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TACC3 is a microtubule plus end-tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types.

Nwagbara BU, Faris AE, Bearce EA, Erdogan B, Ebbert PT, Evans MF, Rutherford EL, Enzenbacher TB, Lowery LA - Mol. Biol. Cell (2014)

Bottom Line: Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215.TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends.Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics.

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Affiliation: Department of Biology, Boston College, Chestnut Hill, MA 02467.

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TACC3 and XMAP215 levels affect each other's protein stability and localization to MT plus ends. (A, B) Representative Western blots showing levels of TACC3 and XMAP215 after TACC3 KD (A) or overexpression (B). The graphs in A′ and B′ are compilations of seven and six individual Western blot experiments, respectively. (C, D) Representative Western blots showing levels of XMAP215 and TACC3 after XMAP215 KD (C) or overexpression (D). The graphs in C′ and C′′ are from six Western blot experiments, and D′ and D′′ are from three experiments. Bars in graphs of Western densitometry denote SE. A Kruskal–Wallis test was performed to assess significance of differences in Western blot densitometry. *p < 0.05. (E) Quantification of fluorescence intensity levels of XMAP215-GFP on MT plus ends in control, TACC3 KD, and TACC3 OE conditions, normalized to cytoplasmic levels. Data represent analysis of ∼100 individual MTs from numerous growth cones for each condition. (F) Quantification of fluorescence intensity levels of GFP-TACC3 on MT plus ends in control and XMAP215 KD conditions, normalized to cytoplasmic levels. Data represent analysis of 149 and 151 individual MTs from numerous growth cones per condition. Box-and-whisker plots indicate the mean (diamond), median, extrema, and quartiles. An unpaired t test was performed to assess significance between conditions. **p < 0.01, ***p < 0.001; ns, not significant.
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Figure 7: TACC3 and XMAP215 levels affect each other's protein stability and localization to MT plus ends. (A, B) Representative Western blots showing levels of TACC3 and XMAP215 after TACC3 KD (A) or overexpression (B). The graphs in A′ and B′ are compilations of seven and six individual Western blot experiments, respectively. (C, D) Representative Western blots showing levels of XMAP215 and TACC3 after XMAP215 KD (C) or overexpression (D). The graphs in C′ and C′′ are from six Western blot experiments, and D′ and D′′ are from three experiments. Bars in graphs of Western densitometry denote SE. A Kruskal–Wallis test was performed to assess significance of differences in Western blot densitometry. *p < 0.05. (E) Quantification of fluorescence intensity levels of XMAP215-GFP on MT plus ends in control, TACC3 KD, and TACC3 OE conditions, normalized to cytoplasmic levels. Data represent analysis of ∼100 individual MTs from numerous growth cones for each condition. (F) Quantification of fluorescence intensity levels of GFP-TACC3 on MT plus ends in control and XMAP215 KD conditions, normalized to cytoplasmic levels. Data represent analysis of 149 and 151 individual MTs from numerous growth cones per condition. Box-and-whisker plots indicate the mean (diamond), median, extrema, and quartiles. An unpaired t test was performed to assess significance between conditions. **p < 0.01, ***p < 0.001; ns, not significant.

Mentions: First, we determined that manipulating overall TACC3 protein levels had a corresponding effect on XMAP215 protein levels in whole embryo lysates. As TACC3 levels were reduced, XMAP215 levels decreased (Figure 7A), whereas adding back TACC3 mRNA to the TACC3 KD rescued XMAP215 levels (Figure 7A). Consistently, overexpressing TACC3 led to increased levels of XMAP215 (Figure 7B). Furthermore, TACC3 levels appeared depend on XMAP215 as well, because reducing XMAP215 led to a reduction in TACC3 protein (Figure 7C). Of note, overexpressing TACC3 in the XMAP215 KD background partially rescued the reduction in XMAP215 levels (Figure 7C′), consistent with a function for TACC3 in stabilizing XMAP215 protein. Alternatively, because TACC3 is a known translation regulator (Stebbins-Boaz et al., 1999), TACC3 could be increasing XMAP215 levels by increasing its protein synthesis. However, increased XMAP215 levels also had a positive effect on TACC3 levels (Figure 7D).


TACC3 is a microtubule plus end-tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types.

Nwagbara BU, Faris AE, Bearce EA, Erdogan B, Ebbert PT, Evans MF, Rutherford EL, Enzenbacher TB, Lowery LA - Mol. Biol. Cell (2014)

TACC3 and XMAP215 levels affect each other's protein stability and localization to MT plus ends. (A, B) Representative Western blots showing levels of TACC3 and XMAP215 after TACC3 KD (A) or overexpression (B). The graphs in A′ and B′ are compilations of seven and six individual Western blot experiments, respectively. (C, D) Representative Western blots showing levels of XMAP215 and TACC3 after XMAP215 KD (C) or overexpression (D). The graphs in C′ and C′′ are from six Western blot experiments, and D′ and D′′ are from three experiments. Bars in graphs of Western densitometry denote SE. A Kruskal–Wallis test was performed to assess significance of differences in Western blot densitometry. *p < 0.05. (E) Quantification of fluorescence intensity levels of XMAP215-GFP on MT plus ends in control, TACC3 KD, and TACC3 OE conditions, normalized to cytoplasmic levels. Data represent analysis of ∼100 individual MTs from numerous growth cones for each condition. (F) Quantification of fluorescence intensity levels of GFP-TACC3 on MT plus ends in control and XMAP215 KD conditions, normalized to cytoplasmic levels. Data represent analysis of 149 and 151 individual MTs from numerous growth cones per condition. Box-and-whisker plots indicate the mean (diamond), median, extrema, and quartiles. An unpaired t test was performed to assess significance between conditions. **p < 0.01, ***p < 0.001; ns, not significant.
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Figure 7: TACC3 and XMAP215 levels affect each other's protein stability and localization to MT plus ends. (A, B) Representative Western blots showing levels of TACC3 and XMAP215 after TACC3 KD (A) or overexpression (B). The graphs in A′ and B′ are compilations of seven and six individual Western blot experiments, respectively. (C, D) Representative Western blots showing levels of XMAP215 and TACC3 after XMAP215 KD (C) or overexpression (D). The graphs in C′ and C′′ are from six Western blot experiments, and D′ and D′′ are from three experiments. Bars in graphs of Western densitometry denote SE. A Kruskal–Wallis test was performed to assess significance of differences in Western blot densitometry. *p < 0.05. (E) Quantification of fluorescence intensity levels of XMAP215-GFP on MT plus ends in control, TACC3 KD, and TACC3 OE conditions, normalized to cytoplasmic levels. Data represent analysis of ∼100 individual MTs from numerous growth cones for each condition. (F) Quantification of fluorescence intensity levels of GFP-TACC3 on MT plus ends in control and XMAP215 KD conditions, normalized to cytoplasmic levels. Data represent analysis of 149 and 151 individual MTs from numerous growth cones per condition. Box-and-whisker plots indicate the mean (diamond), median, extrema, and quartiles. An unpaired t test was performed to assess significance between conditions. **p < 0.01, ***p < 0.001; ns, not significant.
Mentions: First, we determined that manipulating overall TACC3 protein levels had a corresponding effect on XMAP215 protein levels in whole embryo lysates. As TACC3 levels were reduced, XMAP215 levels decreased (Figure 7A), whereas adding back TACC3 mRNA to the TACC3 KD rescued XMAP215 levels (Figure 7A). Consistently, overexpressing TACC3 led to increased levels of XMAP215 (Figure 7B). Furthermore, TACC3 levels appeared depend on XMAP215 as well, because reducing XMAP215 led to a reduction in TACC3 protein (Figure 7C). Of note, overexpressing TACC3 in the XMAP215 KD background partially rescued the reduction in XMAP215 levels (Figure 7C′), consistent with a function for TACC3 in stabilizing XMAP215 protein. Alternatively, because TACC3 is a known translation regulator (Stebbins-Boaz et al., 1999), TACC3 could be increasing XMAP215 levels by increasing its protein synthesis. However, increased XMAP215 levels also had a positive effect on TACC3 levels (Figure 7D).

Bottom Line: Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215.TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends.Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Boston College, Chestnut Hill, MA 02467.

Show MeSH
Related in: MedlinePlus