Kinesin-5–dependent Poleward Flux and Spindle Length Control inDrosophilaEmbryo Mitosis

Abstract

We used antibody microinjection and genetic manipulations to dissect the various roles of the homotetrameric kinesin-5, KLP61F, in astral, centrosome-controlled Drosophila embryo spindles and to test the hypothesis that it slides apart interpolar (ip) microtubules (MT), thereby controlling poleward flux and spindle length. In wild-type and Ncd null mutant embryos, anti-KLP61F dissociated the motor from spindles, producing a spatial gradient in the KLP61F content of different spindles, which was visible in KLP61F-GFP transgenic embryos. The resulting mitotic defects, supported by gene dosage experiments and time-lapse microscopy of living klp61f mutants, reveal that, after NEB, KLP61F drives persistent MT bundling and the outward sliding of antiparallel MTs, thereby contributing to several processes that all appear insensitive to cortical disruption. KLP61F activity contributes to the poleward flux of both ipMTs and kinetochore MTs and to the length of the metaphase spindle. KLP61F activity maintains the prometaphase spindle by antagonizing Ncd and another unknown force-generator and drives anaphase B, although the rate of spindle elongation is relatively insensitive to the motor's concentration. Finally, KLP61F activity contributes to normal chromosome congression, kinetochore spacing, and anaphase A rates. Thus, a KLP61F-driven sliding filament mechanism contributes to multiple aspects of mitosis in this system. INTRODUCTIONMitosis, the process by which identical copies of the replicated genome are distributed to the products of each cell division, involves a highly dynamic sequence of coordinated motility events, mediated by a bipolar protein machine, the mitotic spindle (Karsenti and Vernos, 2001;Mitchison and Salmon, 2001;Gadde and Heald, 2004;Wadsworth and Khodjakov, 2004;Mogilner et al., 2006;Brust-Mascher and Scholey, 2007;Walczak and Heald, 2008). These motility events are driven by molecular-scale forces generated by mitotic kinesins and dyneins, together with dynamic microtubules (MTs), whose activities are controlled by a network of regulatory proteins, e.g., mitotic kinases, phosphatases, and proteolytic enzymes (Sharp et al., 2000c; BettencourtDias et al., 2004;Maiato and Sunkel, 2004;Rogers et al., 2005;Goshima et al., 2007). Among these mitotic proteins, the kinesin-5 motor is thought to play a key role (Cottingham et al., 1999;Valentine et al., 2006a;Civelekoglu-Scholey and Scholey, 2007).Purified kinesin-5 is a slow, modestly processive, plusend-directed bipolar homotetramer capable of cross-linking adjacent MTs and sliding apart antiparallel MTs in motility assays (Sawin et al., 1992;Cole et al., 1994;Kashina et al., 1996a;Kapitein et al., 2005;Tao et al., 2006;Valentine et al., 2006b;Krzysiak et al., 2008;Van den Wildenberg et al., 2008). In yeast cells the homotetrameric structure of kinesin-5 appears to be essential for mitosis (Hildebrandt et al., 2006), and in Drosophila embryos KLP61F displays dynamic properties consistent with an association with spindle MTs (Cheerambathur et al., 2008) ...

Prime movers: the mechanochemistry of mitotic kinesins

“…Completion of chromosome segregation also involves the elongation of the spindle -a process known as anaphase B. Again, the motor requirements in human cells are not known, but experiments in flies 149 and yeast 150 reveal an important role for kinesin-5 family members. Current models require that these motors slide apart anti-parallel spindle microtubules, generating an outward pushing force on the spindle poles 8 .…”

The Chromosomal Passenger Complex Is Required for Meiotic Acentrosomal Spindle Assembly and Chromosome Biorientation

“…Whether the microtubules connect to the chromosomes though traditional endon kinetochore attachments or lateral attachments, we propose that these microtubules are bundled with central spindle microtubules to achieve biorientation. Interactions between central spindle microtubules and the microtubules with attachments to the chromosomes could be mediated by the kinesin-5 KLP61F (van den Wildenberg et al 2008;Brust-Mascher et al 2009) or by the kinesin-14 NCD (McDonald et al 1990). Indeed, we have shown here that NCD is required for homolog biorientation.…”

Cooperation Between Kinesin Motors Promotes Spindle Symmetry and Chromosome Organization in Oocytes

“…SUB stabilizes the central spindle, which promotes spindle bipolarity, while KLP61F provides MT forces that promote spindle symmetry and chromosome organization, which is partially dependent on a stable central spindle. A strong central spindle stabilized by SUB may explain why loss of KLP61F in oocytes did not result in monopolar spindles, although it remains possible the reduction in KLP61F levels after RNAi, while drastic, was not sufficient to elicit monopolar spindles (Kwok et al 2004;Brust-Mascher et al 2009).…”