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 organizati on in spindles assembled in Xenopus egg extracts. Anaphase chromosome movement in these extracts resembled that seen in living vertebrate ce lls. During anaphase chromosomes moved toward the spindle poles (anaph ase A) and the majority reached positions very close to the spindle po les. The average rate of chromosome to pole movement (2.4 mu m/min) wa s 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 u s to observe two novel features of mitosis, First, during anaphase, mu ltiple microtubule organizing centers migrated 40 pm or more away from the spindle poles. Second, in telophase, decondensing chromosomes oft en moved rapidly (7-23 mu m/min) away from the spindle poles toward th e 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, Preventi ng the inactivation of Cdc2/cyclin B complexes by adding nondegradable cyclin B allowed anaphase A to occur at normal velocities, but reduce d the ejection of asters from the spindles, blocked chromosome deconde nsation, 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, demonst rating the role of sister chromatid linkage in maintaining spindle bip olarity.