TACC3 is a microtubule plus end-tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types.
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.
Affiliation: Department of Biology, Boston College, Chestnut Hill, MA 02467.Show MeSH
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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.
Affiliation: Department of Biology, Boston College, Chestnut Hill, MA 02467.