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BACKGROUND: Formation of precise neuronal connections requires proper axon guidance. Microtubules (MTs) of the growth cone provide a critical driving force during navigation of the growing ends of axons. Pioneer MTs and their plus-end tracking proteins (+TIPs) are thought to play integrative roles during this navigation. TACC3 is a + TIP that we have previously implicated in regulating MT dynamics within axons. However, the role of TACC3 in axon guidance has not been previously explored.
RESULTS: Here, we show that TACC3 is required to promote persistent axon outgrowth and prevent spontaneous axon retractions in embryonic Xenopus laevis neurons. We also show that overexpressing TACC3 can counteract the depolymerizing effect of low doses of nocodazole, and that TACC3 interacts with MT polymerase XMAP215 to promote axon outgrowth. Moreover, we demonstrate that manipulation of TACC3 levels interferes with the growth cone response to the axon guidance cue Slit2 ex vivo, and that ablation of TACC3 causes pathfinding defects in axons of developing spinal neurons in vivo.
CONCLUSION: Together, our results suggest that by mediating MT dynamics, the + TIP TACC3 is involved in axon outgrowth and pathfinding decisions of neurons during embryonic development.
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???displayArticle.pmcLink???PMC5312526 ???displayArticle.link???Neural Dev ???displayArticle.grants???[+]
Fig. 1. TACC3 promotes axon outgrowth velocity and prevents spontaneous axon retractions. a, Axon outgrowth velocity is significantly decreased in TACC3-depleted axons by 27% (n = 56) and in TACC3 OE, to a lesser extent, by 11% (n = 106) compared to control (GFP only) conditions (n = 58). b, Retraction rate increased 5 fold in TACC3 KD (n = 107) and decreased 0.6 fold in TACC3 OE (n = 155) in comparison to their corresponding non injected (n = 95) and GFP injected (n = 180) controls respectively. c, d, MT growth velocity (DMSO, n = 9, KHS-101, n = 9) (c) and axon outgrowth length (DMSO, n = 8, KHS-101, n = 12) (d) are significantly reduced by 28 and 26% respectively after acute depletion of TACC3 by the inhibitor KHS101. e, Schematic representation of GFP-tagged TACC3 full-length and deletion constructs, along with plus-end tracking ability (denoted by “+â€) and impact on axon outgrowth length. f, Quantification of axon outgrowth length in cultured neural explants of GFP injected control (n = 997), full-length GFP-TACC3 (1-931aa) (n = 787), GFP-TACC3-δN (133–931) (n = 613), GFP-TACC3- δδN (363–931) (n = 563) and GFP-δTACC domain (1-635aa) (n = 764). *p < 0.05, **p < 0.01, ***p < 0.001. ns not significant. n = axon/growth cone number
Fig. 2. TACC3 antagonizes Nocodazole-induced MT depolymerization but does not affect MT stability. a-c, Quantification of the MT dynamics shows significant reduction in MT growth speed (a), MT lifetime (b) and MT growth length (c) in control (n = 22) and TACC3 OE (n = 21) growth cones in response to 50 pM Nocodazole before and 5 min after drug treatment. However, the effect of Nocodazole on TACC3 OE growth cones is dampened compared to controls. a’-c’, Although not significant, reduction in MT growth speed (a’) is more prominent in control growth cones compared to TACC3 OE growth cones while the reduction in both lifetime (b’) and length (c’) in control growth cones are significantly higher than the TACC3 OE growth cones (d) Representative growth cone images of control, TACC3 KD and TACC3 OE, immunostained for tyrosinated tubulin (red) and detyrosinated tubulin (green) to label dynamic versus stable MTs, respectively. e-f, Quantification of the fluorescence intensity of imaging data in G, with TACC3 KD (n = 38) (e) and TACC3 OE (n = 129) (f) growth cones showing no significant changes in dynamic/stable MTs compared to corresponding control growth cones (n = 37 and n =143, respectively). *p < 0.05, **p < 0.01, ***p < 0.001. ns not significant. n = growth cone number. Scale bar, 2 μm
Fig. 3. TACC3 and XMAP215 interacts to promote axon outgrowth. a, b, Combinatorial reduction or elevation of TACC3 and XMAP215 levels reveals synergistics in axon outgrowth. Knocking down both TACC3 and XMAP215 (n = 312) showed significant reduction in axon length in comparison to control (n = 219), TACC3 KD (n = 487) and XMAP21 KD alone (n = 312) (a). Overexpression of both (n = 654) showed significant increase in axon length in comparison to control (n = 1288) and TACC3 OE (n = 1585), while double overexpression had no additive effect in comparison to XMAP215 OE (n = 1227) (b). c, d, Reduced axon outgrowth in TACC3 KD (n = 289) (c) or XMAP215 KD (n = 299) (d) neurons is rescued by the overexpression of XMAP215 (n = 313) or TACC3 (n = 397), respectively. *p < 0.05, **p < 0.01, ***p < 0.001. ns not significant. n = axon number
Fig. 4. TACC3 affects axon guidance in vivo and ex vivo. a, b, confocal images of laterally-viewed whole-mount Xenopus spinal cord fluorescently labeled for acetylated tubulin, showing peripheral axon outgrowth in control (a) and TACC3 KD (b) embryos at 2 dpf. c, Quantitation of the embryos with motor neuron guidance defects (n = 5 embryos). d, Representative neural tube growth cone images of control and TACC3 OE, before and after addition of 400 ng/ml Slit2. e, Quantification of the percentage of the growth cone collapse events in control (n = 48) and TACC3 OE (n = 82) growth cones show significant reduction in growth cone collapse in TACC3 overexpressing growth cones. f, Cartoon model for the role of TACC3 at MT plus ends during axon outgrowth and guidance. Microtubule (blue) plus-ends decorated by TACC3 (green) promotes axon outgrowth, reduces axon retraction, dampens nocodazole induced reduction in MT dynamics parameters, rescues XMAP215 KD induced axon length reduction and opposes repellent guidance signals effect. *p < 0.05, **p < 0.01, ***p < 0.001. ns not significant. n = growth cone number. Scale bar, 50 μm and 5 μm
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