Quantification of endogenous P60 and P80 katanin levels in cell bodies, processes, and tips of processes of cultured hippocampal neurons at different developmental stages. A-D, Neurons in early stage 4. Microtubule staining (β-tubulin) is shown in red, and katanin staining is shown in green. A, B, P60 katanin. C, D, P80 katanin. Arrowheads point to growth cones, which are notably enriched for P60 but not P80. A and C are lower magnification and B and D are higher magnification of the tip regions (growth cone) of axons. The arrows point to branch points, which were also somewhat enriched for P60 but not P80. Quantification of data obtained from various types of processes and various regions of the neuron are shown in E-J. Data (y-axis) are expressed as the ratio of P60 or P80 obtained with each antibody to β-tubulin calculated using AFUs. Absolute levels of P60 and P80 cannot be compared with this approach. Note that the two subunits of katanin do not show identical changes in the various compartments (see Fig. 4). Stages of development [as defined by Dotti et al. (1988)] are shown as roman numerals in these graphs. There is a gradual increase in P80 levels within the cell body as the neuron transitions into early and late stage 4 (1.6-2.1 times compared with stage 1; p < 0.001). Compared with stage 3 axons, the levels of P80 in early and late stage 4 axons only increased by 1.36-1.5 times (p < 0.05). In late dendrites, the levels are approximately triple those in the early immature processes (p < 0.001). With regard to P60, there is a sharp increase in early stage 4 cell bodies, axons (∼2.5 times; p < 0.001), and dendrites (∼2.25 times; p < 0.001) compared with stage 1. The levels of P60 came down in the late stage 4 cell bodies (1.8 times; p < 0.001), axons (1.25 times; p < 0.05), and dendrites (p > 0.05) compared with stage 1. At all developmental stages, there is more P80 at the tips of the processes compared with the processes themselves (1.6-2.5 times; p < 0.01). With regard to P60, there is no significant difference between the tips and the shafts in the immature processes and the stage 3 axons (p > 0.05), but there is a very dramatic enrichment at the tips of both axons and dendrites in early stage 4 (3-6 times; p < 0.0001). Scale bar: A, C, 30 μm; B, D, 50 μm.