Poleward Kinetochore Fiber Movement Occurs during Both Metaphase and Anaphase-A in Newt Lung Cell Mitosis

Microtubules in the mitotic spindles of newt lung cells were marked using local photoactivation of fluorescence. The movement of marked segments on kinetochore fibers was tracked by digital fluorescence microscopy in metaphase and anaphase and compared to the rate of chromosome movement. In metaphase, kinetochore oscillations toward and away from the poles were coupled to kinetochore fiber shortening and growth. Marked zones on the kinetochore microtubules, meanwhile, moved slowly polewards at a rate of ",,0.5/zm/min, which identifies a slow polewards movement, or "flux" of kinetochore microtubules accompanied by depolymerization at the pole, as previously found in PtK2 cells (Mitchison, 1989b). Marks were never seen moving away from the pole, indicating that growth of the kinetochore microtubules occurs only at their kinetochore ends. In anaphase, marked zones on kinetochore microtubules also moved polewards, though at a rate slower than overall kinetochore-to-pole movement. Early in anaphase-A, microtubule depolymerization at kinetochores accounted on average for 75 % of the rate of chromosome-to-pole movement, and depolymerization at the pole accounted for 25 %. When chromosome-to-pole movement slowed in late anaphase, the contribution of depolymerization at the kinetochores lessened, and flux became the dominant component in some cells. Over the whole course of anaphase-A, depolymerization at kinetochores accounted on average for 63 % of kinetochore fiber shortening, and flux for 37 %. In some anaphase cells up to 45 % of shortening resulted from the action of flux. We conclude that kinetochore microtubules change length predominantly through polymerization and depolymerization at the kinetochores during both metaphase and anaphase as the kinetochores move away from and towards the poles. Depolymerization, though not polymerization, also occurs at the pole during metaphase and anaphase, so that flux contributes to polewards chromosome movements throughout mitosis. Poleward force production for chromosome movements is thus likely to be generated by at least two distinct molecular mechanisms. T HE physical segregation of sister chromatids at mitosis is mediated by the interaction of their kinetochores with microtubules of the mitotic spindle. An important goal of mitosis research is to determine how the forces that drive segregation are generated and regulated at a molecular level. This requires first understanding the mechanics of the spindle to determine where in the spindle the segregation forces are generated. Over the last few years it has become apparent that the kinetochore itself is a major site of force generation for chromosome movement (reviewed in Mitchison 1988, 1989a; Mclntosh and Pfarr, 1991; Rieder, 1991; Salmon, 1989). This is particularly clear early in mitosis, when the mechanics of the kinetochore-microtubule interaction can be very simple. Some chromosomes first attach to the spindle when their kinetochores make a side-on interaction with microtubules that emanate from the pole (Rieder and Alexander, 1990; Hayden et al., 1990). This interaction leads to a rapid polewards sliding of the kinetochore over the microtubule lattice (20-50/xm/min in newt cells). Sliding is thought to be driven by dynein molecules attached to the kinetochore (Rieder and Alexander, 1990; Hyman and Mitchison, 1991) and this phase of chromosome movement thus resembles minus end-directed vesicle transport along microtubules. This rapid poleward sliding, however, is not apparent for the great majority of chromosomes. Instead, chromosome attachment and movement depend primarily on interactions of kinetochores with the ends of polar microtubules. During prometaphase, each kinetochore in a higher eukaryotic spindie becomes attached to the plus end of a bundle of microtubules whose minus ends are mostly located near the spindle pole (Rieder, 1982; McDonald et al., 1992). Since these kinetochore microtubules have been shown in vertebrate spindles to extend continuously between kinetochores and poles, chromosome movement along the spindle axis must be accompanied by a change in their length. In principle, 9 The Rockefeller University Press, 0021-9525/92/11/569/14 $2.00 The Journal of Cell Biology, Volume 119, Number 3, November 1992 569-582 569 on July 22, 2018 jcb.rupress. org Downloaded from http://doi.org/10.1083/jcb.119. 3.569 Published Online: 1 November, 1992 | Supp Info: