region of EB1 molecules governs the plus-end localization of functional EB1 dimers. (A) Schematic drawing of sequence alignment of the linker region of EB1 and EB3 molecules. The conserved residues are presented and highlighted with a gray background. (B) Schematic illustration and precise localization of PACF fused EB1WT/WT, EB1KR5Q/KR5Q, and EB1WT/KR5Q. Only EB1WT/WT dimer but not EB1KR5Q/KR5Q or even EB1WT/KR5Q mutant exhibits typical comet-like plus-end localization in transiently transfected HeLa cells. Scale bars: 5 μm. (C) Statistical analyses of histograms of comet numbers per square micrometer in live MCF7 cells expressing PACF fused EB1WT/WT, EB1KR5Q/KR5Q, and EB1WT/KR5Q. Our analyses show that the numbers of comets per square micrometer are decreased in cells expressing EB1KR5Q/KR5Q and EB1WT/KR5Q mutants compared with those of EB1WT/WT-expressing cells. Error bars indicate SD. *, p < 0.005; NS, no significance. (D) FRET spectra of EB1 CH linker (aa 1–190) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine, which demonstrates the importance of those residues in intramolecular interaction of EB1 molecules. (E) FRET spectra of EB1 linker (aa 131–190) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine. (F) FRET spectra of EB1 linker-EBC (aa 131–268) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine.
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Related In: Results  -  Collection

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Figure 4: The linker region of EB1 molecules governs the plus-end localization of functional EB1 dimers. (A) Schematic drawing of sequence alignment of the linker region of EB1 and EB3 molecules. The conserved residues are presented and highlighted with a gray background. (B) Schematic illustration and precise localization of PACF fused EB1WT/WT, EB1KR5Q/KR5Q, and EB1WT/KR5Q. Only EB1WT/WT dimer but not EB1KR5Q/KR5Q or even EB1WT/KR5Q mutant exhibits typical comet-like plus-end localization in transiently transfected HeLa cells. Scale bars: 5 μm. (C) Statistical analyses of histograms of comet numbers per square micrometer in live MCF7 cells expressing PACF fused EB1WT/WT, EB1KR5Q/KR5Q, and EB1WT/KR5Q. Our analyses show that the numbers of comets per square micrometer are decreased in cells expressing EB1KR5Q/KR5Q and EB1WT/KR5Q mutants compared with those of EB1WT/WT-expressing cells. Error bars indicate SD. *, p < 0.005; NS, no significance. (D) FRET spectra of EB1 CH linker (aa 1–190) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine, which demonstrates the importance of those residues in intramolecular interaction of EB1 molecules. (E) FRET spectra of EB1 linker (aa 131–190) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine. (F) FRET spectra of EB1 linker-EBC (aa 131–268) wild type and KR5Q mutant. FRET efficiency decreases upon mutation of the conserved lysine and arginine.
Mentions: To delineate the structure–function relationship of the linker region, we first performed sequence alignment of EB1 homologues and determined that several proline (P) residues and almost all the lysine (K)/arginine (R) residues were relatively conserved in this low homologue region (Figure 4A). Proline residue often serves at the turn or loop of a protein. However, mutation of proline residues to alanine or glycine did not alter the localization of EB1 to the plus end of the microtubule (unpublished data). We then examined whether the conserved and positively charged residues K/R modulate the localization of EB1. To this end, we took the advantage of EB1-PACF to design dimers with a combination of wild-type and mutant EB1 in which the five positively charged K/R residues were mutated to glutamine (Q; EB1KR5Q). In this regard, we can ascertain the respective contribution of each molecule of the engineered EB1 dimer to stable plus-end localization using EB1-PACF (Figure 4B, left panel). We first examined the protein expression of the PACF-fused EB1 proteins with Western blotting (Supplemental Figure 9). Consistent with our demonstration above, precise localization of EB1WT/WT-PACF exhibited a typical comet-like plus end–tracking character (Figure 4B, top right panel). However, expression of either EB1KR5Q/KR5Q-PACF or EB1WT/KR5Q-PACF failed to reach a stable localization to the plus ends, as evidenced by fewer comet-like structures (Figure 4, B and C, and Supplemental Figure 10). Thus the conserved K/R residues in the linker region are essential for the functional EB1 dimers, and their plus-end tracking property is impaired, even when the linker region of one molecule of the EB1 dimer is altered.

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