Microtubules assemble near most kinetochores during early prometaphase in human cells.

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.