THE FORCE-PRODUCING MECHANISM FOR CENTROSOME SEPARATION DURING SPINDLE FORMATION IN VERTEBRATES IS INTRINSIC TO EACH ASTER

A popular hypothesis for centrosome separation during spindle formatio n and anaphase is that pushing forces are generated between interactin g microtubules (MTs) of opposite polarity, derived from opposing centr osomes. However, this mechanism is not consistent with the observation that centrosomes in vertebrate cells continue to separate during prom etaphase when their MT arrays no longer overlap (i.e., during anaphase -like prometaphase). To evaluate whether centrosome separation during prophase/prometaphase, anaphase-like prometaphase and anaphase is medi ated by a common mechanism we compared their behavior in vivo at a hig h spatial and temporal resolution. We found that the two centrosomes p ossess a considerable degree of independence throughout all stages of separation, i.e., the direction and migration rate of one centrosome d oes not impart a predictable behavior to the other, and both exhibit f requent and rapid (4-6 mum/min) displacements toward random points wit hin the cell including the other centrosome. The kinetic behavior of i ndividual centrosomes as they separate to form the spindle is the same whether or not their MT arrays overlap. The characteristics examined include, e.g., total displacement per minute, the vectorial rate of mo tion toward and away from the other centrosome, the frequency of towar d and away motion as well as motion not contributing to separation, an d the rate contributed by each centrosome to the separation process. B y contrast, when compared with prometaphase, anaphase centrosomes sepa rated at significantly faster rates even though the average vectorial rate of motion away from the other centrosome was the same as in proph ase/prometaphase. The difference in separation rates arises because an aphase centrosomes spend less time moving toward one another than in p rophase/prometaphase, and at a significantly slower rate. From our dat a we conclude that the force for centrosome separation during vertebra te spindle formation is not produced by MT-MT interactions between opp osing asters, i.e., that the mechanism is intrinsic to each aster. Our results also strongly support the contention that forces generated in dependently by each aster also contribute substantially to centrosome separation during anaphase, but that the process is modified by intera ctions between opposing astral MTs in the interzone.