Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize for the first time complete 3-D movements of individual kinetochores throughout mitosis in non-transformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected new details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high-density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.

Controlling the number of its centrioles is vital for the cell as supernumerary centrioles result in multipolar mitosis and genomic instability 1,2 . Normally, just one daughter centriole forms on each mature (mother) centriole 3,4 ; however, a mother centriole can produce multiple daughters within a single cell cycle 5,6 . The mechanisms that prevent centriole 'overduplication' are poorly understood. Here we use laser microsurgery to test the hypothesis that attachment of the daughter centriole to the wall of the mother inhibits formation of additional daughters 7,8 . We show that physical removal of the daughter induces reduplication of the mother in S-arrested cells. Under conditions when multiple daughters simultaneously form on a single mother, all of these daughters must be removed to induce reduplication. Intriguingly, the number of daughter centrioles that form during reduplication does not always match the number of ablated daughter centrioles. We also find that exaggeration of the pericentriolar material (PCM) via overexpression of the PCM protein pericentrin 9 in S-arrested CHO cells induces formation of numerous daughter centrioles. We propose that that the size of the PCM cloud associated with the mother centriole restricts the number of daughters that can form simultaneously.A typical centrosome in an animal cell consists of two microtubule-based cylindrical structures, termed the centrioles, surrounded by a cloud of pericentriolar material (PCM). Most of the components responsible for the major centrosomal functions, for example, the Υ-tubulin ring complex, reside in the PCM. However, in the absence of centrioles, the PCM cloud becomes structurally unstable, and eventually disperses 10 . Thus, the number of centrioles ultimately defines the number of centrosomes in the cell.Normally, in somatic cells new ('daughter') centrioles form in association with mature ('mother') centrioles. This process, known as "centriole duplication", is initiated when cells enter S phase, and the daughter centriole remains associated with its mother, in a strictly orthogonal configuration (i.e., 'diplosome'), until the second half of the ensuing mitosis 11-13 . Recent work suggested that as long as the daughter remains attached to the mother formation of additional daughters is not possible 7,8 . *Correspondence should be addressed to AK (Wadsworth Center, PO Box 509, Albany, NY 12201−0509, phone: (518)486−5339; fax: (518)486−4901; E-mail: khodj@wadsworth.org). Author contributions Experiments were conducted by J.L.; P.H. was responsible for EM preparation and data collection; V.M. designed, assembled, and maintained the laser microsurgery workstation; A.K. directed the work. Experiments were planned by J.L. and A.K. Competing financial interestsThe authors declare that they have no competing financial interests. Fig. S3). NIH Public AccessUpon ablation of the daughter centriole within a diplosome, the remaining mother consistently (20 of 20 experiments) developed a new daughter (Figs. 1A, S4). The interval be...

Mitotic spindle formation relies on the stochastic capture of microtubules at kinetochores. Kinetochore architecture affects the efficiency and fidelity of this process with large kinetochores expected to accelerate assembly at the expense of accuracy, and smaller kinetochores to suppress errors at the expense of efficiency. We demonstrate that upon mitotic entry, kinetochores in cultured human cells form large crescents that subsequently compact into discrete structures on opposite sides of the centromere. This compaction occurs only after the formation of end-on microtubule attachments. Live-cell microscopy reveals that centromere rotation mediated by lateral kinetochore-microtubule interactions precedes formation of end-on attachments and kinetochore compaction. Computational analyses of kinetochore expansion-compaction in the context of lateral interactions correctly predict experimentally-observed spindle assembly times with reasonable error rates. The computational model suggests that larger kinetochores reduce both errors and assembly times, which can explain the robustness of spindle assembly and the functional significance of enlarged kinetochores.