Eg5 activity and density-driven bundling organize the human metaphase mitotic spindle independently of spindle bipolarity

ABSTRACT The mitotic spindle segregates chromosomes through the coordinated actions of microtubules and molecular motors. Classic models propose that microtubules nucleate at spindle poles and grow inward to capture chromosomes; however, recent structural studies show that spindles contain short microtubules that do not span the distance between poles and chromosomes. It is unclear how these short microtubules assemble a bipolar spindle. Using cryo-electron tomography to map microtubule polarity in human metaphase spindles, we find that microtubules form locally antiparallel bundles with consistent 8 nm wall-to-wall spacing. We utilized motor perturbations and centriole depletion, which generated motor-active monopolar spindles, to reveal that the kinesin-5 motor Eg5 organizes local antiparallel overlap independently of spindle bipolarity. We found that bundles are organized by density-driven steric interactions rather than motor-mediated crosslinking. These findings support a self-organized, bottom-up model in which local microtubule-motor interactions within dense bundles generate forces that build the bipolar spindle, challenging pole-centric models. KEY FINDINGS Microtubules in metaphase spindles organize in locally antiparallel bundles with consistent 8 nm wall-to-wall spacing. The Eg5 motor generates antiparallel microtubule overlap independently of spindle bipolarity. Microtubule spacing scales with density through steric interactions, not direct motor crosslinking. Dynein regulates microtubule density to control bundle architecture; balanced Eg5-dynein density regulation maintains spindle bipolarity.