Fig. 5.

The effect of nocodazole treatment on the distributions of tyrosinated α-tubulin and acetylated α-tubulin in growth cones and neurites. A–C, Untreated growth cone and neurite. D–F, 330 nm nocodazole-treated growth cone (left) and a segment of its neurite (right) centered 110 μm proximal to the growth cone. A, B, Pseudocolor renderings of anti-tyrosinated α-tubulin staining (A, fluorescein) and anti-acetylated α-tubulin staining (B, Cy3). C, Color overlay of A(green) and B (red). In the growth cone, the tyrosinated tubulin stains out to the tips of the microtubules (A). Acetylated tubulin staining is faint but detectable in the growth cone (B, arrows) and occasionally labels out to the tips brightly (B,arrowhead). By comparing the neurite for which the growth cone has advanced out the field (**) with the distal neurite segment (*), it is evident that tyrosinated tubulin staining is brighter distally than proximally, and the converse is true for the acetylated tubulin. This is also indicated in C, in which the tyrosinated tubulin staining dominates the growth cone and distal neurite, but the acetylated tubulin staining dominates the proximal neurite. D, E, Pseudocolor renderings of anti-tyrosinated α-tubulin staining (D, fluorescein) and anti-acetylated α-tubulin staining (E, Cy3). F, Color overlay of A (green) andB (red). As for the untreated growth cone, the brightness of the tyrosinated tubulin staining is greatest in the growth cone and distal neurite (compareD, * in left to proximal neurite staining inright). In contrast, the brightness of the acetylated tubulin staining is relatively increased in the growth cone and distal neurite (E, *) such that it is as bright as that in the proximal neurite (right). The tyrosinated tubulin staining is typically less attenuated than the acetylated tubulin staining at the tips of microtubules in the growth cones (D,E, arrows), but there are exceptions (D, E, arrowhead). Note the presence of a sinuous microtubule oriented perpendicular to most of the growth cone microtubules over much of its length that is exclusively labeled by the antibody against acetylated tubulin (open arrowhead). In F, there is much more red staining apparent within the nocodazole-treated growth cone compared with the untreated cone inC. The color spectrum to the right shows how pixel values were mapped to colors (black was mapped tomagenta, white to orange). Scale bar, 5 μm.