We used video-light microscopy and laser microsurgery to test the hypothesis that as a bioriented prometaphase chromosome changes position in PtK1 cells, the kinetochore moving away from its associated pole (AP) exerts a pushing force on the centromere. When we rapidly severed congressing chromosomes near the spindle equator between the sister kinetochores, the kinetochore that was originally "leading" the motion towards a pole (P) always (17/17 cells) continued moving P whereas the "trailing" kinetochore moving AP always stopped moving as soon as the operation was completed. This trailing kinetochore then initiated motion towards the pole it was originally moving away from up to 50 s later. The same result was observed (15/15 cells) when we selectively destroyed the leading (P moving) kinetochore on a congressing chromosome positioned > or = 3 microns from the pole it was moving away from. When we conducted this experiment on congressing chromosomes positioned within 3 microns of the pole, the centromere region either stopped moving, before switching into motion towards the near pole (2/4 cells), or it continued to move AP for 30-44 s (2/4 cells) before switching into P motion. Finally, kinetochore-free chromosome fragments, generated in the polar regions of PtK1 spindles, were ejected AP and often towards the spindle equator at approximately 2 microns/min. From these data we conclude that the kinetochore moving AP on a moving chromosome does not exert a significant pushing force on the chromosome. Instead, our results reveal that, when not generating a P force, kinetochores are in a "neutral" state that allows them to remain stationary or to coast AP in response to external forces sufficient to allow their K-fiber to elongate.

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