Analysis of anaphase in <i>Xenopus</i> egg extracts
In mitosis, chromosomes are segregated during anaphase through a combination of chromosome-to-spindle-pole movement (anaphase A) and spindle pole separation (anaphase B). The roles of spindle components in anaphase processes have not been comprehensively studied. In Xenopus egg extracts, spindles can be assembled lacking centrosomes, kinetochores and even chromosomes. Using these reductionist spindle variants, we can observe which components are required for anaphase A and B. In addition, the localization of specific spindle proteins and the effects of inhibiting them during anaphase can be evaluated.
We analyzed the ability of spindle variants to complete anaphase processes in Xenopus egg extracts. Spindles assembled around chromatin beads, lacking kinetochores and centrosomes, could not undergo anaphase A, but could complete anaphase B pole separation. RanGTP-induced spindles, lacking kinetochores, centrosomes and chromatin as well, are not competent to perform either anaphase A or B. Inhibition of dynactin-dependent pole focusing did not prevent anaphase. Bipolar spindles obtained by co-inhibition of dynactin and the spindle motor Eg5 were also able to elongate and segregate chromosomes, suggesting that these motors may not be essential for anaphase. A number of proteins were localized to the central spindle during anaphase, including the motors Mklp1/CHO1 and Xklp1, and the Aurora B kinase complex. Addition of antibodies or inhibitors to several candidate spindle kinesins did not impair anaphase B.
These results suggest that kinetochores and kinetochore microtubules are not required for anaphase B pole separation. In addition, while focused spindle poles aid anaphase A chromosome segregation, they are likewise not essential for anaphase B. The motor activity responsible for anaphase B remains to be identified. We propose that pole separation may not be based on loss of the microtubule overlap zone, but on sliding forces against a stable midzone bundle.