FIGURE 4:

Microtubule length within pineapples. (A) Assembly reaction run in solution for 1 h, squashed, and imaged immediately for width measurement (40× confocal, tubulin probe only). (B) The same reaction as in A imaged 10 min after squashing, which allows time for assemblies to reorganize. (C) Single microtubules from the same reaction as in A and B after salt dissociation and fixation (60× wide-field imaging). For length comparison, A–C are presented at the same magnification. Note that the longest microtubule in C is similar in length to the widest width in B, but most microtubules in C are shorter. (D) Length histogram for dissociated microtubules from the reaction pictured in A–C. Mean pineapple width, estimated from images like those in A and B, is indicated by the blue arrow. (E) Typical pineapple image at 90 min with NUMA (blue) and Aurora B (red) probes selected for intensity profiling (40× confocal). Each color channel was linearly normalized between 0- and 256-Gy levels over the whole image. The white lines indicate a region of 100 pixels wide selected for an intensity profile parallel to the microtubule axis. (F) Fluorescence intensity profile from the line shown in E. Intensity was averaged over 100 pixels normal to the direction of the linescan to smooth out local variation. Note two NUMA peaks on the outside (blue) and an Aurora B peak on the inside (red). Average tubulin (green) intensity rose sharply just inside the two NUMA peaks and then decreased more gradually toward the center.