Molecular mechanisms of kinesin-14 motors in spindle assemblyand chromosome segregationZhen-Yu She and Wan-Xi Yang*
ABSTRACTDuring eukaryote cell division, molecular motors are crucialregulators of microtubule organization, spindle assembly,chromosome segregation and intracellular transport. The kinesin-14motors are evolutionarily conserved minus-end-directed kinesinmotors that occur in diverse organisms from simple yeasts to highereukaryotes. Members of the kinesin-14 motor family can bind to,crosslink or slide microtubules and, thus, regulate microtubuleorganization and spindle assembly. In this Commentary, wepresent the common subthemes that have emerged from studies ofthe molecular kinetics and mechanics of kinesin-14 motors,particularly with regard to their non-processive movement, theirability to crosslink microtubules and interact with the minus- and plus-ends of microtubules, and with microtubule-organizing centerproteins. In particular, counteracting forces between minus-end-directed kinesin-14 and plus-end-directed kinesin-5 motors haverecently been implicated in the regulation of microtubule nucleation.We also discuss recent progress in our current understanding of themultiple and fundamental functions that kinesin-14 motors familymembers have in important aspects of cell division, including thespindle pole, spindle organization and chromosome segregation.
IntroductionOne of the most fundamental activities of life is the reproduction andthe propagation of cells, which depends on accurate cell division.During cell division, the genetic material of the cell is duplicatedand faithfully segregated into two daughter cells. This processoccurs in virtually all cells, from prokaryotes to higher eukaryotesbut excluding many terminally differentiated cell types (Brust-Mascher and Scholey, 2011; Holland and Cleveland, 2009). Ineukaryotic cells, division follows mitosis, which includes spindleformation and nuclear envelope breakdown in higher eukaryotes−except yeast, which undergo closed mitosis (Güttinger et al., 2009).Once the interactions between microtubules within spindle andchromosomes are established, the chromosomes congress at themetaphase equatorial plate and, ultimately, move towards theopposite spindle poles (Güttinger et al., 2009; Pavin and Tolic,2016; Tanaka and Desai, 2008). The genomic stability of theeukaryotic cell relies on error-free segregation of chromosomesduring mitosis and meiosis (Aguilera and Gómez-González, 2008).Accurate segregation of chromosomes is fulfilled by twocomplementary mechanisms: chromosome-to-pole movements
and elongation of the mitotic spindles (Brust-Mascher andScholey, 2011; Brust-Mascher et al., 2015). Finally, the nuclearenvelope is reformed around chromatin and the cytoplasm ispartitioned to form two daughter cells during cytokinesis(Schellhaus et al., 2016).
Eukaryotes have evolved a specialized microtubule cytoskeletonfor chromosome segregation that is distinct from bacterialchromosome segregation (Nogales et al., 1998; Dye and Shapiro,2007; Scholey et al., 2003). In eukaryotes, specialized tubulinproteins assemble into polar microtubules (Nogales, 2000). Here,ensembles of cytoskeletal proteins at microtubule minus- (Wieseand Zheng, 2006) and plus- (Akhmanova and Steinmetz, 2008)ends together with multiple microtubule-associated proteins(MAPs) (Marx et al., 2006) and motile molecular motors(Lawrence et al., 2004; Vicente and Wordeman, 2015) actcooperatively to regulate mitosis with high fidelity (see Box 1).Molecular motor kinesins and dynein exert complex roles toregulate cytoskeleton dynamics, spindle morphogenesis andchromosome movement (Schliwa and Woehlke, 2003; Vicenteand Wordeman, 2015). The discovery of microtubule-associatedkinesin motors (Vale et al., 1985a,b) and their roles as crucialregulators of microtubule organization and chromosomesegregation during the cell cycle (Gatlin and Bloom, 2010;Scholey et al., 1985; Wordeman, 2010) was the beginning of anew era in our understanding of the molecular mechanisms of celldivision.
Generally speaking, the kinesin superfamily includesconventional kinesins as well as kinesin-like proteins that werediscovered later and include the so-called N-terminal motor andC-terminal kinesin motor proteins (Vale and Fletterick, 1997);however, the morphological complexity of kinesins is much greater(Cross and McAinsh, 2014; Hirokawa et al., 2009; Lawrence et al.,2004; Wordeman, 2010). In contrast to plus-end-directed kinesins,kinesin-14 motors are specific minus-end-directed motors; theyutilize the chemical energy of ATP hydrolysis to move alongmicrotubules from their plus- to their minus-end (Friel and Howard,2012; Schnitzer and Block, 1997; Shimizu et al., 1995). Thekinesin-14 family is ubiquitous to all eukaryotes, with typically twoto three members present that are different in their structure andfunction (Olmsted et al., 2015; Simeonov et al., 2009).
In this Commentary, we discuss the common and distinct rolesof kinesin-14 family motors in model organisms. We focuson microtubule-associated functions, such as non-processivemovement on microtubules and roles in microtubule crosslinkingor sliding. We shed light on the molecular interactions of kinesin-14members and their associated interaction factors, such as γ-tubulin,the plus-end microtubule protein EB1 (also known as MAPRE1 inhumans) and the nuclear import machinery.Wewill also summarizethe mutual antagonisms between kinesin-14 and kinesin-5 familymembers during spindle assembly, and discuss new roles of
The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou310058, China.
kinesin-14/-5 motors in microtubule nucleation. We, therefore, aimto provide a comprehensive summary of the cellular functions ofkinesin-14 family members in various processes, such asmicrotubule nucleation, microtubule focusing, mitotic spindleorganization and chromosome segregation during mitotic andmeiotic events.
Discovery, classification and structure of the minus-end-directed kinesin-14 motorsIn 1929, Alfred Henry Sturtevant observed for the first timedefective chromosome segregation in the claret non-disjunctional(ncd) mutant of Drosophila simulans (Sturtevant, 1929; Davis,1969). Subsequently, this locus was cloned and found to encode amitotic/meiotic kinesin in Drosophila (Endow et al., 1990;McDonald and Goldstein, 1990). The researchers revealed thatDrosophila kinesin-14 Ncd is unusual as it moves frommicrotubuleplus-ends towards the minus-ends in motility assays (McDonaldet al., 1990; Walker et al., 1990). In budding yeast, another kinesin-14, Kar3, was independently identified as a motor crucial fornuclear fusion and meiosis by using genetic approaches (Meluh andRose, 1990).The idea of a standardized kinesin nomenclature (Lawrence et al.,
2004) builds on other lineage studies of kinesins (Dagenbach andEndow, 2004; Kim and Endow, 2000; Lawrence et al., 2002; Mikiet al., 2001; Moore and Endow, 1996) and categorizes the kinesinsuperfamily into 14 families with some outliers. The kinesin-14family consists of two subfamilies kinesin14-A and kinesin14-B,although not all their members have so far been included inphylogenetic studies. Within these subfamilies further functionalspecification of kinesin-14 family members is evident beyond thissimple division (Olmsted et al., 2015). In this Commentary, wefocus on the best-studied kinesins, mostly members of the kinesin-
14A family. To distinguish the different species, we refer to thesekinesin-14 motors as Homo sapiens HSET (officially known asKIFC1), Mus musculus KIFC1, Xenopus laevis XCTK2 (officiallyknown as Kifc1), Drosophila melanogaster Ncd, Arabidposisthaliana ATK5, Schizosaccharomyces pombe Pkl1 and Klp2, andSaccharomyces cerevisiae Kar3. Members of the kinesin-14Bfamily (kinesin family members C2 and C3), such as H. sapiensKIFC2 and KIFC3, respectively; A. thaliana KatD (KIN14G) andthe plant KCBP proteins (Lawrence et al., 2002; Wickstead andGull, 2006) have been less-well studied. Moreover, a functionalcomplexity of kinesins has been predicted in recent computationalstudies, supporting a kinesin diversity that reflects the complexity ofthe microtubule cytoskeleton (Wickstead et al., 2010).
The kinesin-14 proteins comprise three functional domains, anN-terminal tail domain, a central coiled-coil stalk domain and aC-terminal motor domain (Cross and McAinsh, 2014; Vale andFletterick, 1997) (Fig. 1A). The globular motor domain contains the‘catalytic core’ for ATP hydrolysis and the microtubule-bindingsite, both of which generate forces for mechanical movements andmotor processivity (Friel and Howard, 2012; Hirokawa and Noda,2008; Schnitzer and Block, 1997). The short and conserved neckregion proximal to the catalytic domain functions as a mechanicaltransducer to regulate directionality (Case et al., 1997; Yamagishiet al., 2016). In Drosophila, the neck region at the proximal end ofthe coiled-coil stalk domain, adjacent to the motor, is essential forthe regulation of lever-like rotations and step size (Hallen et al.,2011). Moreover, biochemical studies of Ncd stalk peptides (Itoet al., 2006) and additional studies (Makino et al., 2007) indicate thepresence of sequence features that generate reversible andirreversible regions within the Ncd coiled-coil region. It has beenproposed that a partial collapse of the Ncd stalk prevents brakingthat can occur when multiple Ncd motors are bound to amicrotubule but without synchronized ATP hydrolysis andforcestroke generation (Makino et al., 2007). Several lines ofevidence point to the tail domain as providing some of the mostcrucial functional cues to diversify kinesin-14 function. KIFC5Ahas an additional ATP-independent microtubule-binding domain(Zhang and Sperry, 2004) and Ncd has a positively chargedsequence in its tail domain that tethers it to the C-terminal E-hook oftubulin (Furuta and Toyoshima, 2008). Mouse KIFC1 has taildomain sequences that target it to membrane-bound organelles(Zhang and Sperry, 2004); the tail domain of Pkl1 direct it toγ-tubulin (Olmsted et al., 2013, 2014). Additional studies in whichchimeric kinesin-14 proteins of human HSET, Drosophila Ncd andfission yeast Pkl1 were generated also support this paradigm andconfirm that tail domain sequences orchestrate function (Simeonovet al., 2009).
Molecular kinetics of kinesin-14 motors in microtubulebinding, stepping, crosslinking and slidingKinesin motors are highly processive motors that convert thechemical energy of ATP hydrolysis into long-range directedmotility traversing more than 50 steps. They undergo multiplecatalytic cycles along microtubules before their detachment andrelease (Asbury et al., 2003; Yildiz et al., 2004). In a study of humankinesin, in which one of the heads had been mutated, alternating fastand slow stepping of the heterodimeric kinesin was observed insupport of a ‘hand-over-hand’ model versus inchworm movement(Kaseda et al., 2003). In contrast, comparative analysis of themotility of homodimeric Ncd or the heterodimeric kinesin-14motors Kar3−Vik1 and Kar3−Cik1 indicates that they are non-processive as single motors and only move 20−50 nm, which
Box1. General characteristics of microtubules andmitotic spindlesMicrotubules are hollow polymers of α- and β-tubulin heterodimers(Burns, 1991; Weisenberg et al., 1968). Typically, a microtubule iscomposed of 13 linear subunits (protofilaments) and has a diameter of25 nm (Ledbetter and Porter, 1963; Snyder and McIntosh, 1976). Incells, the behaviors of their plus- and minus-ends differ. The plus-end iscapped by β-tubulin and assembles faster, whereas the minus-end iscapped by α-tubulin and depolymerizes slower (Martin et al., 1993). Theplus-ends of microtubules capture the kinetochore on the chromosomesand also interact with the cell cortex (Mitchison and Kirschner, 1985). Incontrast, the minus-ends are more stable in cells and usually cluster inthe centrosome (Mejillano et al., 1990).
The microtubule organizing center (MTOC) utilizes the so-calledγ-TuRC template to regulate the organization of α-tubulin−β-tubulinheterodimers into microtubules (Nogales, 2001). In fibroblast cells orother cell types with a radial array of microtubules, almost all microtubulescluster with their minus-ends embedded in the centrosome (Desai andMitchison, 1997). In several differentiated cells, including epithelial cells orneurons, non-centrosomal microtubules predominate and microtubulesare arranged in parallel or anti-parallel to one another (Akhmanova andHoogenraad, 2015; Keating and Borisy, 1999; Simeonov et al., 2009;Yvon and Wadsworth, 1997).
Mitotic spindles are composed of three main types of dynamicmicrotubule: (i) astral microtubules that are generated from the MTOCand extending towards the cell cortex (Reinsch and Gönczy, 1998);(ii) interpolar microtubules that overlap at the midzone with antiparallelorientation (Cai et al., 2009b; Hentrich andSurrey, 2010); (iii) kinetochorefibers (K-fibers) that are generated by centrosome capture through thekinetochore (Hayden et al., 1990; Maiato et al., 2004).
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– +
– +
Parallel microtubules
+ –
– +
Antiparallel microtubules
Overlap microtubules
Clustering microtubules
+ –
Statically crosslinking Robust sliding
Minus-end clustering and pole focusing
Sliding and align
A
N-terminal tail domain Coiled-coil stalk C-terminal motor domain
Tail domain
Coiled-coil stalk
Motor domain
Kinesin-14 motors Structural domains
B
Minus-end (–)Plus-end (+)
C
EB1
Non-processive
(+)
Hold-and-release
Minus-end clustering
Cargo
Tail-mediated binding Tail-dependent diffusion
-tubulin
MiMi
Team work Cargo Tug-of-war
MT-binding site
SxIP motif
Catalytic core
Eg5
Loading
Neck linker
ATPase cycleDirectional motility
DimerizationCargo binding
NLS
Overlap microtubules
– ++
Overlap microtubules
+ –
Search and capture, and robust sliding
–
Minus-end directed motility Plus-end tracking
Crosslinking, align and powerstroke
+ –
MTOC localization
Microtubule nucleation
MT-binding site
+
+
+
+
+
+–
Fig. 1. The structural features and molecular kinetics of kinesin-14 motors. (A) Schematic representations of the structural domains of kinesin-14 motors.(B) Molecular kinetics of kinesin-14 motors on microtubules. Kinesin-14 proteins are non-processive motors that walk from the plus-end to the minus-end of themicrotubules. Cooperative teamwork of kinesin-14 motors can stimulate their processivity. The tail-mediated interaction between kinesin-14 motors andmicrotubules is required for static binding, diffusional motion, MTOC localization and microtubule nucleation. Not all kinesin-14 members have a microtubule-binding site in their tail domain. Physical interaction between kinesin-14 and γ-tubulin or EB1, or other EB homologs is a general mechanism for the accumulationof kinesin-14 motors at the microtubule minus- or plus-end, respectively. (C) Kinesin-14 motors are enriched at regions where microtubules overlap; this ismediated by binding of both their motor- and tail-domains. Kinesin-14 motors can either statically crosslink parallel microtubules (top left) or slide antiparallelmicrotubules immediately after their attachment (top right). Furthermore, kinesin-14 motors can focus the minus-ends of microtubules to initiate cluster formation(middle left). Kinesin-14motors can search and capture the antiparallel microtubules to mediate robust sliding (middle right). Kinesin-14 motors can also crosslinkand align overlapping microtubule in the same direction (bottom left). Finally, kinesin-14 motors can slide and align overlapping microtubule in different directions(bottom right). Dashed or solid arrows show forces in the same or opposite direction, respectively. Three additional features of kinesin-14 motors − heterodimericstates, checkpoint roles and collapsible stalks − are not illustrated here but described in the main text.
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corresponds to approximately two to six 8-nm steps (Foster andGilbert, 2000; Zhang et al., 2015). In the same study, a hold-and-release mechanism for kinesin-14 motors was proposed in contrastto the hand-over-hand model established for conventional kinesins(Zhang et al., 2015). To date, three main means used by kinesin-14motors to generate weakly processive movements have beendescribed (Fig. 1B). First, the non-motile microtubule-binding siteat the tail domain increases the affinity to the microtubule latticesand, thus, increases both the dwell time and the ATP-independentdiffusion steps of kinesin-14 motors on the microtubules. This isreferred to as the ‘tail-dependent diffusion manner’ that allowsgliding of the tail region along the microtubule due to the thermalfluctuation and the weak interactions that prevent the tail fromsticking to and dissociating from the microtubules (Fink et al.,2009). Second, kinesin-14 motors can work in groups to stimulatetheir processivity along the microtubules. For example, two coupledNcd motors can continually walk along the microtubule for morethan 1 μm (Furuta and Toyoshima, 2008; Furuta et al., 2013). Asingle kinesin-14 Pkl1 is not highly processive and shows one-dimensional diffusion along the microtubules. However, severalPkl1 motors (approximately ten molecules) can work in acooperative manner to move along the microtubules for severalstepping cycles (Furuta et al., 2008) (Fig. 1B). Third, theinteractions with partner proteins can also stimulate theirprocessivity. For example, the budding yeast kinesin-14 Kar3generates processive movement through its interactions with thenon-motor proteins Cik1 or Vik1 (Manning et al., 1999; Miecket al., 2015) as demonstrated by deletion of the non-catalytic domainof Cik1, resulting in increased diffusion of the Kar3−Cik1heterodimer away from the microtubule. Velocity differences werealso noticed dependent on the heterodimeric partner.The kinesin-14 motors are found in dimeric form, and, in most
species, the majority are homodimers (Fig. 1A), with the exceptionof several yeast species, for instance the heterodimers Kar3-Cik1and Kar3-Vik1 in budding yeast (Barrett et al., 2000; Duan et al.,2012) and in Candida glabrata (Joshi et al., 2013).Kinesin-14motors display a slow velocity towards the microtubule
minus-end compared with the plus-end-directed conventional kinesin(42.7±2.19 nm/s) (EndowandWaligora, 1998). For example, a singleNcd−Ncd dimer has a velocity of 15.2±0.3 nm/s (Endow andWaligora, 1998), single Pkl1−Pkl1 homodimer a velocity of33±9 nm/s (Furuta et al., 2008) and Kar3−Cik a velocity of 77±23 nm/s (Mieck et al., 2015). The adjacent neck-motor junction isrequired for kinesin-14 Ncd minus-end movement (Endow andWaligora, 1998). The swinging motion of the C-terminal ‘neckmimic’ (especially the conserved AxxVNxT/C residues; in which xrepresents any amino acid) and the ATPase-dependent conformationalchanges of Ncd also yield a directional bias and movements towardstheminus-end (Yamagishi et al., 2016). Kinesin-14motors also utilizeminus-end-directed forces and ATP-mediated powerstrokes tocrosslink microtubules or to slide antiparallel microtubules(Fig. 1C). In addition, ADP release is slow in Ncd and, thus, is arate-limiting step in its ATPase cycle (Foster and Gilbert, 2000).According to the conventional powerstroke mechanism (i.e. one
kinesin head results in turnover of one ATP molecule) for themotility of a single kinesin-14 motor (Endres et al., 2006; Gonzalezet al., 2013; Wendt et al., 2002), only one motor head needs tointeract with microtubules and turnover of only one ATPmolecule isnecessary for the powerstroke, which combines both rotation andATP turnover to slide the microtubules (Chen et al., 2012).However, a newATP-promoted powerstroke hypothesis that impliestwo kinesin heads and turnover of two ATP molecules has been
proposed for heterodimeric Kar3−Vik1 and Kar3−Cik1, as well ashomodimeric Ncd−Ncd (Zhang et al., 2015). The interactionsbetween both Ncd heads and microtubules depend on the nucleotidestate but interactions between either Vik1 or Cik1 and microtubulesare regulated by strain (Zhang et al., 2015). Furthermore, turnover oftwo ATP molecules is required for interaction with microtubules aswell as force generation in the case of the kinesin-14 motors thatbind to the adjacent microtubule lattices (Endres et al., 2006;Gonzalez et al., 2013; Kocik et al., 2009; Zhang et al., 2015).
For microtubule-organizing functions, such as crosslinking ofparallel microtubules, sliding of antiparallel microtubules andfocusing of the spindle pole, both the motor and tail domains ofkinesin-14 motors are required (Fig. 1C) (Wendt et al., 2002). Anexception to this is Pkl1, for which the tail domain was shown to beable to regulate nucleation without the motor domain in vitro and invivo (Fig. 1B), and to do so in yeast and human cells (Olmsted et al.,2013, 2014). Microtubule-dependent D. melanogaster Ncd motorshave been shown to localize to parallel microtubules where theyexert their movement into the opposite direction, thus creating a‘tug-of-war’ effect. This results in static crosslinking and bundlingof parallel microtubules. However, Ncd can also promote efficientsliding of microtubules that have opposite polarity through theso-called ‘directional sliding’ mechanism (Fink et al., 2009)(Fig. 1C). Furthermore, in case of the X. laevis XCTK2, it hasbeen demonstrated that, if the number of kinesins bound tomicrotubules is too high, gliding speed is significantly reduced,presumably owing to the non-synchronized state of multiple boundmotors (Hentrich and Surrey, 2010). As mentioned previously, thepresence of reversibly stable regions that can collapse in the coiled-coil domain (Hallen et al., 2008, 2011; Ito et al., 2006;Makino et al.,2007) might be able to partially compensate for any braking thatresults from unsynchronized power force generation.
Interactions between kinesin-14 motors and associatedproteins during cell divisionDuring mitotic spindle assembly, a subset of kinesin-14 membersappears to exert two functions: organization of microtubule minus-ends at poles and/or microtubule nucleation, both of which aremediated by direct interactions of kinesin-14 members withcomponents of the microtubule organizing center (MTOC) and theγ-tubulin ring complex (γ-TuRC), although with different outcomes.
Interactions of MTOC proteins and microtubule minus-endsDuring spindle formation in HeLa cells, the γ-TuRC is initiallyrecruited to the pole-distal regions and then moves towardsmicrotubule minus-ends around the spindle poles. This process ismediated by cooperation between kinesin-14 HSET, kinesin-5 Eg5(officially known as KIF11 inmammalian cells) and dynein (Leclandand Lüders, 2014). Both H. sapiens HSET and X. laevis XCTK2affect spindle morphology. RNA interference (RNAi) of HSETresults in broader spindles and less focused poles but without anychanges in duration of mitosis or fidelity of chromosome segregation(Cai et al., 2009b). In mouse oocytes, the broader spindles and polesare more evident (Mountain et al., 1999) and, additionally, it has beenshown that HSET can cluster centrosomes (Kwon et al., 2008).
In S. pombe, Pkl1 physically interacts with γ-TuRC through boththe motor and the tail domain, and suppresses microtubulenucleation to influence spindle structure and function at γ-TuRC(Rodriguez et al., 2008, Olmsted et al., 2013, 2014). In its role atγ-TuRC to regulate nucleation, Pkl1 does not absolutely require itsmotor domain or stalk region, and the tail can be reduced to a 30amino-acid-long region and still function (Olmsted et al., 2013,
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2014). Genetic analysis supports this observation as syntheticinteractions have been observed between Pkl1 and the γ-TuRCproteins Alp4 and Alp6 but not with other fission yeast mitoticmotors, and there is no genetic interaction between the second fissionyeast kinesin-14 Klp2 and any γ-TuRC proteins (Tange et al., 2004).In D. melanogaster oocytes, minus-end-directed motility of Ncd
is essential for focusing the spindle pole in meiosis I spindles thatlack γ-tubulin. During meiosis,γ-tubulin interacts with Ncd to formthe microtubule nucleating center at the spindle pole body (Endowand Komma, 1998).The function of spindle pole organization and microtubule
nucleation does not need to be distinct, and some kinesin-14members, such as HSET, might possess both capabilities of spindlepole organization and microtubule nucleation (Walczak et al., 1997;Simeonov et al., 2009). It is worth noting that in meiotic cells and insomatic cells, spindles can nucleate around chromosomes in thepresence of a Ran gradient and this may explain in part theevolutionary need for kinesin-14 members to have dual roles withγ-tubulin complexes at the different Ran-GTP concentrations(Khodjakov et al., 2000; Khodjakov and Rieder, 2001).
Interactions with the plus-end microtubule protein EB1Surprisingly, minus-end-directed kinesins-14 are sometimes foundat microtubule plus-ends (Ambrose et al., 2005; Braun et al., 2013;Maddox et al., 2003; Sproul et al., 2005); this is made possiblethrough their association with plus-end-binding proteins.Microtubule plus-ends serve as sites crucial for microtubulecapture, stabilization, spindle orientation (Akhmanova andSteinmetz, 2008; Howard and Hyman, 2003; Lee et al., 2000) andanchoring to specific cellular organelles (Vaughan et al., 2002) or tothe cell cortex (Lansbergen and Akhmanova, 2006; Mimori-Kiyosue et al., 2005) (see Box 2).In vitro microtubule-gliding assays demonstrated that H. sapiens
HSET accumulates at the plus-ends of microtubules through directinteractions with EB1 (Braun et al., 2013) (Fig. 1B). Similarly, inthe absence of EB1, D. melanogaster Ncd does not accumulate ateither end of antiparallel microtubules (Fink et al., 2009). The tip-tracking property of Ncd is regulated by two concurrentmechanisms: first, the direct binding of Ncd to microtubule-bound EB1 and, second, the interactions of the Ncd tail domain witha composite site that is generated by EB1 and the microtubulesurface (Szcze sna and Kasprzak, 2016). In S. pombe, kinesin-14Klp2 is localized to the microtubule plus-ends through interaction oftwo of its SxIP motifs with Mal3 (a yeast EB1 homologue) (Mana-Capelli et al., 2012).Taken together, these studies suggest that the EB1-dependent plus-
end tracking, which occurs in diverse organisms, is a generalmechanism for several members of the kinesin-14 family. Anexception is S. cerevisiae Kar3−Vik1 that does not accumulate atmicrotubule plus-ends but, instead, is found along the microtubulelattice (Allinghamet al., 2007;Maddoxet al., 2000;Sproul et al., 2005).
Interactions with the nuclear import machineryA number of kinesins-14, including H. sapiens HSET (Cai et al.,2009b), R. norvegicus KIFC1 (Yang and Sperry, 2003), X. laevisXCTK2 (Cai et al., 2009b), D. melanogaster Ncd (Goshima andVale, 2005) and S. pombe Klp2 (Troxell et al., 2001) contain anuclear import signal (NLS) and show a nuclear localization patternduring interphase. This localization is achieved through interactionsbetween their bipartite NLS at the tail domain with importin-α or -βand nuclear import via a Ran-GTP/GDP-mediated pathway (Caiet al., 2009b).
In Xenopus egg extracts, the small GTPase Ran-GTP mediatesthe interactions of importin-α or -β and XCTK2 through its bipartiteNLS (Clarke and Zhang, 2008; Ems-McClung et al., 2004). Whenthe physical Ran-GTP gradient gradually diminishes from thechromosomes to the spindle pole (Kalab et al., 2002), importin α/βcompetitively binds to XCTK2, reducing its spindle anchoring andincreasing its turnover kinetics during spindle formation (Weaveret al., 2015).
In addition, S. pombe Pkl1 is only a mitotic motor, whereas Klp2has a role in both the mitotic phase and interphase (Carazo-Salasand Nurse, 2006; Daga et al., 2006; Troxell et al., 2001). Moreover,as a heterodimer, the functions of S. cerevisiae Kar3 and itslocalization depends on its binding partner. During mitosis, Cik1selectively dimerizes with Kar3 and is targeted either to the spindlemicrotubules (Hepperla et al., 2014) or to the microtubule plus-ends(Sproul et al., 2005), whereas, in interphase, Kar3−Cik1 localizes tothe nucleus (Manning et al., 1999). By contrast, Vik1 is required forthe localization of Kar3 at the poles of the mitotic spindle duringmitosis (Manning et al., 1999).
In summary, the interaction between kinesin-14 members andmicrotubule minus-ends, microtubule plus-ends and partnerproteins can also influence their subcellular localizations andcellular functions, which are crucial in fulfilling their functions andensure the fidelity of cell division (Yount et al., 2015).
Counterbalancing forces between kinesin-14 motors andkinesin-5 motorsThe bipolar spindle apparatus is maintained by antagonisticrelationships. Accumulating evidence in diverse organisms revealsthat on anti-parallel microtubules, a ‘tug-of-war’ takes placebetween kinesin-14 and kinesin-5 in order to crosslink and slide
Box 2. Microtubule end-binding proteins and plus-endtrackingEnd-binding proteins (EBs), which form characteristic comet-likeaccumulations at microtubule plus-ends (Berrueta et al., 1998;Morrison et al., 1998; Schuyler and Pellman, 2001; Tirnauer andBierer, 2000), are found to be crucial regulators or adaptors of dynamicinteractions of microtubule plus-end-tracking proteins (+TIPs) at thegrowing microtubule ends (Galjart, 2010; Honnappa et al., 2009; Jiangand Akhmanova, 2011). EBs undergo rapid switches of binding andrelease from the microtubule lattices (Jiang and Akhmanova, 2011). Thisis mediated by recognition of specific structural properties of the plus-ends, such as the tubulin sheets (Vitre et al., 2008), the specificprotofilaments (Sandblad et al., 2006) and the GTP cap (Maurer et al.,2012; Zanic et al., 2009).To date, H. sapiens HSET (Braun et al., 2013), D. melanogaster Ncd
(Fink et al., 2009), S. pombe Klp2 (Mana-Capelli et al., 2012) and A.thaliana ATK5 (Ambrose et al., 2005) have shown EB1-dependent plus-end tracking. The interactions between the hydrophobic Ser-x-Ile-Pro(SxIP)motif (in which x can represent any amino acid) ofH. sapiensHSETand the EB homology (EBH) domain of EB1 are responsible for itsmicrotubule plus-end tracking (Braun et al., 2013; Mana-Capelli et al.,2012). In addition, inmanyspecies, an evolutionarily conservedSxIPmotifis located at the tail domain of kinesin-14 motors (Braun et al., 2013).However, the question that remains is: what are the underlying functions
of the minus-end-directed kinesin-14 motors that occur alongside theirmicrotubule plus-end tracking properties during cell division? To date, oneplausible hypothesis is that, inDrosophilaS2cells, the plus-end tracking ofkinesin-14Ncd is required for the search-and-capture of kinetochore fibersor the microtubules that stimulate microtubule crosslinking and bundling,and also facilitate the transport of K-fibers to the spindle pole duringprophase and metaphase (Goshima et al., 2005b). However, theunderlying molecular mechanisms are still unclear.
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microtubules, and is a common mechanism to maintain correctspindle organization, as discussed in detail below (Brust-Mascherand Scholey, 2011; Mountain et al., 1999; Sharp et al., 2000)(Fig. 2). Whereas kinesin-14 motors are dimers, kinesin-5 forms adumbbell-shaped homotetramer with two pairs of motor domainspositioned at the opposite ends (Hentrich and Surrey, 2010; Kashinaet al., 1996; Sawin et al., 1992). The kinesin-5 tetramer utilizes acombination of four motor-domain-binding and four nonmotormicrotubule-binding sites for efficient microtubule crosslinking andsliding (Kapitein et al., 2008; Kaseda et al., 2009; Kwok et al., 2006;Weinger et al., 2011).
The antagonism between kinesin-14 and kinesin-5 also occurson parallel spindle microtubules. First, kinesin-14 motors have theintrinsic ability to focus the minus-ends of microtubules intospindle poles, but kinesin-5 motors counteract focusing of themicrotubule plus-ends at the poles (Hentrich and Surrey, 2010).Second, kinesin-14 motors generate inward pulling forces onspindles, whereas the kinesin-5 motors have been shown togenerate outwards forces on spindles in vitro (Yukawa et al.,2015). Another mechanism by which kinesin-14 and kinesin-5counterbalance their activities does not require the motor domainsbut is mediated primarily by the tail, i.e. the regulation of
A Mammalian KIFC1 vs Eg5 B X. laevis XCTK2 vs Eg5
RanGTP gradient
Importin α/β
Interpolar microtubule
C D. melanogaster Ncd vs KLP61F D S. pombe Pkl1 vs Cut7
Astralmicrotubule
Chromosome
Cell cortex
K-fibers
KIFC1
Eg5
Microtubule aster formation and assembly Directional instability, spindle pole focusing
XCTK2
Eg5
MTOC
Cell cortex
Motor-proteinfriction model
Chromosome
K-fibers
Ncd KLP61F
Fully stochasticforce-balance model
MTOC
Classic force-balance model
Powerstroke
In vitro microtubule gliding, prometaphase pole-polespacing, metaphase spindle assembly
+
++
++
–
Spindle pole body
+++++++++++
Pkl1 Cut7
Microtubule nucleation, spindle pole focusing,mitotic spindle organization
Pkl1 blocks microtubulenucleation
Cut7 opposesthe roles of Pkl1
Klp2
MTOC dependent Microtubule dependent
–
–
––
Fig. 2. Counterbalance between kinesin-14 motors and kinesin-5 motors in diverse organisms. (A) In mammalian, kinesin-14 HSET and kinesin-5 Eg5cooperatively regulate microtubule aster formation, centrosome separation and spindle assembly. (B) In X. laevis oocytes, a directional instability, rather than astable balance of forces, is generated by kinesin-14 XCTK2 and kinesin-5 Eg5 between antiparallel microtubules. A Ran-GTP gradient (yellow) spatially regulatesthe importin-α/-β−kinesin-14 released-bound state during spindle formation. (C) In D. melanogaster cells, three models are used to describe the antagonisticactions of kineisn-14 Ncd and kinesin-5 KLP61F: the classic ‘force-balance’model, the ‘motor-protein friction’model and the new ‘fully stochastic force-balance’model. (D) In S. pombe, the balance of push-and-pull forces (denoted by arrows) between kinesin-14 Pkl1 or Klp2 and kinesin-5 Cut7 in correct spindleorganization either depend on the MTOC or on microtubules. Push-and-pull within the midzone is attributed to Klp2 and Cut7, whereas counteracting forces atminus-ends are mediated by both Pkl1 and Klp2, with those occurring at the spindle pole only generated by Pkl1.
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microtubule nucleation by γ-TuRC, as discussed below (Olmstedet al., 2013, 2014) (Fig. 2).It is worth pointing out that only kinesin-14 is able to focus
spindle poles, but not kinesin-5. In vitro self-organizationexperiments indicate that X. laevis XCTK2 is able to focus theminus-ends of parallel microtubules to asters without the need foradditional factors but kinesin-5 Eg5 is unable to focus themicrotubule minus-ends. Additionally, Eg5 antagonizes XCTK2in focusing of the spindle pole, which slows down the accumulationof XCTK2. Together, this results in the formation of microtubuleasters through an interconnected microtubule network and preventspole formation by the plus-ends of parallel microtubules duringspindle assembly (Hentrich and Surrey, 2010) (Fig. 2B).
Tug-of-war between kinesin-14 and kinesin-5 in spindle assemblyA tug-of-war between kinesin-14 and kinesin-5 motors is a commonmechanism to maintain correct spindle organization in diverseorganisms. For example, inhibition of HSET leads to the disruption ofmicrotubule aster assembly but when kinesin-5 Eg5 is also inhibited,microtubule aster formation, centrosome separation and spindleorganization are all restored. Therefore, a balance between HSET andEg5 is responsible for microtubule aster assembly both in vitro and invivo (Mountain et al., 1999; Kim and Song, 2013) (Fig. 2A).Directional instability, whereby antiparallel microtubules undergomicrometer-range movements back and forth owing to the forcesmediated by other kinesins, rather than a stable balance of forces, isexerted by XCTK2 and Eg5 on antiparallel microtubules due to theasymmetric roles of these antagonistic motors (Hentrich and Surrey,2010) (Fig. 2B). This implies that other motors and spindlemechanisms can work together to form spindles when the kinesin-14–kinesin-5 pair is removed (Mountain et al., 1999). This notion issupported by an observation in fission yeast in which the kinesin-5Cut7 that was thought to be essential (Hagan and Yanagida, 1999) is– together with Pkl1 – dispensable (Olmsted et al., 2013, 2014).In fly, the classic ‘force-balance’ model for bipolar mitotic
spindle formation suggests that spindles are maintained by thebalance between outwards- and inward-sliding forces that aregenerated by kinesin-5 (KLP61F) and kinesin-14 motors (Ncd),respectively (Fink et al., 2009). The force-balance model gainssupports from the findings that both Ncd and KLP61F can crosslinkantiparallel microtubules and slide them to the opposite directions incompetitive microtubule gliding experiments (Fink et al., 2009;Oladipo et al., 2007; Tao et al., 2006) (Fig. 2C).In addition, an increase in the ratio between kinesin-14 and
kinesin-5 motors can give rise to a scenario, whereby passiveresistance slows down the velocity of the advantage motors and thatof their relative movements to each other compared to prior,balanced steady-state. This has been raised in the so-called ‘motorprotein friction’ model for competitive motility (Tao et al., 2006).Kinesin-14 and kinesin-5 family motors balance sliding but slidingis not a continuous process and results in episodes of unstable,oscillatory swings versus balanced braking and sliding by twomotors (Tao et al., 2006). However, this motor protein frictionmodel does not provide a good fit for the phenomenon of a balancepoint in the force-balance model (Fig. 2C).More recently, a new, ‘fully stochastic force-balance’ model’ for
prometaphase spindles in vitro and in vivo has been proposed(Civelekoglu-Scholey et al., 2010). This model incorporatesmicrotubule-motor kinetics and dynamic stability of interpolarmicrotubules, and appears to fit the experimental and theoreticaldata better than the previous motor protein friction model. In thismodel, kinesin-14 Ncd and kinesin-5 KLP61F act synergistically,
thereby providing opposing powerstrokes with load-dependentdetachment, whereby the motors exert a constant force when theyare loaded on microtubules and maintain the correct spacing betweenthe poles. Furthermore, a specific ratio between Ncd and KLP61F isrequired for maintaining the steady-state of prometaphase spindles(Brust-Mascher et al., 2009; Civelekoglu-Scholey et al., 2010; Kwonet al., 2004; Sharp et al., 2000) (Fig. 2C).
In centrosome-controlled Drosophila embryo spindles, kinesin-5KLP61F crosslinks and slides anti-parallel microtubules to mediatethe poleward flux of interpolar and kinetochore microtubules; this isantagonized by kinesin-14 Ncd through mediating spindle collapses(Brust-Mascher et al., 2004; Brust-Mascher et al., 2009). In anastral(i.e. mitotic spindle formation without centrioles) X. laevis eggextracts, kinesin-5 Eg5 slides microtubules to poles and contributesto poleward flux and spindle length control (Miyamoto et al., 2004;Peterman and Scholey, 2009).
Tail-mediated antagonism between kinesin-14 and kinesin-5in S. pombeIn S. pombe, Pkl1 has been shown to directly block microtubulenucleation at γ-TuRC, while the kinesin-5 Cut7 binds to γ-tubulinthrough its motor and BimC domains; this opposes the effect of Pkl1in inhibiting microtubule nucleation. In this case, the microtubule-binding domain of Pkl1 is not required for the regulation ofmicrotubule nucleation (Olmsted et al., 2014) (Fig. 2D).
Another study indicated that Pkl1 cooperates with the spindle-pole-associated protein mitotic spindle disanchored 1 (Msd1) tointeract with γ-TuRC, before focusing the minus-ends ofmicrotubules at the spindle pole body (Syrovatkina and Tran,2015). Here, the deletion of pkl1 results in unfocused microtubuleminus-ends at the spindle poles and in longmicrotubule protrusions.This can be rescued by the ablation of cut7 (Syrovatkina and Tran,2015) (Fig. 2D). In the mitotic spindle pole body, Pkl1 is requiredfor the localization of Msd1 and the mitosis-specific spindle polebody component Wdr8; it cooperatively interacts with these twoproteins to mediate the spindle anchoring, where it counteracts theoutward pushing forces that are generated by Cut7 (Yukawa et al.,2015). In addition, Pkl1 also functions as the spindle assemblycheckpoint, which regulates chromosome bi-orientation bymediating the spindle pole assembly (Grishchuk et al., 2007).Collectively, there are at least three roles for kinesin-14 Pkl1 inyeast. One is the crosslinking and sliding of microtubules. Its secondrole is to mediate microtubule nucleation at the spindle pole infission yeast and its third role is at the checkpoint.
Compartmentalized mitotic and meiotic roles of kinesin-14In this section, we discuss the roles of kinesin-14 motors incentrosome clustering within wild-type cells, solid tumors and inhematologic malignancies, in the regulation of spindle length andmorphology, as well as in chromosome alignment and segregation(Fig. 3).
Acentriolar spindle-pole-focusing in meiotic germ cells or in cellswithout centrosomesIn the oocytes of many species – including human, mouse, frog andfly – meiotic spindles organize in the absence of centrosomes(Dumont and Desai, 2012; Szollosi et al., 1972). In mouse oocytes,which lack conventional centrosomes, KIFC1 inhibition results inan aberrant barrel-shaped spindle during metaphase, as well as inbroad spindle poles, reduced number of astral microtubules and,finally, arrest during metaphase II of meiosis (Mountain et al.,1999). In X. laevis egg extracts, which also lack centrosomes,
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XCTK2 is localized at mitotic spindles, crosslinks parallelmicrotubule and focuses the microtubule minus-ends to mediatemitotic spindle assembly in vitro (Walczak et al., 1997, 1998).
In Drosophila oocytes, the meiosis II spindle forms byreorganization of the meiosis I spindle fibers, rather than by itsdisassembly or breakdown. Here, Ncd stabilizes the newly
++
++
+
++
++
+
A Kinesin-14 in wild-type cells
Metaphase Anaphase Daughter cell
B Kinesin-14 ablation in wild-type cells
D S. pombe kinesin-14 Pkl1 and chromosome motions
C Kinesin-14 ablation in cancer cells with multiple centrosomes
-
++
+
Nucleus
Nucleus Micronucleus
Multinucleated cell
Multiplenuclei
Metaphase Anaphase Daughter cell
Metaphase Anaphase
Lagging chromosome
MidzoneEquatorial plate
Aberrant central spindleShorter, broaderspindle
Multipolarspindle
Prophase Anaphase pkl1∆/∆, anaphase
Chromosomecut
-++++
Bipolar spindle
+
+
++
+
+
+
-
-+
+
+
+
++
+-
-
- +-
--
+-
++
+++
++
+++
------
------
+
- - --
LaggingchromosomeSpindle pole body
Centrosomeamplification
Aneuploidy
Asymmetriccell division
- --
Pkl1
+++++++++++++++++++++++++++++++++++++++++
K-fibers
MTOCγ-tubulin
Spindle polefocusing
Interphase
Misalignedchromosome
Spindlemultipolarity
Correct karyotype
Pkl1 Klp2
+
+
+
+
+
++
+
+
+
+
+ +
+
+
+
+ ++
+
+
+
+
+
+
+
+
+
+
++
+
+
+
+
+
+
+
++
+
+ + +
+
+
+
+
+
-
--
-
-
-
--
-- -
--
--
-
--
-
-
-
--
-
-- - - --
--
-
---
-
-
--
---
Fig. 3. Multiple functions of kinesin-14 motors during cell division. (A) Kinesin-14 motors are important for correct spindle assembly, stable spindle polefocusing and chromosome segregation during mitosis. For meiosis in human and fly, HSET and Ncd, respectively, is required for spindle-pole-focusing andcentrosome clustering through interactions with γ-tubulin. (B) In wild-type cells, kinesin-14 deletion results in defects in bipolar spindle formation, laggingchromosomes, micronuclei formation, aneuploidy and genomic instability. (C) In cancer cells with multiple centrosomes, the ablation of kinesin-14 results in asubstantial increase in multipolar spindles, centrosome clustering defects as well as in multinucleated cells. (D) S. pombe kinesin-14 Pkl1 and Klp2 motors havedistinct or overlapping roles in microtubule nucleation, the focusing of spindle poles and in the crosslinking of microtubules. Deletion of pkl1 results in unequalchromosome distribution, lagging chromosomes and chromosome loss. Defects in chromosome segregation do neither occur with the same frequencies, nor arethey necessarily attributable to the motor function of Pkl1 because it also has a role in the spindle assembly checkpoint. In addition, Klp2 localizes to kinetochoresand also crosslinks microtubules, thus alleviates some of the effects following Plk1 ablation (Gachet et al., 2008).
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nucleated microtubule minus-ends at the spindle poles and alsoaccounts for recruitment of γ-tubulin to the spindle poles(Baumbach et al., 2015; Endow and Komma, 1997). γ-Tubulinand Ncd stabilize the association of the γ-tubulin-recruiting augmincomplex with the polar regions of the spindles and facilitatemicrotubule organization and chromosome congression (i.e. theprocess of aligning chromosomes on the spindle) in oocytes(Colombié et al., 2013).
The roles of kinesin-14 in the regulation of spindle length andmorphologyIn different cell types, the length of the metaphase spindle istypically maintained at a characteristic constant length throughsliding and microtubule−microtubule interactions, as well asthrough microtubule nucleation and dynamics. A constant spindlelength is crucial for accurate chromosome-to-microtubuleattachment and the fidelity of chromosome separation (Goshimaand Scholey, 2010; Goshima et al., 2005b; Hepperla et al., 2014;Sharp et al., 1999; Syrovatkina et al., 2013).In HeLa cells, ablation of HSET function leads to broader and
shorter spindles, with an 18% decrease in distance between thepoles compared to that of wild type. Mechanistically, HSETlengthens the spindle by the applying outward forces to slide themicrotubules and modulates spindle organization by thecrosslinking and sliding of mitotic spindles (Cai et al., 2009b)(Fig. 3A-C; Table 1).However, other studies indicate that kinesins-14 generate a pull
force on spindles. D. melanogaster Ncd is required for themaintenance of spacing between the duplicated centrosomes atprometaphase and metaphase through the pull forces, and also forthe positioning of centrosomes at anaphase and telophase (Kwonet al., 2004; Sharp et al., 1999, 2000) (Table 1). Similarly, inA. thaliana, the mitotic spindles of atk5−/− mutants exhibit greaterdistances in mean midzone length, splayed spindle poles, thespindle broadening phenotype and an increased duration duringprometaphase (Ambrose and Cyr, 2007; Ambrose et al., 2005)(Table 1).A recent study suggested a new model based on the deletion of
Kar3, Cik1 or Vik1 in budding yeast, which results in shorter andseverely misaligned spindles, and a dysfunctional spindle midzoneat metaphase. Kar3−Cik1 is thought to improve the alignmentefficiency of the interpolar microtubules in the midzone. This thenallows the plus-end-directed kinesin-5 Cin8 to exert the outwardforces on anti-parallel microtubules that are required for correctspindle bipolarity (Hepperla et al., 2014) (Table 1).
The roles of kinesin-14 motors in chromosome congression,alignment and segregationHuman HSET has three possible roles in chromosome movementand dynamics during mitosis (Fig. 3A-C). The first is that HSET isnot essential for the mitotic spindle assembly in cultured cells withcentrosomes and that the role of kinesin-14 HSET is substituted bythe centrosomes and NuMA (nuclear mitotic apparatus protein).The perturbation of HSET only causes a slightly prolongedprometaphase, which does not show obvious disruptions tocharacteristic chromosome dynamics, including those ofchromosome movement toward spindle equator, congression,oscillation and separation (Gordon et al., 2001; Mountain et al.,1999).The second possible role for HSET is in the regulation of
chromosome alignment and segregation during mitosis (Kim andSong, 2013; Zhu et al., 2005). Depletion of HSET in HeLa
epithelial cells or IMR-90 fibroblasts leads to several misalignedchromosomes at the spindle equator and, owing to the formation ofmultipolar spindles, results in lagging chromosomes, micronuclei,genomic instability in daughter cells (Kim and Song, 2013; Zhuet al., 2005) (Fig. 3B,C).
The third possible role for HSET is to facilitate the correct end-onattachment of kinetochores to K-fibers through a search-and-capturemechanism. This is fulfilled by interactions with regulatory subunitsof the B56 family of protein phosphatase 2A (B56-PP2A; officiallyknown as PTPA) or with kinetochore protein NDC80 homolog(NDC80) at the kinetochores in Hela cells under conditions whereany end-on kinetochore attachments are lacking (Cai et al., 2009a;Xu et al., 2014).
During mitosis and meiosis in D. melanogaster, kinesin-14 Ncdis necessary for correct chromosome distribution and configuration,and Ncdmutants show separated or scattered chromosomes. Duringmeiosis, nucleation occurs from Ncd-associated microtubule astersto generate spindle-associated bivalent chromosomes (Hatsumi andEndow, 1992; Matthies et al., 1996; Sköld et al., 2005).
Deletion of pkl1 in fission yeast only partially interferes withchromosome congression in the spindle midzone and abrogates themitotic checkpoint that mediates chromosome bi-orientation(Grishchuk et al., 2007). Mutation of pkl1 or simultaneousmutation of pkl1 and kinesin-5 encoding cut7 lead to three maindefects in chromosome segregation (1) unequal chromosomedistribution, (2) lagging chromosomes and (3) chromosome loss(Olmsted et al., 2014). In the absence of Pkl1, Cut7-dependentsliding forces mediate the long spindle microtubules protrusion atthe minus-ends and, so, push segregated chromosomes to the site ofcell division, resulting in chromosome breaks and chromosome loss(Syrovatkina and Tran, 2015) (Fig. 3D). Another S. pombe kinesin-14, Klp2, is also involved in nuclear positioning during interphase,organization of bipolar microtubules, chromosome alignment andanaphase spindle elongation (Akera et al., 2015; Braun et al., 2009;Mana-Capelli et al., 2012) (Fig. 3D).
In many solid tumors and in hematological malignancies, anincrease in centrosome numbers, a common feature referred to ascentrosome amplification, often strongly correlates withaneuploidy, chromosomal instability and malignant behaviors(Acilan and Saunders, 2008; Chandhok and Pellman, 2009; Nigg,2002; Kwon et al., 2008). A crucial cellular mechanism forminimizing the deleterious consequences of multiple centrosomesis the clustering of the extra centrosomes into bipolar spindles toavoid the adverse levels of aneuploidy during mitosis (Acilan andSaunders, 2008; Basto et al., 2008; Holland and Cleveland, 2009).
In Drosophila S2 cells, depletion of kinesin-14 Ncd results indefects of centrosome clustering and in the numbers of multipolarspindles, especially in cells with supernumerary centrosomes (Bastoet al., 2008; Kwon et al., 2008). There is a similar mechanism inmammalian cancer cells because human HSET is indispensable forfocusing of acentrosomal spindle poles, bipolar spindleorganization, bipolar cell division and the survival of cancer cellswith extra centrosomes, although it has no obvious effect on wild-type diploid cells (Chavali et al., 2016; Kleylein-Sohn et al., 2012;Kwon et al., 2008; Mittal et al., 2016) (Fig. 3A-C).
Conclusions and future directionsIn this Commentary, we mainly focused on the molecular kineticsand mechanisms of the minus-end-directed kinesin-14 motors incell division. We have also discussed the counterbalance betweenkinesin-14 and kinesin-5 motors that is prevalent on both paralleland antiparallel microtubules, as well as at the MTOC during
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Table1.
Kines
in-14motorsregu
late
spindleleng
than
dmorph
olog
y
Kines
inOrgan
ism
Celltyp
e/system
Metho
d
Cha
nges
insp
indleleng
thCha
nges
inMT
dyna
mics(lo
ss-of-
func
tion)
Effe
ctson
thesp
indle
morph
olog
y(lo
ss-of-
func
tion)
Referen
ces
Wild
type
Loss-of-func
tion
HSET(KIFC1)
H.s
apiens
HeL
aRNAi
14.3±0.5μm
11.7±0.4μm
Red
uced
outward
force
Sho
rter,thinn
ersp
indles
;broa
dersp
indlewidth
Cai
etal.,20
09a,b;
Cai
etal.,
2010
.
XCTK2(Kifc1)
X.lae
vis
XL1
77mito
tic/m
eiotic
extrac
t
Antibod
yinhibitio
n60
μm,
48/30−
32μm
Not
detected
Spind
leinstab
ility
∼70
%redu
ctionof
bipo
lar
spindles
,abn
ormal
spindlestructure
Gos
himaan
dSch
oley
,201
0;Walczak
etal.,19
97;W
alczak
etal.,19
98.
Ncd
D.m
elan
ogas
ter
S2
RNAi
11.5±2.0μm
3−17
%increa
seSplay
ingof
kine
toch
orefib
ers
Splay
edsp
indles
,slight
increa
sein
spindleleng
thGos
himaet
al.,20
05a;
Gos
himaet
al.,20
05b;
Sha
rpet
al.,19
99;S
harp
etal.,
2000
.
ATK5
A.tha
liana
Meristems
prom
etap
hase
metap
hase
anap
hase
Deletion
9.3±
0.3μm,
5.80
±0.18
μm,
5.42
±0.19
μm
11.0±0.1μm,
6.58
±0.12
μm,
6.48
±0.17
μm
Red
uced
spindle
polarity,
defectsin
cros
slinking
Elong
ated
spindles
,broa
denmidzo
ne,
bent
morph
olog
y,sp
laye
dpo
les
Ambros
ean
dCyr,2
007;
Ambros
eet
al.,20
05.
Kar3
S.c
erev
isiae
Deletion
1.35
3±0.01
8μm
1.17
7±0.03
2μm
Inefficient
spindle
microtubu
lealignm
ent
Sho
rter
andmisaligne
dsp
indles
,wider
metap
hase
midzo
ne
End
owet
al.,19
94;H
eppe
rlaet
al.,20
14;H
uyette
tal.,
1998
;Meluh
andRos
e,19
90.
Pkl1
S.p
ombe
Deletion
2.03
±0.02
1μm
1.81
±0.01
7μm
Microtubu
lenu
clea
tion,
defectsin
MTcros
slinking
Sho
rter
andbroa
der
spindles
Olm
sted
etal.,20
14;T
roxe
llet
al.,20
01.
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spindle assembly. Finally, we have provided a detailed view of theroles of kinesin-14 motors in different model systems that reflect thecomplexities of the mitotic or meiotic spindles and the cellularenvironment.Furthermore, the studies of kinesins-14 across several model
organisms have led to an understanding of the evolution of mitoticmechanisms and co-evolution of kinesins-14 (Braun et al., 2013;Olmsted et al., 2014), indicating that these motor proteins shouldnot be considered as being constraint to exert a few standardcapabilities. An important focus for future work will be to study theantagonistic interactions between kinesin-14 and kinesin-5 motorsunder more complex conditions, for example within multiplemicrotubule bundles or in the context of their associated partnerproteins, as well as in vivo in a complex cellular environment.Further detailed in vitro and in vivo analyses are also required toelucidate the molecular kinetics and precise mechanisms of kinesin-14 motors at the both plus- and minus-ends of microtubules. Inaddition, the roles of kinesin-14 motors during development inmodel organisms remain largely unexplored. This raises the keyscientific questions of whether loss-of-function of kinesin-14motors results in defects of cell metabolism or morphogenesis,developmental abnormalities, or gives rise to any associateddiseases.
AcknowledgementsThe authors thank all members in the Sperm Laboratory at Zhejiang University fortheir helpful discussions. We sincerely thank our colleague Christopher RaymondWood for the careful editing of the manuscript.
Competing interestsThe authors declare no competing or financial interests.
FundingThis work was supported in part by the following grants: National Natural ScienceFoundation of China (grant numbers: 31572603 and 41276151).
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