Kinetochores bind microtubules laterally in a transient fashion and stably, by insertion of plus ends. These pathways may exist to carry out distinct tasks during different stages of mitosis and likely depend on distinct molecular mechanisms. On isolated chromosomes, we found microtubule nucleation/binding depended additively on both dynein/dynactin and on the Ndc80/Hec1 complex. Studying chromosome movement in living Xenopus cells within the simplified geometry of monopolar spindles, we quantified the relative contributions of dynein/dynactin and the Ndc80/Hec1 complex. Inhibition of dynein/dynactin alone had minor effects but did suppress transient, rapid, poleward movements. In contrast, inhibition of the Ndc80 complex blocked normal end-on attachments of microtubules to kinetochores resulting in persistent rapid poleward movements that required dynein/dynactin. In normal cells with bipolar spindles, dynein/dynactin activity on its own allowed attachment and rapid movement of chromosomes on prometaphase spindles but failed to support metaphase alignment and chromatid movement in anaphase. Thus, in prometaphase, dynein/dynactin likely mediates early transient, lateral interactions of kinetochores and microtubules. However, mature attachment via the Ndc80 complex is essential for metaphase alignment and anaphase A.

Summary Human Papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes – E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis.

Summary Kinesins in the mitotic spindle play major roles in determining spindle shape, size, and bipolarity, although specific regulation of these kinesins at distinct locations on the spindle is poorly understood. To balance the forces that are required for spindle bipolarity, microtubule-depolymerizing kinesins are tightly regulated. Aurora B kinase phosphorylates the neck regions of the kinesin-13 family microtubule depolymerases Kif2a and MCAK (Mitotic Centromere Associated Kinesin), and inhibits their depolymerase activities. How they are reactivated, and how this is controlled independently on different kinetochore fibers is unknown. We show that Inner Centromere Kin-I Stimulator (ICIS), which stimulates the related depolymerase MCAK, can reactivate Kif2a after Aurora B inhibition. When antibodies that block the ability of ICIS to activate Kif2a are injected into cells, monopolar spindles are generated. This phenotype is rescued by co-injection of anti-Nuf2 antibodies. We have performed a structure-function analysis of the ICIS protein, and find that the N-terminus of ICIS binds Aurora B and its regulators INCENP and TD60, while a central region binds MCAK, Kif2a and microtubules, suggesting a scaffold function for ICIS. These data argue that ICIS and the CPC (Chromosomal Passenger Complex) regulate Kif2a depolymerase activity.

Balanced chromosome segregation requires coordination of chromosome movement on the spindle with regulation of cell cycle progression through mitosis. Initial attachments of chromosomes to spindle microtubules often appear due to kinetochores of chromosomes attaching and moving on the lateral surfaces of microtubules. These mature into end‐on microtubule attachments. Microtubule attachment and mechanical tension induced by bipolar attachment of sister kinetochores serve to bring chromosomes to alignment at the metaphase plate and simultaneously silence the spindle checkpoint. We have found that inhibition of the Ndc80 complex at kinetochores by antibody microinjection results in loss of metaphase alignment. Inhibition of the Ndc80 complex also results in abrogation of the mitotic spindle checkpoint. Thus in such cells, chromatids separate but completely fail to undergo anaphase movements. We hypothesize that the residual chromosome movements seen in prometaphase cells lacking Ndc80 function are due to dynein/dynactin at kinetochores modulating lateral attachments of kinetochores to spindle microtubules. We are studying chromosome movements on monopolar spindles induced with the drug monastrol to dissect the mechanisms that mediate chromosome movement and checkpoint protein dynamics in mitosis.