KINETOCHORES MOVING AWAY FROM THEIR ASSOCIATED POLE DO NOT EXERT A SIGNIFICANT PUSHING FORCE ON THE CHROMOSOME

We used video-light microscopy and laser microsurgery to test the hypo thesis that as a bioriented prometaphase chromosome changes position i n PtK1 cells, the kinetochore moving away from its associated pole (AP ) exerts a pushing force on the centromere. When we rapidly severed co ngressing chromosomes near the spindle equator between the sister kine tochores, the kinetochore that was originally ''leading'' the motion t owards a pole (P) always (17/17 cells) continued moving P whereas the ''trailing'' kinetochore moving AP always stopped moving as soon as th e operation was completed. This trailing kinetochore then initiated mo tion towards the pole it was originally moving away from up to 50 s la ter. The same result was observed (15/15 cells) when we selectively de stroyed the leading (P moving) kinetochore on a congressing chromosome positioned greater than or equal to 3 mu m from the pole it was movin g away from. When we conducted this experiment on congressing chromoso mes positioned within 3 mu m 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 s witching 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 similar to 2 mu M/min. From thes e data we conclude that the kinetochore moving AP on a moving chromoso me does not exert a significant pushing force on the chromosome. Inste ad, our results reveal that, when not generating a P force, kinetochor es are in a ''neutral'' state that allows them to remain stationary or to coast Af in response to external forces sufficient to allow their K-fiber to elongate.