THE KINETOCHORE MICROTUBULE MINUS-END DISASSEMBLY ASSOCIATED WITH POLEWARD FLUX PRODUCES A FORCE THAT CAN DO WORK

During metaphase and anaphase in newt lung cells, tubulin subunits wit hin the kinetochore microtubule (kMT) lattice flux slowly poleward as kMTs depolymerize at their minus-ends within in the pole. Very little is known about how and where the force that moves the tubulin subunits poleward is generated and what function it serves during mitosis. We found that treatment with the drug taxol (10 mu M) caused separated ce ntrosomes in metaphase newt lung cells to move toward one another with an average velocity of 0.89 mu m/min, until the interpolar distance w as reduced by 22-62%. This taxol-induced spindle shortening occurred a s kMTs between the chromosomes and the poles shortened. Photoactivatio n of fluorescent marks on kMTs revealed that taxol inhibited kinetocho re microtubule assembly/disassembly at kinetochores, whereas minus-end MT disassembly continued at a rate typical of poleward flux in untrea ted metaphase cells. This poleward flux was strong enough to stretch t he centromeric chromatin between sister kinetochores as much as it is stretched in control metaphase cells. In anaphase, taxol blocked kMT d isassembly/assembly at the kinetochore whereas minus-end disassembly c ontinued at a rate similar to flux in control cells (similar to 0.2 mu m/min). These results reveal that the mechanism for kMT poleward flux 1) is not dependent on kMT plus-end dynamics and 2) produces pulling forces capable of generating tension across the centromeres of biorien ted chromosomes.