Supplemental Material for Vicars et al., 2024

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 congress and 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 the presence of spindles with disrupted interpolar microtubules 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. Taken together these studies suggest that plus-end forces mediated by the outer interpolar microtubules contribute significantly to acentric congression and alignment. Surprisingly, we observe disrupted telomere pairing and alignment of sister acentrics indicating that the kinetochore is required to ensure proper gene-to-gene alignment of sister chromatids. Finally, we demonstrate that like mammalian cells, the Drosophila congressed chromosomes on occasion exhibit a toroid configuration.