During anaphase identical sister chromatids separate and move towards opposite poles of the mitotic spindle. In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochore and their minus ends at the spindle pole. Two models have been proposed to account for the movement of chromatids during anaphase. In the 'Pac-Man' model, kinetochores induce the depolymerization of kinetochore microtubules at their plus ends, which allows chromatids to move towards the pole by 'chewing up' microtubule tracks. In the 'poleward flux' model, kinetochores anchor kinetochore microtubules and chromatids are pulled towards the poles through the depolymerization of kinetochore microtubules at the minus ends. Here, we show that two functionally distinct microtubule-destabilizing KinI kinesin enzymes (so named because they possess a kinesin-like ATPase domain positioned internally within the polypeptide) are responsible for normal chromatid-to-pole motion in Drosophila. One of them, KLP59C, is required to depolymerize kinetochore microtubules at their kinetochore-associated plus ends, thereby contributing to chromatid motility through a Pac-Man-based mechanism. The other, KLP10A, is required to depolymerize microtubules at their pole-associated minus ends, thereby moving chromatids by means of poleward flux.

Kinesin-14 family proteins are minus-end directed motors that cross-link microtubules and play key roles during spindle assembly. We showed previously that the Xenopus Kinesin-14 XCTK2 is regulated by Ran via the association of a bipartite NLS in the tail of XCTK2 with importin ␣/␤, which regulates its ability to cross-link microtubules during spindle formation. Here we show that mutation of the nuclear localization signal (

Kif18B is a newly discovered plus-tip-tracking protein that is enriched on astral microtubule (MT) ends during early mitosis. Kif18B binds directly to EB1, and this interaction is required for proper localization of Kif18B and to control astral MT length.

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin ␣ and/or importin ␤ to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin ␣ and ␤ (␣/␤) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin ␣/␤-mediated regulation of XCTK2 microtubule binding. Our data show that importin ␣/␤ directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport. INTRODUCTIONThe process of chromosome congression and segregation is mediated by the mitotic spindle, which is comprised of microtubules (MTs) and their associated proteins. Proper spindle assembly requires the activities of both plus-endand minus-end-directed MT motors, nonmotor MT-associated proteins, and other essential non-MT-associated proteins (Compton, 2000). Minus-end-directed cytoplasmic dynein and plus-end-directed Eg5 play key roles in the organization of spindle MTs in Xenopus egg extracts and in cells (Gaglio et al., 1996;Heald et al., 1996;Merdes et al., 1996;Walczak et al., 1998). In addition, the mitotic minus-enddirected C-terminal kinesins function as MT cross-linkers to promote proper spindle assembly in many organisms (Endow and Komma, 1996;Walczak et al., 1997Walczak et al., , 1998Matuliene et al., 1999;Mountain et al., 1999;Ovechkina and Wordeman, 2003) and KIFC1 and KIFC5 C-terminal kinesins may serve a similar cross-linking function during spermatogenesis (Navolanic and Sperry, 2000;Yang and Sperry, 2003). The mechanism that regulates this MT cross-linking is currently unknown.Recently, the GTPase Ran was shown to be sufficient to induce MT aster formation as well as bipolar spindle assembly in egg extracts (Carazo-Salas et al., 1999;Kalab et al., 1999;Ohba et al., 1999;Wilde and Zheng, 1999;Nachury et al., 2001). Subsequently, it was reported to be involved in regulating numerous mitotic spindle assembly processes, including MT nucleation and dynamics (Wilde and Zheng, 1999;Carazo-Salas et al., 2001;Wilde et al., 2001), MT motor activity , and nuclear envelope assembly (Bamba et al., 2002;Hetzer et al., 2002;Zhang et al., 2002). It is believed that Ran provides positional cues by the generation of a steep gradient of Ran-GTP between the condensed chromosomes and the surrounding cytoplasm (Kalab et al., 2002;Trieselmann and Wilde, 2002). After nuclear envelope breakdown, the localized concentration of Ran-GTP around chromatin is thought to contribute to proper spind...

SUMMARY Background Proper spindle assembly and chromosome segregation relies on precise microtubule dynamics, which are governed in part by the Kinesin-13 MCAK. MCAK microtubule depolymerization activity is inhibited by Aurora B-dependent phosphorylation, but the mechanism of this inhibition is not understood. Results Here we develop the first FRET-based biosensor for MCAK and show that MCAK in solution exists in a closed conformation mediated by an interaction between the C-terminal domain (CT) and the neck. Using fluorescence lifetime imaging (FLIM) we show that MCAK bound to microtubule ends is closed relative to MCAK associated with the microtubule lattice. Aurora B phosphorylation at S196 in the neck opens MCAK conformation and diminishes the interaction between the CT and the neck. Using FLIM and TIRF imaging we found that changes in MCAK conformation are associated with a decrease in MCAK affinity for the microtubule. Conclusions Unlike motile kinesins, which are open when doing work, the high affinity binding state for microtubule depolymerizing kinesins is in a closed conformation. Phosphorylation switches MCAK conformation, which inhibits its ability to interact with microtubules and reduces its microtubule depolymerization activity. This work shows that the conformational model proposed for regulating kinesin activity is not universal and that microtubule depolymerizing kinesins utilize a distinct conformational mode to regulate affinity for the microtubule, thus controlling their catalytic efficiency. Furthermore, our work provides a mechanism by which the robust microtubule depolymerization activity of Kinesin-13s can be rapidly modulated to control cellular microtubule dynamics.