Christopher B. O'Connell

A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function

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.

The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly

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 complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected 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.

Cooperative mechanisms of mitotic spindle formation

Cooperativity is well known to promote the speed of some biochemical reactions by accelerating the activity of enzymes. Recent studies have shown that cooperative interactions also function during the formation of a complex cellular structure, the mitotic spindle. Capture of kinetochores by dynamic astral microtubules was originally proposed as the basis of spindle formation. However, mounting evidence indicates that a more complex series of events occurs. It is now clear that there are multiple microtubule nucleation and capture sites throughout the spindle. Kinetochores, centrosomes and microtubules play multiple roles in establishing connections between spindle components and integrating them into a common structure. These data support a modified search-and-capture model that incorporates additional assembly pathways coordinated by a RanGTP gradient.

Transferrin receptor recycling in the absence of perinuclear recycling endosomes

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Rab11, suggesting that cytoplasts assembled functional REs. These results suggest that although perinuclear REs are not essential components of the Tfn recycling pathway, they are dynamic structures which preexist in the peripheral cytoplasm or can be regenerated from EE- and cytosol-derived components such as Rab11.

Myosin-IXb Is a Single-headed and Processive Motor

Class IX myosins are unique among the many classes of known actin-based motors in that the tail region of these myosins contains a GTPase-activating protein domain for the small GTP-binding protein, Rho. Previous studies on human myosin-IXb indicate that this myosin is mechanochemically active and exhibits actin-binding properties similar to the processive motor, myosin-Va. Motility analysis of antibody-tethered myosin-IXb performed using the sliding actin filament assay indicates that this myosin does exhibit properties characteristic of a processive motor. Like myosin-Va, the velocity of myosin-IXb remains constant (38.2 ± 1.2 nm/s) even at single motor/filament densities. At low motor densities, filaments can be seen passing through and pivoting about single points on the motility surface. Analysis of filament landing rates as a function of motor density also indicates that a single motor is sufficient for filament movement. However, in contrast to myosin-Va, which uses coordinated motion of its two heads to move processively along the filament, hydrodynamic and chemical cross-linking studies indicate that under the conditions tested, myosin-IXb is a single-headed motor consisting of a single heavy chain and associated light chains.

Distinct roles of the equatorial and polar cortices in the cleavage of adherent cells

Over the past 100 years, many models have been proposed and tested for cytokinesis [1]. There is strong evidence that the equator represents a unique region that receives cleavage signals from the mitotic spindle [2, 3]. The nature of such a signal and the mechanism of cleavage, however, remain poorly understood. To probe the contribution of different cortical regions in the cleavage of cultured epithelial cells, we applied cytochalasin D (CD), a known inhibitor of cytokinesis [4], in a highly localized manner to different regions of dividing NRK cells. Surprisingly, equatorial application of CD not only allowed cytokinesis to complete but also appeared to facilitate the process. Conversely, local application of CD near the polar region caused inhibition of cytokinesis. Our results suggest a mechanism that involves global coordination of cortical activities, including controlled cortical disassembly along the equator and possibly global cortical contraction.

Relative contributions of chromatin and kinetochores to mitotic spindle assembly.

Its the kinetochores, not the DNA, that initiate spindle assembly.

The spindle assembly checkpoint is satisfied in the absence of interkinetochore tension during mitosis with unreplicated genomes

The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The SAC is thought to monitor two distinct events: attachment of kinetochores to microtubules and the stretch of the centromere between the sister kinetochores that arises only when the chromosome becomes properly bioriented. We examined human cells undergoing mitosis with unreplicated genomes (MUG). Kinetochores in these cells are not paired, which implies that the centromere cannot be stretched; however, cells progress through mitosis. A SAC is present during MUG as cells arrest in response to nocodazole, taxol, or monastrol treatments. Mad2 is recruited to unattached MUG kinetochores and released upon their attachment. In contrast, BubR1 remains on attached kinetochores and exhibits a level of phosphorylation consistent with the inability of MUG spindles to establish normal levels of centromere tension. Thus, kinetochore attachment to microtubules is sufficient to satisfy the SAC even in the absence of interkinetochore tension.

Native Myosin-IXb is a plus-, not a minus-end-directed motor

Myosin-IXb (Myo9b) is a single-headed, processive motor that contains a Rho-GTPase-activating protein (GAP) domain within its tail. Although tail-less myosin-IXb motor domain moves towards the minus end of the actin filament, we show here that full-length myosin-IXb is a plus-end-directed motor. This suggests that the tail domain of myosin-IXb regulates motor directionality.

BubR1 is modified by sumoylation during mitotic progression.

Background: The molecular basis by which BubR1 is post-translationally modified during the cell cycle remains poorly understood. Results: BubR1 is modified by sumoylation and lysine 250 is crucial for its SUMO-modification. Conclusion: A new type of post-translational modification is identified that is essential for BubR1 function. Significance: An important molecular mechanism is identified that inactivates the spindle checkpoint. BubR1 functions as a crucial component that monitors proper chromosome congression and mitotic timing during cell division. We investigated molecular regulation of BubR1 and found that BubR1 was modified by an unknown post-translation mechanism during the cell cycle, resulting in a significant mobility shift on denaturing gels. We termed it BubR1-M as the nature of modification was not characterized. Extended (>24 h) treatment of HeLa cells with a microtubule disrupting agent including nocodazole and taxol or release of mitotic shake-off cells into fresh medium induced BubR1-M. BubR1-M was derived from neither phosphorylation nor acetylation. Ectopic expression coupled with pulling down analyses showed that BubR1-M was derived from SUMO modification. Mutation analysis revealed that lysine 250 was a crucial site for sumoylation. Significantly, compared with the wild-type control, ectopic expression of a sumoylation-deficient mutant of BubR1 induced chromosomal missegregation and mitotic delay. Combined, our study identifies a new type of post-translational modification that is essential for BubR1 function during mitosis.