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. 2020 Nov 30;11:6131. doi: 10.1038/s41467-020-19914-3

Fig. 9. Chemogenetic activation of DRGs increases the ratio of tyrosinated to total tubulin in the distal axon to improve growth on inhibitory substrates.

Fig. 9

a–f Representative images of the distal-most portion of axons from mCherry+ (a, b) or hM3Dq+ (c, d) DRGs stained for βIII-tubulin (green) and tyrosinated tubulin (red in a, c; standard fire scale pseudo-color in b, d) 24 h after replating and CNO treatment. There was no difference in the fluorescence intensity for βIII-tubulin in the distal-most 50 µm of axons growing from mCherry+ neurons and chemogenetically activated, hM3Dq+ neurons (e). However, the ratio of tyrosinated tubulin to total βIII-tubulin was higher in the axons from CNO-activated, hM3Dq+ DRG neurons (f), indicating an increase of labile microtubules in axons extending from chemogenetically-activated neurons. g, h Cultures of mCherry+ or hM3Dq+ DRGs on ChABC-treated aggrecan spots were treated with CNO and either parthenolide to inhibit tubulin carboxypeptidase or DMSO control. Adding parthenolide to mCherry+ DRGs increased the ratio of tryosinated tubulin to total tubulin in the distal axon end (g) and improved their ability to extend axons across the inhibitory rim of the aggrecan spot. The number of axon crossings from parthenolide-treated, mCherry+ DRGs was similar to activated, hM3Dq+ neurons (h). N = 86 mCherry+ neurons and 87 hM3Dq+ neurons in e, f. N = 87 mCherry++vehicle neurons, 72 hM3Dq++vehicle neurons, 72 mCherry++parthenolide neurons, and 73 hM3Dq++parthenolide neurons in g. N = 16 aggrecan spots (4 coverslips)/group in h. Mean ± SEM. Two-tailed unpaired t-test in f and one-way ANOVA and post-hoc multiple comparisons testing using the two-stage step-up method of Benjamini, Krieger, and Yekutieli in g, h. *p < 0.05 (p = 0.0195 in f; mCherry+vehicle vs. hM3Dq+vehicle p = 0.0480 in h), **p < 0.01 (mCherry+vehicle vs. hM3Dq+vehicle p = 0.0066, mCherry+Vehicle vs. hM3Dq+Parthenolide p = 0.0071 in g; mCherry+Vehicle vs. hM3Dq+parthenolide p = 0.0062 in h), ****p < 0.0001. Scale bar: 10 µm. Source data are provided as a Source Data file.