Distribution of a thiophosphorylated spindle midzone antigen during spindle reactivation in vitro

Mitotic spindles isolated from the diatom Stephanopyxis turris will elongate in vitro in the presence of ATP with a concurrent decrease in the width of the zone of microtubule overlap. A spindle-associated phosphoprotein that co-localizes with the zone of microtubule overlap in isolated spindles serves as a convenient marker for midzone-associated proteinsother than microtubules. We have used a monoclonal antibody that labels this protein when it is artificially thiophosphorylated and studied its redistribution during spindle reactivation in vitro. As the spindle elongates midzone label accumulates in a successively narrower and brighter ring at the spindle midpoint with increasing time in ATP. Biotinylated bovine microtubule segments polymerized onto the ends of the diatom microtubules increase the overall width of the zone of microtubule overlap and serve as a marker for the boundary of the original diatom overlap zone. During elongation in ATP, the biotinylated segments move into the area marked by the monoclonal antibody, which does not decrease in width until the spindle has elongated to the point at which the zone of microtubule overlap delineated by the newly polymerized microtubules is smaller than the original overlap zone. We use these results to develop a model to explain the behaviour of nonmicrotubule midzone-associated proteins during spindle elongation.