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* Wadsworth Center, Division of Molecular Medicine, New York State Department of Health, Albany, New York 12201-0509;
and Kinetochore microtubules (kMts) are a subset of spindle microtubules that bind directly to the kinetochore to form the kinetochore fiber (K-fiber). The
K-fiber in turn interacts with the kinetochore to produce chromosome motion toward the attached spindle
pole. We have examined K-fiber maturation in PtK1
cells using same-cell video light microscopy/serial section EM. During congression, the kinetochore moving
away from its spindle pole (i.e., the trailing kinetochore) and its leading, poleward moving sister both
have variable numbers of kMts, but the trailing kinetochore always has at least twice as many kMts as the
leading kinetochore. A comparison of Mt numbers on
sister kinetochores of congressing chromosomes with
their direction of motion, as well as distance from their
associated spindle poles, reveals that the direction of
motion is not determined by kMt number or total kMt
length. The same result was observed for oscillating
metaphase chromosomes. These data demonstrate that
the tendency of a kinetochore to move poleward is not
positively correlated with the kMt number. At late
prometaphase, the average number of Mts on fully congressed kinetochores is 19.7 ± 6.7 (n = 94), at late
metaphase 24.3 ± 4.9 (n = 62), and at early anaphase
27.8 ± 6.3 (n = 65). Differences between these distributions are statistically significant. The increased kMt
number during early anaphase, relative to late
metaphase, reflects the increased kMt stability at
anaphase onset. Treatment of late metaphase cells with
1 µM taxol inhibits anaphase onset, but produces the
same kMt distribution as in early anaphase: 28.7 ± 7.4 (n = 54). Thus, a full complement of kMts is not sufficient to induce anaphase onset. We also measured the
time course for kMt acquisition and determined an initial rate of 1.9 kMts/min. This rate accelerates up to 10fold during the course of K-fiber maturation, suggesting
an increased concentration of Mt plus ends in the vicinity of the kinetochore at late metaphase and/or cooperativity for kMt acquisition.
Department of Biomedical Sciences, State University of New York, Albany, New York 12222
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