Proper segregation of chromosomes during mitosis depends on "amphitelic attachments"-load-bearing connections of sister kinetochores to the opposite spindle poles via bundles of microtubules, termed as the "K-fibers." Current models of spindle assembly assume that K-fibers arise largely from stochastic capture of microtubules, which occurs at random times and locations and independently at sister kinetochores. We test this assumption by following the movements of all kinetochores in human cells and determine that most amphitelic attachments form synchronously at a specific stage of spindle assembly and within a spatially distinct domain. This biorientation domain is enriched in bundles of antiparallel microtubules, and perturbation of microtubule bundling changes the temporal and spatial dynamics of amphitelic attachment formation. Structural analyses indicate that interactions of kinetochores with microtubule bundles are mediated by non-centrosomal short microtubules that emanate from most kinetochores during early prometaphase. Computational analyses suggest that momentous molecular motor-driven interactions with antiparallel bundles rapidly convert these short microtubules into nascent K-fibers. Thus, load-bearing connections to the opposite spindle poles form simultaneously on sister kinetochores. In contrast to the uncoordinated sequential attachments of sister kinetochores expected in stochastic models of spindle assembly, our model envisions the formation of amphitelic attachments as a deterministic process in which the chromosomes connect with the spindle poles synchronously at a specific stage of spindle assembly and at a defined location determined by the spindle architecture. Experimental analyses of changes in the kinetochore behavior in cells with perturbed activity of molecular motors CenpE and dynein confirm the predictive power of the model.

For proper segregation during cell division, each chromosome must connect to the poles of the spindle via microtubule bundles termed kinetochore fibers (K-fibers). K-fibers form by two distinct mechanisms: (1) capture of astral microtubules nucleated at the centrosome by the chromosomes' kinetochores or (2) attachment of kinetochores to noncentrosomal microtubules with subsequent transport of the minus ends of these microtubules toward the spindle poles. The relative contributions of these alternative mechanisms to normal spindle assembly remain unknown. In this study, we report that most kinetochores in human cells develop K-fibers via the second mechanism. Correlative light electron microscopy demonstrates that from the onset of spindle assembly, short randomly oriented noncentrosomal microtubules appear in the immediate vicinity of the kinetochores. Initially, these microtubules interact with the kinetochores laterally, but end-on attachments form rapidly in the first 3 min of prometaphase. Conversion from lateral to end-on interactions is impeded upon inhibition of the plus end-directed kinetochore-associated kinesin CenpE.