Figure 3.

Labeled anti-TPX2 as a probe for spindle poles and flux. Cycled bipolar spindles were labeled with Alexa 488 anti-TPX2 (top panels in each pair) and rhodamine tubulin at speckle levels (bottom panels in each pair). All kymographs were taken on a line through both poles, with the time and scale bar shown in D. (A) Example of a bipolar spindle. The anti-TPX2 probe accumulates at the poles (p). It also labels puncta and fibrils throughout the spindle, which are partly coaligned with tubulin (e.g., arrows). Faint accumulation of the probe on chromatin can also been seen in this example (ch)aa. Bar, 5 μm. See Supplementary Video M3_1. (B) Kymograph of the spindle shown in A. Top panel, anti-TPX2; bottom panel, tubulin. Anti-TPX2 puncta and tubulin speckles move continuously toward the poles at the same rate, indicated by the diagonal steaks. This is diagnostic of poleward flux. Time and scale bar for B–D shown in D. See Supplementary Video M3_1. (C) Kymograph of a spindle treated with 2 mM AMPPNP. This treatment blocks poleward flux (Sawin and Mitchison, 1991b). Note that both anti-TPX2 puncta and tubulin speckles are static, indicated by vertical lines. In the supplementary video, the anti-TPX2 signal is much more tightly coaligned with tubulin in AMPPNP compared with control. See Supplementary Video M3_2. (D) Kymograph of a spindle assembled in the presence of p50 dynamitin (0.9 mg/ml), a dynactin antagonist that blocks pole assembly but not flux. Note poleward movement of anti-TPX2 puncta and tubulin speckles at the same rate. Note also that anti-TPX2 does not accumulate at the unfocussed poles. The rate of flux is slower in this example than B, but this was not always the case for p50 spindles. We are currently developing better tools to measure flux rate and explore its variability. See Supplementary Video M3_3.