Figure 3.

EB1 is acquired at the kinetochore microtubule attachment site in late prometaphase and requires microtubule attachment to remain at kinetochores. (a) PtK1 cell microinjected with Alexa⁵⁹⁴-labeled EB1 during prophase. Images from a time-lapse series of EB1 fluorescence (green) and phase contrast (contrast inverted and red) are shown; time (minutes) is from an arbitrary start point. Before nuclear envelope breakdown and during early prometaphase, no EB1 was seen at kinetochores (t = 0 and 5 min). By late prometaphase (t = 8 min), the first kinetochore (arrow) acquired EB1 fluorescence, and additional kinetochores (t = 19 min) subsequently acquired EB1 (t = 37 and 39 min). (b–d) EB1 requires microtubule attachment to persist at kinetochores. Indirect immunofluorescence of EB1 and CREST antigens in a PtK1 cell treated with nocodazole. EB1 (b), CREST (c) and merged image of EB1 (green) and CREST (red) (d). There is no EB1 at kinetochores. Bar, 10 μm.