SUPPLEMENTARY FIGURE LEGENDS
Supplemental Figure 1 Localization of eGFP-positive signals to nodes of Ranvier using an alternative visualization strategy.
We sought to confirm the axonal localization of eGFP-L10a using an alternate visualization method. In these experiments, we used an immunogold protocol to label eGFP-L10a.
(a) Anti-eGFP signal is not present in the nodes of Ranvier in wild-type animals. Corticospinal tracts of Glt25d2::eGFP-L10a mice and wild type controls were incubated with anti-eGFP mAb and visualized with a secondary antibody conjugated to 1 nm gold particles. Gold signal was intensified by incubating in a silver solution for 5 min. Nodes are recognized by characteristic myelin loops (green). Scale bar, 500 nm.
(b) eGFP signal colocalizes to nodes of Ranvier in Glt25d2::eGFP-L10a mice visualized with gold-conjugated secondary antibody. Gold particles (orange arrows) localize beneath the membrane of nodes of Ranvier in Glt25d2::eGFP-L10a mice. Due to the modest penetration of gold particles through tissue, signal intensity with gold visualization is lower than that of immunoperoxidase. eGFP signal, visualized with either immunogold in this panel, or immunoperoxidase (shown in Fig 1), specifically localizes below the membrane at the node of Ranvier, which are recognized by characteristic loops of myelin (green). The detection of eGFP-L10a by two different labeling methodologies helps to rule out the possibility that the nodal labeling is an artifact of the labeling protocol. Scale bar, 500 nm.
Supplemental Figure 2 Localization of eGFP signals in nodes of Ranvier after axonal injury.
(a) Anti-eGFP signal at nodes of Ranvier at different time points after injury. In these experiments, Glt25d2::eGFP-L10a mice were subjected to a dorsal hemisection injury, and the corticospinal tract 500 µm rostral to the site of transection was harvested for immunoelectron microscopy at the indicated time points. At all time points, eGFP signal consistently localizes to nodes of Ranvier. Although there are subtle differences in labeling in the images shown above, there was no consistent increase in the intensity of eGFP-positive labeling at nodes within 500 µm of the injury site at either 24 h or 7 d. The image in the 7 d post-injury section shows a mitochondrion (orange arrow) within the node, which is occasionally observed in either the injured or uninjured sections, and consistent with previous reports that mitochondria can be observed in nodes42. Myelin labeling is shown in green. The node is the area that is not surrounded by myelin. Scale bar, 500 nm.
(b) Quantification of percent positive nodes in the corticospinal tract of injured and uninjured Glt25d2::eGFP-L10a mice. Incidence of positive nodes in injured Glt25d2::eGFP-L10a mice (*P < 0.640, n = 2 animals in each group) as compared with uninjured control. Positive signal was defined as the presence of a 67 nm2 area below the nodal membrane with average signal intensity at least two standard deviations higher than the average signal intensity in wild-type controls. Comparisons were made with a one-way analysis of variance. Data are expressed as mean ± s.e.m.
Supplemental Figure 3 eGFP-L10a in growth cone of a regenerating adult axon.
We sought to determine if ribosomes are found in growth cones after injury. As described in the text, eGFP-L10a-expressing mice were subjected to dorsal hemisection. Spinal cords sections within 500 µm of the injury site were harvested 4 days after injury and subjected to immunoelectron microscopy. Due to their low abundance, growth cones are technically challenging to locate. The growth cone that was identified was emanating from a myelinated axon, consistent with corticospinal tract axons. The growth cone was identified based on its similarity in morphology and composition to previous electromicroscopic descriptions, such as that by Tennyson50. This regenerating growth cone is characteristically enriched in mitochondria and vesicular bodies, and it contains eGFP labeling (orange arrows). Myelin is colored green. Scale bar, 2 µm.
Supplemental Figure 4 Additional examples of eGFP-sACt expression in axons in the corticospinal tract.
In Figure 4C, we showed an example of an axon in the CST which expresses eGFP-sACt. In these experiments, the Sindbis-IRES-eGFP or Sindbis-IRES-eGFP-sACt virus (1 µl) was stereotaxically injected into spinal cords at T7. In each of the animals (n = 3), myelinated axons in the CST were found to be positive for eGFP labeling. Shown are additional examples of Tuj1 positive axons (red) within the CST expressing eGFP immunoreactivity (green) after injection of the Sindbis-IRES virus. Scale bar, 5 µm.