Acentric chromosome congression and alignment on the metaphase plate occur via kinetochore-independent forces

Abstract

Chromosome congression and alignment on the metaphase plate involves lateral and microtubule plus-end interactions with the kinetochore. Here we take advantage of our ability to efficiently generate a GFP-marked acentric X chromosome fragment in Drosophila neuroblasts to identify forces acting on chromosome arms that drive congression and alignment. We find acentrics efficiently align on the metaphase plate, often more rapidly than kinetochore-bearing chromosomes. Unlike intact chromosomes, the paired sister acentrics oscillate as they move to and reside on the metaphase plate in a plane distinct and significantly further from the main mass of intact chromosomes. Consequently, at anaphase onset acentrics are oriented either parallel or perpendicular to the spindle. Parallel-oriented sisters separate by sliding while those oriented perpendicularly separate via unzipping. This oscillation, together with the fact that in monopolar spindles acentrics are rapidly shunted away from the poles, indicates that distributed plus-end directed forces are primarily responsible for acentric migration. This conclusion is supported by the observation that reduction of EB1 preferentially disrupts acentric alignment. In addition, reduction of Klp3a activity, a gene required for the establishment of pole-to-pole microtubules, preferentially disrupts acentric alignment. Taken together these studies suggest that plus-end forces mediated by the outer pole-to-pole microtubules are primarily responsible for acentric metaphase alignment. Surprisingly, we find that a small fraction of sister acentrics are anti-parallel aligned indicating that the kinetochore is required to ensure parallel alignment of sister chromatids. Finally, we find induction of acentric chromosome fragments results in a global reorganization of the congressed chromosomes into a torus configuration. Article SummaryThe kinetochore serves as a site for attaching microtubules and allows for successful alignment, separation, and segregation of replicated sister chromosomes during cell division. However, previous studies have revealed that sister chromosomes without kinetochores (acentrics) often align to the metaphase plate, undergo separation and segregation, and are properly transmitted to daughter cells. In this study, we discuss the forces acting on chromosomes, independent of the kinetochore, underlying their successful alignment in early mitosis.