Mutations in the LIS1 gene cause gross histological disorganization of the developing human brain, resulting in a brain surface that is almost smooth. Here we show that LIS1 protein co-immunoprecipitates with cytoplasmic dynein and dynactin, and localizes to the cell cortex and to mitotic kinetochores, which are known sites for binding of cytoplasmic dynein. Overexpression of LIS1 in cultured mammalian cells interferes with mitotic progression and leads to spindle misorientation. Injection of anti-LIS1 antibody interferes with attachment of chromosomes to the metaphase plate, and leads to chromosome loss. We conclude that LIS1 participates in a subset of dynein functions, and may regulate the division of neuronal progenitor cells in the developing brain.

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.

In animal cells, positioning of the mitotic spindle is crucial for defining the plane of cytokinesis and the size ratio of daughter cells. We have characterized this phenomenon in a rat epithelial cell line using microscopy, micromanipulation, and microinjection. Unmanipulated cells position the mitotic spindle near their geometric center, with the spindle axis lying roughly parallel to the long axis of the cell. Spindles that were initially misoriented underwent directed rotation and caused a delay in anaphase onset. To gain further insight into this process, we gently deformed cells with a blunted glass needle to change the spatial relationship between the cortex and spindle. This manipulation induced spindle movement or rotation in metaphase and/or anaphase, until the spindle reached a proper position relative to the deformed shape. Spindle positioning was inhibited by either treatment with low doses of nocodazole or microinjection of antibodies against dynein, apparently due to the disruption of the organization of dynein and/or astral microtubules. Our results suggest that mitotic cells continuously monitor and maintain the position of the spindle relative to the cortex. This process is likely driven by interactions among astral microtubules, the motor protein dynein, and the cell cortex and may constitute part of a mitotic checkpoint mechanism. INTRODUCTIONCorrect placement of the cleavage furrow is essential for the successful conclusion of mitosis and meiosis. During typical cell division, a centrally placed cleavage plane ensures that the two daughter cells receive a similar share of organelles and molecular components. During embryonic development, regulated asymmetric division coupled with localization of signaling molecules or organelles functions as an effective means for determining cell fate (Strome, 1993). Although it has been well established that the plane of cytoplasmic division is determined by the position of the mitotic spindle (reviewed by Fishkind and Wang, 1995;Glotzer, 1997;Field et al., 1999;Hales et al., 1999), little is known about how the location of the spindle itself is regulated.What little evidence there is comes mostly from invertebrates and suggests that astral microtubules, which link the spindle with the cortex, are involved in bringing the spindle to defined sites within the cytoplasm (Shaw et al., 1997). In addition, studies with Saccharomyces cerivisae and embryos of Caenorhabditis elegans and Drosophila melanogaster indicate that dynein interacts with its accessory protein, dynactin, to generate forces for positioning the nucleus or mitotic spindle (Carminati and Stearns, 1997;McGrail and Hays, 1997;Skop and White, 1998). However, because little work has been done to identify an active spindle-positioning mechanism in other cell types, it was not clear whether these activities represent a general cellular function or specialized processes in yeast or large embryos to bring the spindle to specific destinations. Furthermore, although there is some evidence that molecular s...

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.