Stability of microtubuie attachment to metaphase kinetochores in PtICi cells

Kinetochore microtubules are known to be differentially stable to a variety of microtubuie depolymerization agents compared to the non-kinetochore polar microtubules, but the dynamics of microtubuie attachment to the kinetochore is currently controversial. We have examined the stability of kinetochore microtubules in metaphase PtKx spindles at 23 °C when microtubuie assembly is abruptly blocked with the drug nocodazole. Metaphase cells were incubated in medium containing 34/iM nocodazole for various times before fixation and processing either for immunofluorescence light microscopy or serial-section electron microscopy. Microtubules not associated with kinetochore fibers disappeared completely in less than 1 min. Kinetochore fibers persisted and shortened, as the spindle poles moved close to the chromosomes over a 10—20 min interval. During this shortening process, the number of kinetochore microtubules decreased slowly. The mean number of kinetochore microtubules was 24 ±5 in control cells and zero in cells incubated with nocodazole for 20 min. The half-time of microtubuie attachment to the kinetochore was ~7.5 min. These results show that when microtubuie assembly is blocked, kinetochore microtubules shorten more slowly and persist about 10 times longer than the labile polar microtubules. If kinetochore microtubules shorten by tubulin dissociation at their plus-ends like the non-kinetochore polar microtubules, then the microtubuie surface lattice must be able to translocate through the kinetochore attachment site without frequent detachment occurring.