DIRECTIONAL INSTABILITY OF KINETOCHORE MOTILITY DURING CHROMOSOME CONGRESSION AND SEGREGATION IN MITOTIC NEWT LUNG-CELLS - A PUSH-PULL MECHANISM

Most models of mitotic congression and segregation assume that only po leward pulling forces occur at kinetochores. However, there are report s for several different cell types that both mono-oriented and bi-orie nted chromosomes oscillate toward and away from the pole throughout mi tosis. We used new methods of high resolution video microscopy and com puter-assisted tracking techniques to measure the positions over time of individual kinetochores with respect to their poles during mitosis in living newt lung cells. The results show that kinetochores oscillat e throughout mitosis when they are tethered to spindle poles by attach ment to the plus-ends of kinetochore microtubules (kMTs). Oscillations were not sinusoidal. Instead, kinetochores abruptly (as quick as 6 s or less) switched between persistent (approximately 1.5 min average du ration) phases of poleward (P) and away from the pole (AP) movement. T his kinetochore ''directional instability'' was a property of motility at the plus-ends of kMTs since fluorescent marks on the lattice of kM Ts have previously been observed to exhibit only relatively slow P mov ement. Each P and AP phase consisted of one or a few constant velocity domains (approximately 1.7 mum/min average velocity). Velocities of P and AP phases were similar from prometaphase through mid-anaphase. Ki netochores occasionally switched to an indeterminant (N) phase of no o r confused motion, which was usually brief compared to the durations o f P and AP phases. Net chromosome displacements that occurred during c ongression to the equator or poleward movement during anaphase were pr imarily generated by differences in the durations and not the velociti es of P and AP movements. Careful analysis of centromere deformation s howed that kinetochore P movement produced pulling forces while kineto chore AP movement produced pushing forces. These data show that kineto chore directional instability is fundamental to the processes of chrom osome congression and segregation. We argue that tension at the kineto chore attachment site is a key factor which controls the switching bet ween P and AP phases of kinetochore motion.