Limits...
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.

View Article: PubMed Central - PubMed

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

Show MeSH

Related in: MedlinePlus

TACC3 regulates MT dynamics in X. laevis growth cones and neural crest cells. (A–C) Representative micrographs of EB1-GFP comets in control (A), TACC3 KD (B), and TACC3 OE (C) conditions. See Figure 2 Supplemental Movies S1–S4. Bar, 5 μm. (D–F) Quantification of MT growth track parameters in cultured embryonic neuronal growth cones (GC) after TACC3 manipulation. EB1-GFP localizes to the ends of growing MTs and is thus a marker for MT polymerization. Automated tracking of EB1-GFP comets calculate MT growth-track velocity (D), MT growth-track lifetime (E), and MT growth-track length (F). Examples of actual mean values for a single experiment: GC MT velocity, control, 6.4 μm/min; TACC3 KD, 5.7 μm/min; TACC3 OE, 7.1 μm/min. GC MT lifetime, control, 12.6 s; KD, 13.3 s; OE, 14.1 s. GC MT length, control, 1.4 μm; KD, 1.3 μm; OE, 1.7 μm. NCC MT velocity, control, 7.5 μm/min; KD, 6.0 μm/min; OE, 8.5 μm/min. NCC MT lifetime, control. 11.4 s; KD. 13.0 s; OE, 12.1 s. NCC MT length, control, 1.3 μm; KD, 1.3 μm; OE, 1.8 μm. For each independent experiment (six were performed in total), measurements of MT parameters were normalized to their respective experimental control means due to the significant day-to-day fluctuations in control MT dynamics (in part, due to room temperature changes). Control data represent the means of 44 growth cones, representing a total of 1228 analyzed tracks; TACC3 KD represents 49 growth cones with 964 tracks; TACC3 OE represents 24 growth cones with 524 tracks. (G–I) Quantification of MT growth-track parameters in cultured embryonic neural crest cells. Control data represent the means of 20 neural crest cells, representing a total of 2876 analyzed tracks; TACC3 KD represents 13 cells with 1313 tracks; TACC3 OE represents 10 cells with 1386 tracks. Box-and-whisker plots indicate the mean (diamond), median, extrema, and quartiles. An unpaired t test was performed to assess significance between conditions. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4214782&req=5

Figure 2: TACC3 regulates MT dynamics in X. laevis growth cones and neural crest cells. (A–C) Representative micrographs of EB1-GFP comets in control (A), TACC3 KD (B), and TACC3 OE (C) conditions. See Figure 2 Supplemental Movies S1–S4. Bar, 5 μm. (D–F) Quantification of MT growth track parameters in cultured embryonic neuronal growth cones (GC) after TACC3 manipulation. EB1-GFP localizes to the ends of growing MTs and is thus a marker for MT polymerization. Automated tracking of EB1-GFP comets calculate MT growth-track velocity (D), MT growth-track lifetime (E), and MT growth-track length (F). Examples of actual mean values for a single experiment: GC MT velocity, control, 6.4 μm/min; TACC3 KD, 5.7 μm/min; TACC3 OE, 7.1 μm/min. GC MT lifetime, control, 12.6 s; KD, 13.3 s; OE, 14.1 s. GC MT length, control, 1.4 μm; KD, 1.3 μm; OE, 1.7 μm. NCC MT velocity, control, 7.5 μm/min; KD, 6.0 μm/min; OE, 8.5 μm/min. NCC MT lifetime, control. 11.4 s; KD. 13.0 s; OE, 12.1 s. NCC MT length, control, 1.3 μm; KD, 1.3 μm; OE, 1.8 μm. For each independent experiment (six were performed in total), measurements of MT parameters were normalized to their respective experimental control means due to the significant day-to-day fluctuations in control MT dynamics (in part, due to room temperature changes). Control data represent the means of 44 growth cones, representing a total of 1228 analyzed tracks; TACC3 KD represents 49 growth cones with 964 tracks; TACC3 OE represents 24 growth cones with 524 tracks. (G–I) Quantification of MT growth-track parameters in cultured embryonic neural crest cells. Control data represent the means of 20 neural crest cells, representing a total of 2876 analyzed tracks; TACC3 KD represents 13 cells with 1313 tracks; TACC3 OE represents 10 cells with 1386 tracks. Box-and-whisker plots indicate the mean (diamond), median, extrema, and quartiles. An unpaired t test was performed to assess significance between conditions. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Mentions: Axon outgrowth is regulated in large part by MT dynamics within the neuronal growth cone, which is the dynamic structure at the tip of the growing axon (Tanaka et al., 1995; Lowery and Van Vactor, 2009). Thus we sought to examine whether MT plus end dynamics were specifically disrupted within growth cones after TACC3 mani­pulation. To test this, we acquired high-resolution live images of tagged end-binding protein 1 (EB1), which binds all growing MT plus ends (Stepanova et al., 2003), and we then quantified parameters of MT polymerization dynamics using the Matlab-based open-source software plusTipTracker (Applegate et al., 2011). This software program has been validated for accurate tracking of EB1-GFP comets in X. laevis growth cones, using identical imaging conditions to those used in the present study (Stout et al., 2014). EB1-GFP comet number and morphology were similar in control, TACC3 knockdown (KD), and TACC3 OE growth cones (Figure 2, A–C, and Figure 2 Supplemental Movies 1–4). However, compared with controls, EB1-GFP comet velocity was reduced by 11% upon TACC3 KD and increased by 11% upon TACC3 OE (Figure 2D). MT growth-track lifetime (which measures the number of seconds of MT polymerization in a given growth track before pausing or catastrophe) was not affected by TACC3 manipulation (Figure 2E). Conversely, MT growth-track length (which measures the distance of persistent MT polymerization growth before pausing or catastrophe) was significantly decreased in KD and increased in OE. TACC3 KD led to a 14% reduction in MT growth-track length, whereas TACC3 OE led to an 18% increase in MT growth-track length (Figure 2F). To summarize, although the time persistence of MT polymerization is independent of TACC3, the velocity and length of MT forward progression trend with the level of TACC3, suggesting that TACC3 promotes more efficient MT polymerization.


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 regulates MT dynamics in X. laevis growth cones and neural crest cells. (A–C) Representative micrographs of EB1-GFP comets in control (A), TACC3 KD (B), and TACC3 OE (C) conditions. See Figure 2 Supplemental Movies S1–S4. Bar, 5 μm. (D–F) Quantification of MT growth track parameters in cultured embryonic neuronal growth cones (GC) after TACC3 manipulation. EB1-GFP localizes to the ends of growing MTs and is thus a marker for MT polymerization. Automated tracking of EB1-GFP comets calculate MT growth-track velocity (D), MT growth-track lifetime (E), and MT growth-track length (F). Examples of actual mean values for a single experiment: GC MT velocity, control, 6.4 μm/min; TACC3 KD, 5.7 μm/min; TACC3 OE, 7.1 μm/min. GC MT lifetime, control, 12.6 s; KD, 13.3 s; OE, 14.1 s. GC MT length, control, 1.4 μm; KD, 1.3 μm; OE, 1.7 μm. NCC MT velocity, control, 7.5 μm/min; KD, 6.0 μm/min; OE, 8.5 μm/min. NCC MT lifetime, control. 11.4 s; KD. 13.0 s; OE, 12.1 s. NCC MT length, control, 1.3 μm; KD, 1.3 μm; OE, 1.8 μm. For each independent experiment (six were performed in total), measurements of MT parameters were normalized to their respective experimental control means due to the significant day-to-day fluctuations in control MT dynamics (in part, due to room temperature changes). Control data represent the means of 44 growth cones, representing a total of 1228 analyzed tracks; TACC3 KD represents 49 growth cones with 964 tracks; TACC3 OE represents 24 growth cones with 524 tracks. (G–I) Quantification of MT growth-track parameters in cultured embryonic neural crest cells. Control data represent the means of 20 neural crest cells, representing a total of 2876 analyzed tracks; TACC3 KD represents 13 cells with 1313 tracks; TACC3 OE represents 10 cells with 1386 tracks. Box-and-whisker plots indicate the mean (diamond), median, extrema, and quartiles. An unpaired t test was performed to assess significance between conditions. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4214782&req=5

Figure 2: TACC3 regulates MT dynamics in X. laevis growth cones and neural crest cells. (A–C) Representative micrographs of EB1-GFP comets in control (A), TACC3 KD (B), and TACC3 OE (C) conditions. See Figure 2 Supplemental Movies S1–S4. Bar, 5 μm. (D–F) Quantification of MT growth track parameters in cultured embryonic neuronal growth cones (GC) after TACC3 manipulation. EB1-GFP localizes to the ends of growing MTs and is thus a marker for MT polymerization. Automated tracking of EB1-GFP comets calculate MT growth-track velocity (D), MT growth-track lifetime (E), and MT growth-track length (F). Examples of actual mean values for a single experiment: GC MT velocity, control, 6.4 μm/min; TACC3 KD, 5.7 μm/min; TACC3 OE, 7.1 μm/min. GC MT lifetime, control, 12.6 s; KD, 13.3 s; OE, 14.1 s. GC MT length, control, 1.4 μm; KD, 1.3 μm; OE, 1.7 μm. NCC MT velocity, control, 7.5 μm/min; KD, 6.0 μm/min; OE, 8.5 μm/min. NCC MT lifetime, control. 11.4 s; KD. 13.0 s; OE, 12.1 s. NCC MT length, control, 1.3 μm; KD, 1.3 μm; OE, 1.8 μm. For each independent experiment (six were performed in total), measurements of MT parameters were normalized to their respective experimental control means due to the significant day-to-day fluctuations in control MT dynamics (in part, due to room temperature changes). Control data represent the means of 44 growth cones, representing a total of 1228 analyzed tracks; TACC3 KD represents 49 growth cones with 964 tracks; TACC3 OE represents 24 growth cones with 524 tracks. (G–I) Quantification of MT growth-track parameters in cultured embryonic neural crest cells. Control data represent the means of 20 neural crest cells, representing a total of 2876 analyzed tracks; TACC3 KD represents 13 cells with 1313 tracks; TACC3 OE represents 10 cells with 1386 tracks. Box-and-whisker plots indicate the mean (diamond), median, extrema, and quartiles. An unpaired t test was performed to assess significance between conditions. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.
Mentions: Axon outgrowth is regulated in large part by MT dynamics within the neuronal growth cone, which is the dynamic structure at the tip of the growing axon (Tanaka et al., 1995; Lowery and Van Vactor, 2009). Thus we sought to examine whether MT plus end dynamics were specifically disrupted within growth cones after TACC3 mani­pulation. To test this, we acquired high-resolution live images of tagged end-binding protein 1 (EB1), which binds all growing MT plus ends (Stepanova et al., 2003), and we then quantified parameters of MT polymerization dynamics using the Matlab-based open-source software plusTipTracker (Applegate et al., 2011). This software program has been validated for accurate tracking of EB1-GFP comets in X. laevis growth cones, using identical imaging conditions to those used in the present study (Stout et al., 2014). EB1-GFP comet number and morphology were similar in control, TACC3 knockdown (KD), and TACC3 OE growth cones (Figure 2, A–C, and Figure 2 Supplemental Movies 1–4). However, compared with controls, EB1-GFP comet velocity was reduced by 11% upon TACC3 KD and increased by 11% upon TACC3 OE (Figure 2D). MT growth-track lifetime (which measures the number of seconds of MT polymerization in a given growth track before pausing or catastrophe) was not affected by TACC3 manipulation (Figure 2E). Conversely, MT growth-track length (which measures the distance of persistent MT polymerization growth before pausing or catastrophe) was significantly decreased in KD and increased in OE. TACC3 KD led to a 14% reduction in MT growth-track length, whereas TACC3 OE led to an 18% increase in MT growth-track length (Figure 2F). To summarize, although the time persistence of MT polymerization is independent of TACC3, the velocity and length of MT forward progression trend with the level of TACC3, suggesting that TACC3 promotes more efficient MT polymerization.

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