Current Biology Vol 24 No 19 R968 humans heterozygous for Rb Myrmeleotettix maculatus found 10. Pardo-Manuel de Villena, F., and Sapienza, C. (2001). Female meiosis drives karyotypic chromosomes can incur fitness costs that the meiotic spindle was evolution in mammals. Genetics 159, in the form of reduced fertility [17,18]. asymmetric — fibers from the egg pole 1179–1189. This sets up an intriguing tension to the equator were measured to be 11. Malik, H.S., and Henikoff, S. (2009). Major evolutionary transitions in centromere in populations, in which Rb approximately three times as long as complexity. Cell 138, 1067–1082. chromosomes are propagated those from the polar body pole [20]. 12. Nanda, I., Schneider-Rasp, S., Winking, H., and Schmid, M. (1995). Loss of telomeric sites in because of their female meiotic Taking advantage of this asymmetry, the chromosomes of Mus musculus advantage but are impeded because of B chromosomes in this species drive domesticus (Rodentia: Muridae) during the associated fitness disadvantages. by positioning themselves on the Robertsonian rearrangements. Chrom. Res. 3, 399–409. Such fitness tradeoffs are likely to eggward side of the spindle. Similar 13. Black, B.E., and Cleveland, D.W. (2011). rapidly select for modifiers that act in to their insights into centromere Epigenetic centromere propagation and the nature of CENP-a nucleosomes. Cell 144, centromere specification or meiosis to strength, driving Rb chromosomes 471–479. alleviate the fitness disadvantage. may help further dissect the 14. Malik, H.S., and Henikoff, S. (2001). Adaptive Indeed, many essential proteins mysterious cell biology of female evolution of Cid, a centromere-specific histone in Drosophila. 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Chromosomal heterozygosity and fertility In these situations, hybrids between 3. Chma´ tal, L., Gabriel, S.I., Mitsainas, G.P., in house mice (Mus musculus domesticus) populations fixed for different Rb Martı´nez-Vargas, J., Ventura, J., Searle, J.B., from Northern Italy. Genetics 150, 1143–1154. Schultz, R.M., and Lampson, M.A. (2014). 18. Daniel, A. (2002). Distortion of female meiotic chromosomes could unleash Centromere strength provides the cell segregation and reduced male fertility in deleterious effects in meiosis, resulting biological basis for meiotic drive and human Robertsonian translocations: karyotype evolution in mice. Curr. Biol. 24, consistent with the centromere model of in chromosomal speciation [19]. 2295–2300. co-evolving centromere DNA/centromeric The results of Chmatal et al. highlight 4. Sandler, L., and Novitski, E. (1957). Meiotic histone (CENP-A). Am. J. Med. Genet. 111, the insight that can be revealed by drive as an evolutionary force. Am. 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Mitotic Kinesins: A Reason to Delve mitotic spindle is an attractive approach for anti-tumor therapeutics, into Kinesin-12 this functional redundancy reduces the probability of finding effective single-target drugs. One promising The failure of kinesin-targeting cancer drugs is thought to result from functional target is kinesin-5 (KIF11 or Eg5), a redundancy of mitotic kinesins. A new study provides mechanistic insights into tetrameric kinesin that plays a key kinesin-12 that help to explain its targeting to kinetochore fibers and its ability role in spindle formation by generating to compensate for inhibition of kinesin-5. forces that separate the two poles. In cell culture, inhibition of Eg5 William O. Hancock redundancy is natural, given the vital results in monopolar spindles and importance of faithfully separating mitotic arrest [1]. The trouble is that The intricate dynamics of mitotic duplicated chromosomes, it hampers in clinical trials, Eg5 inhibitors are spindle morphogenesis involves efforts toward a detailed less effective than hoped, and a many proteins that possess understanding of spindle dynamics. principal reason is thought to be overlapping functions. While this Further, because targeting the this problem of redundancy — other Dispatch R969 motors taking over the function of Eg5, In their new work, Sturgill et al. characterization, in mitotic cells lacking allowing cells to escape mitotic propose an alternative explanation for K-fibers, full-length KIF15 localized to block [2]. the K-fiber localization of KIF15 [6]. pharmacologically-induced non-K- The kinesin-12 motor KIF15 is Instead of localizing through fiber microtubule bundles. Thus, their thought to share some properties of interactions with TPX2, they conclude model is that KIF15 autonomously Eg5, making it a potential target for a that KIF15 naturally localizes to targets microtubule bundles and TPX2 combination therapy with Eg5. Cells microtubule bundles because it is involved in forming or maintaining the are able to complete mitosis if Eg5 is possesses both a secondary (non- bundles rather than direct recruitment inhibited after spindle formation, but motor) microtubule binding domain of the motor. inhibiting both Eg5 and KIF15 results in and an autoinhibitory domain. While this work provides an spindle collapse and mitotic arrest Structurally, KIF15 can be subdivided important dissection of the different [3,4]. In human cells, KIF15 into three domains: the heads, the functional domains of KIF15, there are overexpression confers the ability to proximal coiled-coil (coil-1) and the a number of remaining questions. The undergo cell division following distal coiled-coil (coil-2). Their first question involves the mechanism pharmacological disruption of Eg5, model is that the motor is normally of microtubule sliding. The best providing a clue as to how tumor cells autoinhibited by coil-2, but in understood model is tetrameric Eg5, may develop resistance to Eg5 microtubule bundles the motor which slides antiparallel microtubules inhibitors [5]. These studies have becomes activated such that the heads apart by virtue of its two pairs of provided significant motivation to bind one microtubule and coil-1 binds a motor domains located at each end understand (to delve into) kinesin-12 second microtubule, resulting in of the molecule [12]. One might ask: (KIF15), and in this issue of Current relative sliding of the microtubules in why don’t both pairs of heads just bind Biology, Sturgill, Ohi and colleagues the bundle. to the same microtubule? In other uncover important mechanistic In support of this autoinhibition dimeric kinesins the tail and the heads details of KIF15 that help to understand mechanism, they show in interphase do bind to the same microtubule, its role in spindle formation and cells that deleting coil-2 causes the resulting in enhanced motor maintenance, as well as how it may motor to localize to interphase processivity [9,10]. In Eg5, the answer functionally replace Eg5 under some microtubules, and in vitro the addition appears to be that the stiff tetrameric conditions [6]. of an antibody that binds to coil-2 coiled-coil prevents the two pairs of In mitotic cells, KIF15 localizes to results in robust processive motility. heads from binding to the same K-fibers, bundles of microtubules Finally, hydrodynamic analysis microtubule [13]. But what about that span between the kinetochore suggests the motor can transition KIF15? Sequence analysis and electron and the spindle poles [3,4]. Because between a compact and an extended microscopy suggest that KIF15 has a these K-fibers are the mechanical form. These results all point to coil-2 long discontinuous coiled-coil with a elements through which poleward acting as an autoinhibitory domain, hinge between coil-1 and coil-2. forces on duplicated chromosomes akin to the tail domain of kinesin-1 [8]. Previous studies of PRC1-generated are exerted, they are crucial to the Sturgill et al. also identify a microtubule bundles found that delicate force balance that exists in non-motor microtubule-binding inter-microtubule distances in them are the mitotic spindle. KIF15 knockdown domain in KIF15, with the surprise roughly 35 nm [11]. By sequence causes a decrease in spindle being that instead of it being located analysis, this is well within the length of length, suggesting that KIF15 may at the carboxy-terminal tail of the KIF15 between the heads and the end elongate K-fibers by sliding parallel motor like other kinesins [9,10],itis of coil-1. Perhaps the reason that the microtubules relative to one located in coil-1, between the motor second microtubule binding site in another [3,5]. domains and the autoinhibition KIF15 is located in coil-1 rather than at The new study by Sturgill et al. domain. Supporting this contention, the end of coil-2 is that this central requires a reevaluation of the isolated coil-1 binds microtubules in position provides steric constraints mechanism by which KIF15 localizes pelleting assays, and a construct in that prevent binding to the same to K-fibers. Until now, the consensus which the autoinhibitory coil-2 domain microtubule to which the heads are in the field has been that KIF15 is deleted bundles microtubules in vitro bound, while a distal position would localizes to K-fibers through its in the absence of ATP and slides allow no such constraints. interactions with TPX2 (Targeting microtubules relative to one another in The second question involves the Protein for Xklp2, the Xenopus KIF15 the presence of ATP. mechanism of regulation. In kinesin-1, ortholog) [3,5]. In support of this idea, To address the question of why the motor is proposed to be activated TPX2 is in the nucleus in interphase KIF15 localizes to K-fibers in cells, by cargo binding to the tail domain, when KIF15 does not localize to Sturgill et al. generated microtubule which relieves autoinhibition [8]. cytoplasmic microtubules, and bundles in vitro using a completely Sturgill and colleagues favor a following nuclear envelope breakdown, different protein, PRC1, which mechanism by which there is an TPX2 localizes first to K-fibers, crosslinks antiparallel microtubules equilibrium between the open followed by KIF15 localization [3,5]. into bundles [11]. Full-length KIF15 (activated ) and closed (inhibited) Furthermore, TPX2 knockdown strongly labels PRC1-mediated conformations of the motor that eliminates KIF15 spindle localization microtubule bundles at moderate under normal conditions is shifted [3]. However, evidence is lacking for a motor concentrations and walks strongly towards the inhibited state. direct interaction between KIF15 and processively along the bundles at At the high local microtubule TPX2 in the absence of single-molecule motor concentrations. concentrations found in microtubule microtubules [3,6,7]. Complementing this in vitro bundles, microtubule binding (by the Current Biology Vol 24 No 19 R970 heads or coil-1 or both) is proposed to with their minus-ends located at the References 1. Mayer, T.U., Kapoor, T.M., Haggarty, S.J., lock the motor in an open (activated) poles and plus-ends abutting the King, R.W., Schreiber, S.L., and Mitchison, T.J. conformation. Thus, this is a case of kinetochore [15]. A simple explanation (1999). Small molecule inhibitor of mitotic conformational selection rather than for why knockdown of KIF15 results spindle bipolarity identified in a phenotype-based screen. Science 286, induced fit [14]. A more complete in shorter spindles is that KIF15 971–974. understanding of KIF15 regulation will elongates K-fibers [3,5]. However, this 2. Rath, O., and Kozielski, F. (2012). Kinesins and cancer. Nat. Rev. Cancer 12, 527–539. require further tests of the precise is a difficult geometrical problem. 3. Vanneste, D., Takagi, M., Imamoto, N., and inhibition mechanism as well as Antiparallel microtubule sliding makes Vernos, I. (2009). The role of Hklp2 in exploration of other modes of geometric sense because forces the stabilization and maintenance of spindle bipolarity. Curr. Biol. 19, regulation such as phosphorylation or generated by motors on opposite 1712–1717. binding of coil-2 by activating proteins. microtubules are additive. However, 4. Tanenbaum, M.E., Macurek, L., Janssen, A., Geers, E.F., Alvarez-Fernandez, M., and Before accepting all of the the mechanism by which a motor that Medema, R.H. (2009). Kif15 cooperates with conclusions of Sturgill et al., the spans two parallel microtubules could eg5 to promote bipolar spindle assembly. Curr. experimental results must be reliably slide one with respect to the Biol. 19, 1703–1711. 5. Sturgill, E.G., and Ohi, R. (2013). Kinesin-12 reconciled with a recently published other is more perplexing. If one differentially affects spindle assembly study of KIF15 from the McAinsh lab microtubule has more motors bound to depending on its microtubule substrate. Curr. Biol. 23, 1280–1290. that used similar approaches but it through their tail domains, this would 6. Sturgill, E.G., Das, D.K., Takizawa, Y., Shin, Y., arrived at starkly different conclusions produce a net sliding force between the Collier, S., Ohi, M.D., Hwang, W., Lang, M.J., regarding the quaternary state of KIF15 microtubules. But without this bias, and Ohi, R. (2014). Kinesin-12 Kif15 targets kinetochore-fibers via an intrinsic two-step [7]. Using native gels, gel filtration and motors on opposing microtubules mechanism. Curr. Biol., 2307–2313. photobleaching, Dreschler et al. found should generate equal and opposing 7. Drechsler, H., McHugh, T., Singleton, M.R., Carter, N.J., and McAinsh, A.D. (2014). The that KIF15 tetramerizes in an ionic forces, resulting in zero net sliding Kinesin-12 Kif15 is a processive strength and concentration-dependent force. track-switching tetramer. eLife 3, e01724. manner, while Sturgill et al. used gel Relevant to this, Sturgill et al. 8. Hackney, D.D., Levitt, J.D., and Suhan, J. (1992). Kinesin undergoes a 9 S to 6 S filtration, ultracentrifugation, electron observed sliding in microtubule pairs, conformational transition. J. Biol. Chem. 267, microscopy and photobleaching to but the relative microtubule 8696–8701. 9. Stumpff, J., Du, Y., English, C.A., Maliga, Z., show that KIF15 exists as a dimer. orientations were not specified. Wagenbach, M., Asbury, C.L., Wordeman, L., While experimental conditions differed Drechsler et al. claimed that in the rare and Ohi, R. (2011). A tethering mechanism to some extent, there is no simple way events when unidirectional sliding was controls the processivity and kinetochore- microtubule plus-end enrichment of to account for the dimer/tetramer observed the activity represented the kinesin-8 Kif18A. Mol. Cell 43, discrepancy between the two studies. parallel microtubule sliding, but 764–775. 10. Gudimchuk, N., Vitre, B., Kim, Y., Kiyatkin, A., This is no small point because the because the microtubule orientations Cleveland, D.W., Ataullakhanov, F.I., and different structures lead to contrasting were not verified the result is not Grishchuk, E.L. (2013). Kinetochore kinesin interpretations of how KIF15 might convincing. This question of parallel CENP-E is a processive bi-directional tracker of dynamic microtubule tips. Nat. Cell Biol. 15, generate inter-microtubule forces in versus antiparallel microtubule sliding 1079–1088. cells. Another difference between the was addressed in detail by the Diez 11. Subramanian, R., Wilson-Kubalek, E.M., Arthur, C.P., Bick, M.J., Campbell, E.A., studies was the character of the group for the kinesin-14 motor ncd, Darst, S.A., Milligan, R.A., and Kapoor, T.M. motility. Sturgill et al. observed which also possesses a non-motor (2010). Insights into antiparallel microtubule unidirectional single-molecule microtubule binding site in its tail [16]. crosslinking by PRC1, a conserved nonmotor microtubule binding protein. Cell 142, processivity and robust microtubule By carefully identifying microtubule 433–443. sliding when the motor was activated polarities, they clearly showed that ncd 12. Kapitein, L.C., Peterman, E.J., Kwok, B.H., Kim, J.H., Kapoor, T.M., and Schmidt, C.F. either by truncating coil-2 or by a coil-2 can robustly slide apart antiparallel (2005). The bipolar mitotic kinesin Eg5 moves antibody. In contrast, in single- microtubule pairs, but parallel on both microtubules that it crosslinks. Nature molecule assays Dreschler et al. microtubules only slide short distances 435, 114–118. 13. Scholey, J.E., Nithianantham, S., Scholey, J.M., observed runs in both directions as well before being locked together by and Al-Bassam, J. (2014). Structural basis for as numerous pauses, and observed motors bridging the two filaments. This the assembly of the mitotic motor Kinesin-5 into bipolar tetramers. eLife 3, e02217. no clear sliding between pairs of type of investigation needs to be 14. Hammes, G.G., Chang, Y.C., and Oas, T.G. microtubules. Based on the Sturgill undertaken for KIF15. (2009). Conformational selection or induced work, this lack of microtubule sliding What seems clear is that KIF15 fit: a flux description of reaction mechanism. Proc. Natl. Acad. Sci. USA 106, may result from coil-2 inhibiting motor possesses both a non-motor 13737–13741. activity. A conservative conclusion microtubule binding domain and an 15. Groen, A. (2013). Microtubule motors: a new hope for kinesin-5 inhibitors? Curr. Biol. 23, from theses studies is that KIF15 is inhibitory domain, and due to this R617–R618. able to tetramerize under some modularity it can autonomously target 16. Fink, G., Hajdo, L., Skowronek, K.J., conditions, but a simple dimer is microtubule bundles such as K-fibers. Reuther, C., Kasprzak, A.A., and Diez, S. (2009). The mitotic kinesin-14 Ncd drives directional sufficient to generate microtubule While we await development of small microtubule-microtubule sliding. Nat. Cell Biol. sliding. Resolving these discrepancies molecule inhibitors of KIF15 that can 11, 717–723. should be at the top of the experimental be tested in combination with Eg5 to-do list. inhibitors, these mechanistic insights Department of Biomedical Engineering, Another intriguing question is what into KIF15 will further our Pennsylvania State University, 229 Hallowell KIF15 is actually doing in K-fibers, or understanding of how motors, Building, Pennsylvania State University, more specifically: can KIF15 slide microtubules and regulatory proteins University Park, PA, USA. E-mail: [email protected] apart both parallel and antiparallel work together to create the beautiful microtubules? K-fibers are thought to and vital structure that is the mitotic be bundles of parallel microtubules spindle. http://dx.doi.org/10.1016/j.cub.2014.09.011