During mitosis (cell division) in animal cells many chromosomes initially attach to microtubules (MTs) from a single spindle pole and move toward that pole. When the unattached kinetochore (K) on these chromosomes attaches to MTs from the other pole, the sister Ks begin near unidirectional movement away from the closest pole towards the spindle equator. During these """"""""congression"""""""" movements the most recently attached K exhibits MT minus-end directed motion towards its pole (P motion), while the other K exhibits MT plus-end directed motion away from its pole (AP motion). Based on in vitro observations that phosphorylation and a greater number of associated MTs favor plus-end directed motion, Hyman and Mitchison (C.H.S.Q.B. (1991)56:745) have postulated that the accumulation of MTs biases the K toward AP motion, and that unequal rates of MT accumulation by sister Ks contributes to congression. We are using same-cell correlative video-enhanced DIC LM/3D EM techniques to study congressing Ks in PtK1 cells. In support of the above hypothesis, we have found to date that the AP moving K of congressing chromosomes contains at least twice as many MTs as its sister. By contrast, only 3 of 13 fully congressed sister Ks had comparable disparities in MT numbers. The data contain considerable variability in the number of MTs bound to each K (1-33), and in the ratio of MTs bound to sister Ks (1:1 to 3:1). This variability is consistent with the idea that attached Ks continue to acquire MTs throughout prometaphase until they become saturated (approximately 25-33 MTs), and that many of the Ks we observed were unsaturated. We are evaluating this by counting numbers of MTs on Ks of late metaphase cells. Since current evidence suggests that the direction of K motion is regulated independently at each individual MT docking site in the K, the progressive acquisition of MTs by sister Ks could stabilize chromosome position near the spindle equator by making force coordination between individual K MTs more difficult. This work has been abstracted to the 1995 ASCB meeting.
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