Fig. 2: Cytoskeletal organization and membrane tension dynamics during EpoB-induced axon regeneration.

a, Colocalization of microtubule (SiR-tubulin) and membrane (CellMask) signals at regenerating axon tips 1 h after axotomy and EpoB treatment. Scale bar, 10 µm. b, Actin organization (SPY555-FastAct) at the tip of axons under control (Ax⁻EpoB⁻) and regenerating (Ax⁺EpoB⁺) conditions. Scale bars, 3 µm. c–e, Membrane tension analysis by FLIM. c, Membrane tension is increased at tips relative to shafts (>15 µm downstream from the tip) 1 h after axotomy (n = 12). d, Tension normalizes at 4 h (n = 14). e, Control neurons show no difference between tip and shaft (n = 20). Data are mean ± s.e.m.; two-tailed Mann–Whitney test. f, Cryo-EM images of regenerating axons. The grid view gives an overview of the location (left; scale bar, 200 µm), with a magnified view showing an axotomy site (right; scale bar, 5 µm). Arrow indicates axonal growth direction and dashed line and scissors indicate the axotomy site. g, Distribution of microtubule lengths in cryo-EM snapshots 1 h after axotomy. Ax⁺EpoB⁺: n = 17, median = 19.1; Ax⁺EpoB⁻: n = 21, median = 0. The centre line shows the median; two-tailed Mann–Whitney test. h, Time course of axonal extension by cryo-EM snapshots, in the presence of EpoB after axotomy. n = 6 (15 min), n = 17 (1 h), n = 6 (3 h) and n = 12 (6 h) axons. Data are mean ± s.e.m. i, High-magnification montage of a regenerating axon 1 h after axotomy with EpoB. Top, cryo-EM montage; dashed line indicates the axotomy site. Scale bar, 400 nm. Bottom, segmentation depicting microtubules (green), membranes (vesicles and endoplasmic reticulum (ER)) (orange) and actin (magenta). Arrow depicts axonal growth direction.

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