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FIGURE 1:. TACC2 can act as a +TIP that localizes in front of EB1 in many embryonic cell types. (A–J) Representative images of mKate2-tubulin, GFP-TACC2, and merged in cultured embryonic mesenchymal cells derived from neural tube (A–C) and neuronal growth cone (F–H). See Supplemental Movies S1 and S2. (D, I) Magnified time-lapse montage of the boxed regions in C and H. The time interval between frames is ∼2–3 s. (E, J) Fluorescence intensity profile of GFP-TACC2. (K–T) Images of mKate2-EB1, GFP-TACC2, and merged in cultured embryonic mesenchymal cell (K–M) and neuronal growth cone (P–R). See Supplemental Movie S3. (N, S) Magnified time-lapse montage of the boxed region in M and R. For N, the green channel was translated left by ∼0.2 μm to account for the different acquisition times of the red and green channels (MT plus-end velocity ∼0.24 μm/s in this series, and the green channel was imaged 600 ms after the red channel). This allows for a more accurate visualization of +TIP colocalization. For S, the green channel was translated left by ∼0.1 μm (plus end was moving at 0.085 μm/s, and the green channel was imaged 600 ms after the red channel). For all fluorescence intensity profiles, ∼20 MTs for each condition were quantified by intensity line scans. The MT plus end is toward the right, and error bars represent SDs. Scale bars, 5 μm (montages), 10 μm (all others).
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FIGURE 2:. TACC2 localizes to the plus end through its C-terminal domain and overlaps with both TACC1 and TACC3. (A–C) Representative images of mKate2-TACC3, GFP-TACC2, and merged in cultured neuronal growth cones. (D) Fluorescence intensity profiles of GFP-TACC2 and mKate2-TACC3. (E–G) mKate2-TACC1, GFP-TACC2, and merge with GFP-TACC2 fluorescence intensity plot profile (H). (I–K) Images of mKate2-EB1, GFP-TACC2 N-term (aa 1–540), and merged with the plot of fluorescence intensity profiles (L). (M–O) Images of mKate2-EB1, GFP-TACC2 C-term (aa 923-1168), and merged with the plot showing their fluorescence intensity profiles (P). Arrow in N points to lattice binding of TACC protein. Time interval between all frames is ∼2 s. For D, the green plot profile was translated ∼0.2 μm left (MT plus-end velocity ∼0.124 μm/s, green channel imaged 1.5 s after red) to accurately represent relative localization. For H, the green profile was translated left based on the velocity of each comet measured to account for difference in acquisition time points between channels (green channel imaged 1.5 s after red). In P, the green profile was translated 0.1 μm (MT plus-end velocity ∼0.148 μm/s, red channel imaged 0.96 s after green). For all fluorescence intensity profiles, ∼10 MTs for each condition were quantified by intensity line scans, and SDs are shown in error bars. MT plus end is at the right. Scale bars, 10 μm.
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FIGURE 3:. TACC2 can regulate microtubule plus-end dynamics through its C-terminal domain in a cell type–specific manner. All raw data of automated tracking of mKate2-EB1 comets were normalized to the same-day control means, and all reported values are relative ones combined over at least three separate experiments. (A–C) Quantification of mean values of MT parameters (velocity, lifetime, length) upon TACC2 OE in embryonic mesenchymal cells. (D–F) MT parameters in growth cones (GC). (G–I) MT parameters in mesenchymal cells on overexpression of C- or N-terminal-truncated TACC2. (J–L) Quantification of MT parameters in mesenchymal cells when multiple combinations of TACC family members were expressed. Bars on dot plots show mean and SEM. To assess statistical significance, ANOVA tests were first performed (for multiple conditions), followed by an unpaired t test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s., not significant.
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FIGURE 4:. TACC family members are differentially expressed during embryonic development. Cell type–specific expression of TACC mRNAs shown by whole-mount in situ hybridization using full-length antisense probes to TACC1, TACC2, and TACC3. TACC1 is expressed uniformly at the late blastula stage (A), in somites, neural tube, and hindbrain at stage 23 (B), and in somites, eye, and anterior neural tube at stage 34 (C, D). TACC2 expression is similar to that of TACC1 at the blastula stage (E) and also increases in a tissue-specific manner after neurulation, apparent in the neural tube, early brain, and presomitic mesoderm at stage 23 (F). At stage 34, TACC2 is expressed in the somitic mesoderm, the ocular lens, and the anterior neural tube (G, H). Arrows in D and H indicate somitic mesoderm. TACC3 expression resembles that of TACC1 and TACC2 at the blastula stage (I), but its expression becomes more neural specific by stage 23, showing expression along the entire length of the neural tube, although strongest anteriorly near the brain (J). At stage 34, TACC3 is strongly expressed in the pharyngeal arches (L, arrows), otic vesicle, and trunk neural crest (K, L). Neural tube and trunk neural crest are indicated by the box in L. Scale bars, 150 μm.
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tacc2 (transforming, acidic coiled-coil containing protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior right, dorsal up.
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tacc1 (transforming, acidic coiled-coil containing protein 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior right, dorsal up.
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FIGURE 1:. TACC2 can act as a +TIP that localizes in front of EB1 in many embryonic cell types. (A–J) Representative images of mKate2-tubulin, GFP-TACC2, and merged in cultured embryonic mesenchymal cells derived from neural tube (A–C) and neuronal growth cone (F–H). See Supplemental Movies S1 and S2. (D, I) Magnified time-lapse montage of the boxed regions in C and H. The time interval between frames is ∼2–3 s. (E, J) Fluorescence intensity profile of GFP-TACC2. (K–T) Images of mKate2-EB1, GFP-TACC2, and merged in cultured embryonic mesenchymal cell (K–M) and neuronal growth cone (P–R). See Supplemental Movie S3. (N, S) Magnified time-lapse montage of the boxed region in M and R. For N, the green channel was translated left by ∼0.2 μm to account for the different acquisition times of the red and green channels (MT plus-end velocity ∼0.24 μm/s in this series, and the green channel was imaged 600 ms after the red channel). This allows for a more accurate visualization of +TIP colocalization. For S, the green channel was translated left by ∼0.1 μm (plus end was moving at 0.085 μm/s, and the green channel was imaged 600 ms after the red channel). For all fluorescence intensity profiles, ∼20 MTs for each condition were quantified by intensity line scans. The MT plus end is toward the right, and error bars represent SDs. Scale bars, 5 μm (montages), 10 μm (all others).
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FIGURE 2:. TACC2 localizes to the plus end through its C-terminal domain and overlaps with both TACC1 and TACC3. (A–C) Representative images of mKate2-TACC3, GFP-TACC2, and merged in cultured neuronal growth cones. (D) Fluorescence intensity profiles of GFP-TACC2 and mKate2-TACC3. (E–G) mKate2-TACC1, GFP-TACC2, and merge with GFP-TACC2 fluorescence intensity plot profile (H). (I–K) Images of mKate2-EB1, GFP-TACC2 N-term (aa 1–540), and merged with the plot of fluorescence intensity profiles (L). (M–O) Images of mKate2-EB1, GFP-TACC2 C-term (aa 923-1168), and merged with the plot showing their fluorescence intensity profiles (P). Arrow in N points to lattice binding of TACC protein. Time interval between all frames is ∼2 s. For D, the green plot profile was translated ∼0.2 μm left (MT plus-end velocity ∼0.124 μm/s, green channel imaged 1.5 s after red) to accurately represent relative localization. For H, the green profile was translated left based on the velocity of each comet measured to account for difference in acquisition time points between channels (green channel imaged 1.5 s after red). In P, the green profile was translated 0.1 μm (MT plus-end velocity ∼0.148 μm/s, red channel imaged 0.96 s after green). For all fluorescence intensity profiles, ∼10 MTs for each condition were quantified by intensity line scans, and SDs are shown in error bars. MT plus end is at the right. Scale bars, 10 μm.
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FIGURE 3:. TACC2 can regulate microtubule plus-end dynamics through its C-terminal domain in a cell type–specific manner. All raw data of automated tracking of mKate2-EB1 comets were normalized to the same-day control means, and all reported values are relative ones combined over at least three separate experiments. (A–C) Quantification of mean values of MT parameters (velocity, lifetime, length) upon TACC2 OE in embryonic mesenchymal cells. (D–F) MT parameters in growth cones (GC). (G–I) MT parameters in mesenchymal cells on overexpression of C- or N-terminal-truncated TACC2. (J–L) Quantification of MT parameters in mesenchymal cells when multiple combinations of TACC family members were expressed. Bars on dot plots show mean and SEM. To assess statistical significance, ANOVA tests were first performed (for multiple conditions), followed by an unpaired t test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s., not significant.
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FIGURE 4:. TACC family members are differentially expressed during embryonic development. Cell type–specific expression of TACC mRNAs shown by whole-mount in situ hybridization using full-length antisense probes to TACC1, TACC2, and TACC3. TACC1 is expressed uniformly at the late blastula stage (A), in somites, neural tube, and hindbrain at stage 23 (B), and in somites, eye, and anterior neural tube at stage 34 (C, D). TACC2 expression is similar to that of TACC1 at the blastula stage (E) and also increases in a tissue-specific manner after neurulation, apparent in the neural tube, early brain, and presomitic mesoderm at stage 23 (F). At stage 34, TACC2 is expressed in the somitic mesoderm, the ocular lens, and the anterior neural tube (G, H). Arrows in D and H indicate somitic mesoderm. TACC3 expression resembles that of TACC1 and TACC2 at the blastula stage (I), but its expression becomes more neural specific by stage 23, showing expression along the entire length of the neural tube, although strongest anteriorly near the brain (J). At stage 34, TACC3 is strongly expressed in the pharyngeal arches (L, arrows), otic vesicle, and trunk neural crest (K, L). Neural tube and trunk neural crest are indicated by the box in L. Scale bars, 150 μm.
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