Real time observation of anaphase in vitro

We used digital fluorescence microscopy to make real-time observations of anaphase chromosome movement and changes in microtubule organization in spindles assembled in Xenopus egg extracts. Anaphase chromosome movement in these extracts resembled that seen in living vertebrate cells. During anaphase chromosomes moved toward the spindle poles (anaphase A) and the majority reached positions very close to the spindle poles. The average rate of chromosome to pole movement (2.4 microns/min) was similar to earlier measurements of poleward microtubule flux during metaphase. An increase in pole-to-pole distance (anaphase B) occurred in some spindles. The polyploidy of the spindles we examined allowed us to observe two novel features of mitosis. First, during anaphase, multiple microtubule organizing centers migrated 40 microns or more away from the spindle poles. Second, in telophase, decondensing chromosomes often moved rapidly (7-23 microns/min) away from the spindle poles toward the centers of these asters. This telophase chromosome movement suggests that the surface of decondensing chromosomes, and by extension those of intact nuclei, bear minus-end-directed microtubule motors. Preventing the inactivation of Cdc2/cyclin B complexes by adding nondegradable cyclin B allowed anaphase A to occur at normal velocities, but reduced the ejection of asters from the spindles, blocked chromosome decondensation, and inhibited telophase chromosome movement. In the presence of nondegradable cyclin B, chromosome movement to the poles converted bipolar spindles into pairs of independent monopolar spindles, demonstrating the role of sister chromatid linkage in maintaining spindle bipolarity.