Fig. 12.
Quantitative analyses of the time course of nocodazole effects on the amount of MT polymer in the axon. Cultures were treated with 5 μg/ml nocodazole for 0, 10, or 20 min and processed by combined extraction and fixation using procedure 3. The cells were then double-stained for tubulin and tau using staining condition 1. Images were obtained from the resulting cells and subjected to the segmented mask procedure to quantify tubulin and tau staining along the length of the axon. We then computed the total staining intensity of tubulin and tau for the entire axon, and for a 100 μm segment located proximally, and a 100 μm segment located distally (10 or 11 axons were measured for each drug treatment condition). The proximal segment was situated between 50 and 150 μm from the cell body and contains the polymer that has the lowest average content of tau in the axon. The distal segment included the most distal 100 μm of the axon and contains polymer that has a much higher tau content than the polymer in the proximal segment, and includes the axonal polymer with the highest average content of tau. The staining intensity for tubulin and tau of drug-treated axons is expressed as a percentage of the intensity in control axons. These analyses show that the time course of polymer loss is the same in the proximal and distal axonal regions in spite of the severalfold difference in tau content of the polymer in these regions.