A Bundle of Antiparallel Microtubules Connects Sister K-Fibers and Balances Forces within the Metaphase Spindle

Abstract

Chromosome segregation is driven by forces generated by motor proteins and microtubules (MTs). Although MTs are highly dynamic, the metaphase spindle can be considered as a stable state as all forces acting within it are accurately balanced. Here, we propose that a new class of spindle microtubules exists which contributes to the force map of the mitotic spindle. We named this population of MTs bridging microtubules (bMTs) as they, being a bundle of antiparallel MTs, connect two sister k-fibers. To confirm the connection of the bMT bundle with the sister k-fibers, a laser cutting assay was developed, where the outermost k-fiber of the spindle was severed. After the cut, sister k-fibers moved together with the bMT bundle and behaved as a single object, revealing their connection. To test whether the forces in the spindle can be explained by including the bMT bundle, a theoretical model was developed. The model predicts that the thickness of the bMT fiber defines the magnitude of the forces within the spindle. To test this prediction, the thickness of the bMT fiber was increased by overexpression of tubulin and antiparallel MT crosslinking protein PRC1. This perturbation of the system resulted in a much faster movement of the severed k-fibers, confirming that the force map within the spindle was affected by thickening the bMT fiber. Furthermore, the experiments showed that the bMT bundle participates in anaphase A movement of the chromosomes. When the severed k-fiber did not reconnect to the spindle pole before the onset of anaphase, the sister chromatids, which were connected only to one pole, were able to move apart along the bMT. This finding reveals an alternative mechanism of chromosome segregation.