KINETOCHORE FIBER MATURATION IN PTK1 CELLS AND ITS IMPLICATIONS FOR THE MECHANISMS OF CHROMOSOME CONGRESSION AND ANAPHASE ONSET

Kinetochore microtubules (kMts) are a subset of spindle microtubules t hat 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 fr om its spindle pole (i.e., the trailing kinetochore) and its leading, poleward moving sister both have variable numbers of kMts, but the tra iling kinetochore always has at least twice as many kMts as the leadin g kinetochore. A comparison of Mt numbers on sister kinetochores of co ngressing chromosomes with their direction of motion, as well as dista nce 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 anaph ase 27.8 +/- 6.3 (n = 65). Differences between these distributions are statistically significant. The increased kMt number during early anap hase, relative to late metaphase, reflects the increased kMt stability at anaphase onset. Treatment of late metaphase cells with 1 mu M taxo l inhibits anaphase onset, but produces the same kMt distribution as i n early anaphase: 28.7 +/- 7.4 (n = 54). Thus, a full complement of kM ts is not sufficient to induce anaphase onset. We also measured the ti me course for kMt acquisition and determined an initial rate of 1.9 kh kMs/min. This rate accelerates up to 10-fold during the course of K-f iber maturation, suggesting an increased concentration of Mt plus ends in the vicinity of the kinetochore at late metaphase and/or cooperati vity for kMt acquisition.