How Essential Kinesin-5 Becomes Non-Essential: Force Balance and Microtubule Dynamics Matter

The bipolar mitotic spindle drives accurate chromosome segregation by capturing the kinetochore and pulling each set of sister chromatids to the opposite poles. In this review, we describe recent findings on the multiple pathways leading to bipolar spindle formation in fission yeast and discuss these results from a broader perspective. Roles of four mitotic kinesins (Kinesin-5, Kinesin-6, Kinesin-12 and Kinesin-14) in spindle assembly are depicted, and how a group of microtubule-associated proteins, sister chromatid cohesion and the kinetochore collaborates with these motors is shown. We have paid special attention to the molecular pathways that render otherwise essential Kinesin-5 to become non-essential: how cells build bipolar mitotic spindles without the need for Kinesin-5 and where the alternate forces come from are considered. We highlight the force balance for bipolar spindle assembly and explain how outward and inward forces are generated by various ways, in which the proper fine-tuning of microtubule dynamics plays a crucial role. Overall, these new pathways have illuminated remarkable plasticity and adaptability of spindle mechanics. Kinesin molecules are regarded as prospective targets for cancer chemotherapy and many specific inhibitors have been developed. However, several hurdles have arisen against their clinical implementation. This review provides insight into possible strategies to overcome these challenges.


Bipolar mitotic spindles and kinesin motor proteins
The bipolar mitotic spindle is a dynamic ensemble of core microtubule polymers and a cohort of microtubule-associated proteins (MAPs). It attaches to the kinetochore on sister chromatids to align chromosomes at the spindle equator in metaphase, and pulls each pair of sister chromatids towards the opposite poles in anaphase A. During anaphase B, spindles further elongate to ensure equal partition of each set of chromosomes, which is followed by cytokinesis [1,2]. MAPs are required for spindle assembly and coordinate the multiple events of mitosis in a spatiotemporal manner. Kinesin motors comprise one of the major MAPs and couple the energy of ATP hydrolysis to force generation [3]. Kinesin superfamilies comprise at least 15 families, which are further structurally categorised into three groups, called N-kinesin (N-terminal), M-kinesin (middle) and C-kinesin (C-terminal), depending on the location of the motor domain within each molecule [4][5][6]. Mitotic kinesins include

Conditions under which cells do not need Kinesin-5 for survival
As aforementioned, the main means in which Kinesin-5 becomes dispensable is by simultaneous inactivation of opposing Kinesin-14 or Dynein. In order to explore the genetic network that plays a role in conferring the non-essentiality of Kinesin-5, we conducted systematic screening for suppressors against cut7 temperature sensitive (ts) mutants in fission yeast. Spontaneous survivors of cut7 mutant strains grown at the restrictive temperature (36°C) were isolated. After standard genetic analyses and nucleotide sequencing, suppressor genes (designated skf=suppressor of kinesin five) were identified (Table 1) [21]. Suppressors can be classified into three main groups: Kinesin-14s, non-motor MAPs and tubulins.

Suppression by mutations in Kinesin-14s or their cofactors
In fission yeast, two Kinesin-14s, Pkl1 and Klp2, form distinct complexes with specific cofactors and are localised to different sites on the microtubule to play non-redundant roles in spindle assembly and mitotic progression [22][23][24][25]. Pkl1 forms a ternary complex with Msd1 and Wdr8 (referred to as the MWP complex) and is localised predominantly to the mitotic SPB, thereby anchoring the minus end of the spindle microtubule to the SPB [26][27][28]. During early mitosis, when the duplicated SPBs start to separate that is driven by the Cut7-mediated outward force, the SPBtethered MWP complex is loaded onto the spindle microtubule that nucleates from the other SPB, where this complex exerts an antagonistic inward force. Thus, the reason for suppression of cut7 mutants by pkl1∆, msd1∆ or wdr8∆ is that the two SPBs can separate because the overwhelming inward force exerted by the MWP Kinesin-14 complex disappears ( Figure 1A).  When spindle bipolarity starts to be established upon, SPB-tethered Pkl1 (the MWP complex) engages with the spindle microtubule that emanates from the opposite SPB. Minus end-directed motility of Pkl1 generates an inward force (blue arrows). This pulling force antagonises an outward force exerted by Cut7 (red arrows) that is also localised in the vicinity of the SPB and promotes interdigitation of microtubules emanating from the two SPBs. In addition to inward force generation, the MWP complex plays a crucial role in anchoring the minus end of the spindle microtubule to the mitotic SPB as a barrier [28]. (B) Once bipolar spindles are formed, Klp2 and Cut7 are localised on antiparallel microtubules. These two kinesins antagonistically generate an inward force (blue arrows) and an outward force (red arrows), respectively. + and -stand for the microtubule plus and minus ends, respectively. Note that Cut7 is reportedly localised to the two other sites. One is a medial microtubule contact site. This localisation is seen when bipolar spindles are depolymerised first and then allowed for repolymerisation. Under this condition, Cut7 is required for interpolar bundle formation [116]. The other site is the kinetochore, to which Cut7 is recruited when chromosomes are misaligned. Under this condition, Cut7 is required for chromosome gliding towards the spindle equator [117].
By contrast, Klp2 mainly is localised along spindles in a punctate manner [29]. Spindle-localising Klp2 crosslinks the antiparallel microtubule bundles, which produces an inward force by exploiting minus-end motility, and this force acts antagonistically with the Cut7-driven outward force on the spindle microtubule [30]. The reason for suppression of cut7 mutants by klp2∆ is that antiparallel microtubules can elongate in the absence of Cut7, as Klp2-driven inhibitory inward force is lost ( Figure 1B). Collectively, Pkl1 acts mainly during the early stages of bipolar spindle assembly, while Klp2 plays a role in spindle elongation at later stages of mitosis. These distinct modes of the spatiotemporal regulation between Pkl1 and Klp2 underlie the collaborative actions of these two Kinesin-14s.
The deletion of either pkl1 or klp2 suppresses the temperature sensitivity caused by the cut7 mutations [23,25,31]. Intriguingly, gene deletion of pkl1, but not klp2, is capable of rescuing a complete deletion of cut7 [30,[32][33][34]. Despite apparent ordinary growth, cut7∆pkl1∆ cells display mitotic delay in which cells spend a longer period of time with short spindles. This implies that in the absence of Cut7 and Pkl1, an excessive inward force is imposed during early mitosis. This inward force at least in part is generated by Klp2, as slower spindle elongation rate is significantly ameliorated in the cut7∆pkl1∆klp2∆ triple mutant [30].
Apart from its role in microtubule stabilisation, Mal3/EB1 is known to interact with Klp2, which is a prerequisite for this motor to be loaded on the spindle microtubule [29]. Thus, suppression of the cut7 ts mutation by the mal3 mutation could be ascribable to the loss of Klp2 function [21]. Overall, the common features of suppressor genes encoding tubulins and MAPs are that all these mutations lead to the destabilisation of the spindle microtubules. It is worth noting that in cut7 mutant cells, intensities of spindle microtubules are augmented [21]. Importantly, in these mutant cells, intensities of Klp2 on the spindle microtubule are also substantially increased. Notably, in the double mutant containing cut7 ts and any of the suppressor mutations, the intensities of Klp2 are reduced. Given these observations, we propose that the rescue of cut7 mutants by these suppressors is derived from quantitative downregulation of Klp2 activity. In fact, the overproduction of Klp2 in the double mutants between cut7 and suppressor mutations restored a ts phenotype similar to a single cut7 mutant [21], corroborating the notion that the reduced localisation/activity of Klp2 is the primary, if not the sole, reason for the rescue of the cut7 mutation. In summary, suppressor analyses have uncovered that multiple factors that regulate microtubule structures are involved by which Kinesin-5/Cut7 becomes dispensable and importantly, inactivation of Kinesin-14s, Pkl1 and Klp2, is the main means for the rescue of the cut7 mutation by these suppressors.

Suppression by microtubule-destabilising drugs
As previously mentioned, cut7 mutants exhibit increased intensities of the spindle microtubule accompanied by more Klp2 proteins on the spindle microtubule. In line with this observation, these cells display hyper-resistance against microtubule-depolymerising drugs, such as thiabendazole (TBZ) or methyl 2-benzimidazolecarbamate (MBC), and interestingly, treatment of cut7 ts mutants with TBZ or MBC rescues temperature sensitivity [21]. Under this condition, Klp2 levels are lessened as in the other suppressor mutations. Remarkably, drug treatment rescues even a complete deletion of cut7. Collectively, the impairment of microtubule stability and/or dynamics by either suppressor mutations or treatment with microtubule-destabilising drugs renders fission yeast cells viable in the absence of Kinesin-5 ( Figure 2).
It is worth pointing out that in cultured human cells (HeLa or U2OS), microtubule destabilisation can also effectively rescue monopolar spindle phenotypes induced by Kinesin-5/KIF11 inhibition [52,53]. This underscores the evolutionary conservation of Kinesin-5 function and its regulation from fission yeast to human beings. Microtubule stability and dynamics promoted by a cohort of MAPs play positive roles in Klp2 activity by enhancing its localisation to the spindle microtubule, which Kinesin-5/Cut7 opposes. In cut7 ts or cut7∆ cells (middle), Kinesin-14-mediated inward forces dominate, leading to the formation of monopolar spindles. In double mutants between cut7 and pkl1/klp2 or the cut7 mutant treated with microtubule-depolymerising drugs (bottom), loss of inward forces or the compromised microtubule dynamics, respectively, renders Cut7 to become dispensable for bipolar spindle assembly and therefore the survival.

Suppression through downregulation of the cAMP/PKA pathway
In many organisms, the extracellular environment such as nutritional cues regulates microtubule dynamics through the intracellular signal transduction pathways. In yeasts, glucose in the media activates the cAMP/PKA pathway, by which it controls a diverse set of downstream events [54][55][56]. Deletion of the pka1 gene rescues the cut7 ts mutant [57]. Pka1 reportedly fine-tunes microtubule dynamics at least in part through downregulating the Cls1/Peg1/CLASP MAP [58][59][60], and consistent with this notion, overproduction of Cls1/Peg1 is capable of rescuing the cut7 ts mutant like pka1∆ [57]. Cls1/Peg1 is shown to promote microtubule bundling [61]. It is possible that enhanced bundling activity by overproduced Cls1/Peg1 would help convert the monopolar spindle in the cut7 mutation to a bipolar spindle, leading to rescue of this mutant.

Outward force generators in the absence of Kinesin-5
Cells without Kinesin-5 become viable if Kinesin-14 is defective (e.g. cut7∆pkl1∆) or if microtubules are destabilised (e.g. cut7∆ treated with microtubule-destabilising drugs). This finding poses the following important question: how do bipolar spindles assemble in the absence of Kinesin-5-mediated outward force? Detailed genetic and cell biological analyses have unravelled this puzzle; at least 11 gene products are capable of generating outward forces instead of Kinesin-5 (Table 2).

Outward forces exerted by Kinesin-6
One possibility of the survival of cut7∆pkl1∆ cells is that the other kinesin motors exert an outward force in place of Kinesin-5, thereby promoting spindle bipolarity. The fission yeast genome contains in total 9 genes encoding kinesin motors. Genetic crosses indicate that only one kinesin, Klp9, becomes essential when combined with cut7∆pkl1∆; cut7∆pkl1∆klp9∆ triple mutants are inviable. Klp9 belongs to the N-kinesin Kinesin-6. Interestingly, like Cut7, it moves on the microtubule towards the plus end and forms homotetramers, thereby crosslinking antiparallel microtubules [62,63]. Previous work showed that this kinesin accumulates at the spindle midzone upon onset of anaphase B and promotes spindle elongation during late mitosis [62], though it appears to also play additional roles during earlier stages of mitosis [64][65][66]. Detailed analysis shows that Klp9 accelerates spindle elongation only during anaphase B in both wild type and cut7∆pkl1∆ cells and that inviable cut7∆pkl1∆klp9∆ cells are in fact capable of assembling bipolar spindles. However, these spindles are shorter comparted to those in wild type or cut7∆pkl1∆ cells, and upon mitotic exit, the nucleus and chromosomes are intersected by the cytokinetic actomyosin contractile ring and the septum, resulting in cell death imposed by a catastrophic "cut" (cell untimely torn) phenotype [63,67].
How do the two N-kinesins, Kinesin-5/Cut7 and Kinesin-6/Klp9, act in concert to drive spindle elongation in wild type cells? Differential localisation patterns of these two kinesins on the spindle microtubule may give us a clue. While Cut7 mainly accumulates near the SPB, Klp9 is localised exclusively to the spindle midzone where antiparallel microtubules interdigitate ( Figure 3A). Given these different localisations, we posit that during anaphase B, Cut7 crosslinks mainly parallel microtubules near the SPBs, while Klp9 bundles antiparallel microtubules at the spindle midzone ( Figure 3B). DeltaVision image acquisition software (softWoRx 6.5.2; GE Healthcare, Chicago, IL) equipped with a charge-coupled device camera (CoolSNAP HQ2; Photometrics, Tucson, AZ) was used. Time-lapse live imaging was performed, in which pictures were taken at 1 min intervals after incubation of cultures at 27°C. Images were taken as 16 sections along the z-axis at 0.2 μm intervals. The sections of images acquired at each time point were compressed into a 2D projection using the DeltaVision maximum intensity algorithm. Deconvolution was applied before the 2D projection. Captured images were processed with Photoshop CS6 (version 13.0; Adobe, San Jose, CA). Scale bar, 10 μm. (B) A schematic showing localisations of Cut7 and Klp9 on anaphase B spindles. Cut7 bundles parallel microtubules in the vicinity of the SPB, while Klp9 bundles antiparallel microtubules at the spindle midzone. Note that Klp9 bundles antiparallel microtubules at the spindle midzone independent of its motor activity [63]. + and -stand for the microtubule plus and minus ends, respectively.

Outward forces exerted by the microtubule crosslinker and stabiliser
Fission yeast cells are capable of forming nearly normal bipolar spindles in the presence of only Kinesin-6 Klp9, which acts in spindle elongation later in mitosis [57]. How then could spindle bipolarity be established in the first place under this condition? It transpires that two conserved MAPs, Ase1/PRC1 [68,69] and Cls1/Peg1/CLASP, in concert, play an indispensable role in this process. Ase1 and Cls1/Peg1 bundle and stabilise antiparallel spindle microtubules ( Figure 4). Notably, theoretical modelling supports bipolar spindle assembly by these two factors; Brownian dynamics-kinetic Monte Carlo simulations show that Ase1 and Cls1/Peg1 activity are sufficient for initial bipolar spindle formation [57,70,71].  . Outward force generation through the spindle midzone. Short microtubules nucleating from the two SPBs are crosslinked in an antiparallel manner and stabilised by Ase1/PRC1 and Cls1/Peg1/CLASP. This provides an outward force (green arrows) towards the SPB that is sufficient to form short bipolar spindles in the absence of Kinesin-5 and Kinesin-14 [57]. + and -stand for the microtubule plus and minus ends, respectively.
Unlike in other species, C. elegans does not require Kinesin-5/BMK-1 for bipolar spindle formation [72]. During embryonic division of this organism, the spindle midzone could produce outward forces in concert with cortical pulling forces, thereby promoting chromosome segregation [18]. Interestingly, in this process, the midzone components including SPD-1/Ase1 and CLASP play vital roles in force generation, though SPD-1 seems to antagonise CLASP-mediated spindle elongation [18,73]. Taken together, the spindle midzone could produce outward forces in which the microtubule crosslinking and stabilising MAPs are key players.
Mutations in dis1, alp7 or alp14 rescue the cut7 ts mutation, while rather contradictorily, the same mutations become indispensable in the cut7∆pkl1∆ background; defects in the microtubule polymerisation confer both positive and negative impacts on the cut7 mutation. In a single cut7 mutation, microtubule polymerisation is negative for cell survival, while in cut7∆pkl1∆, it plays a positive role. This illuminates a remarkable mechanistic plasticity of bipolar spindle assembly; cells could generate either inward or outward forces using microtubule polymerases in a contextdependent manner.
Intriguingly, in interphase fission yeast cells, the nucleus is centred by pushing forces that are generated as growing cytoplasmic microtubules hit the cell tip at each end [80,81] (Figure 5B). Hence, pushing forces generated through the polymerising microtubule plus ends play important roles in both interphase and mitosis: nuclear positioning during interphase and SPB separation/bipolar spindle assembly during mitosis. The generation of an outward pushing force by the polymerising microtubule plus end is widely observed in other systems. For instance, during embryonic divisions of animal cells, the plus ends of astral microtubules physically interact with and push the cell cortex, and this force ensures proper spindle positioning [82,83].

Outward forces exerted by the kinetochore and sister chromatid cohesion
Recent work has identified a fourth class of outward force generators [84]. This force is elicited through the kinetochore, a several-MDa-sized proteinaceous structure assembled on a specialised region of the chromosome called the centromere [85]. The spindle microtubule attaches to the kinetochore for accurate sister chromatid segregation. Mutations in genes encoding the conserved Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2020 doi:10.20944/preprints202004.0375.v1 kinetochore component (Nuf2) [86] or those required for centromere-mediated sister chromatid cohesion (Rad21/Scc1/Mcd1 and Swi6/HP1) [87][88][89][90] confer a severe synthetic growth defect to cut7∆pkl1∆. These triple mutant cells impair proper spindle assembly and display largely monopolar spindles. These results show that the kinetochore is captured by the plus end of the spindle microtubule, thereby producing outward forces that support bipolar spindle assembly ( Figure 6) [84].

Figure 6.
Outward force generation through the kinetochore and sister chromatid cohesion. The kinetochore and sister chromatid cohesion contribute to the generation of outward forces for SPB separation in mitosis. In the absence of Kinesin-5 and Kinesin-14, outward forces (purple arrows) derived from the kinetochore and/or sister chromatid cohesion are capable of separating the SPBs, thereby promoting bipolar spindle assembly [84]. + and -stand for the microtubule plus and minus ends, respectively.
It is noteworthy that a similar result was reported in human cells, in which stable kinetochoremicrotubule attachment plays a crucial role in centrosome separation [91] and becomes essential for maintenance of the bipolar spindle in the absence of Kinesin-5 [92]. Taking all these findings together, the balance between inward and outward forces generated by opposing motor proteins, multiple MAPs, the kinetochore and sister chromatid cohesion underlies mechanisms by which bipolar spindles are formed and maintained.

Outward force generation by Kinesin-12 in human cells
In fission yeast, Kinesin-6/Klp9 collaborates with Kinesin-5/Cut7 to generate an outward force. However, Klp9 acts in spindle elongation only during late mitosis irrespective of the presence or absence of Cut7, and furthermore, Klp9 cannot be substituted for Cut7 [57,79]. By contrast, in human cells, a similar role appears to be executed by the N-kinesin Kinesin-12/KIF15/HKLP2, which does not exist in yeasts. It has been shown that Kinesin-12 functions redundantly with Kinesin-5 to promote spindle bipolarity [52,[93][94][95], and curiously, the overproduction of Kinesin-12 can drive bipolar spindle assembly even when Kinesin-5 activity is fully inhibited. This indicates that Kinesin-12 has the potential to execute all essential functions of Kinesin-5. Therefore, functions of fission yeast Kinesin-6/Klp9 and human Kinesin-12/KIF15/HKLP2 appear similar but mechanistically different.

Force generation in human prophase cells
Human cells undergo centrosome separation through two temporally distinct pathways, the prophase pathway and the prometaphase pathway [96][97][98]. By contrast, yeasts have adopted only the prometaphase pathway. Recent analysis has uncovered key players acting in the prophase pathway and their individual roles [97]. Duplicated centrosomes in human cells remain closely linked during interphase, in a process called centrosome cohesion. Centrosome cohesion is maintained through dual mechanisms. The first mechanism depends upon a structural linker composed of two proteins, Rootletin and C-NAP1. This linker physically joins the two centrosomes in a side-by-side configuration. The second mechanism involves the Kinesin-14/KIFC3-mediated inward force. KIFC3 forms homotetramers and interconnects a special centrosome-associated microtubule network, thereby producing pulling forces towards the centrosomes. Upon mitotic entry, KIFC3 is inactivated by the NEK2 protein kinase, which also promotes the dissolution of the linker [97]. Antagonising outward forces are produced by Kineisn-5/KIF11 in both prophase and prometaphase pathways.
We contemplate that the difference between human beings and yeasts, in which human cells have developed more complex regulatory mechanisms, stem from different modes of mitosis: an open mitosis in higher eukaryotes vs a closed mitosis in yeasts. As the nuclear membrane disassembles upon mitotic onset, human cells have acquired an additional regulatory process (the prophase pathway), which ensures the temporal order of bipolar spindle assembly to be synchronised with mitotic onset. Implementation of dual, redundant pathways might also be beneficial for the robustness of the system; disrupting one pathway would not result in a catastrophic impact on spindle formation and therefore the fidelity of chromosome segregation.

Force generation in the acentrosomal cells
In higher eukaryotes and plants, the bipolar spindle is formed independent of the centrosome through the pathway referred to as the acentrosomal pathway [99][100][101]. Historically, the acentrosomal pathway is extensively characterised in vitro using extracts prepared from Xenopus oocytes, where the Ran GTPase acts as a master regulator [102,103]. This pathway is functional in vivo in several cell types which are naturally devoid of centrosomes (eg. vertebrate oocytes and plants) or animal somatic cells which are experimentally (chemically, genetically or physically) manipulated to eliminate their centrosomes [104][105][106]. Interestingly, in this acentrosomal pathway, both in vitro and in vivo, Kinesin-5 also plays a major role in the formation of antiparallel microtubule bundles and the generation of an outward force. However, in contrast with the centrosome-dependent pathway, Kinesin-14 and Dynein appear to act collaboratively, rather than antagonistically, with Kinesin-5 [105,107]. Therefore, the importance of the force balance for bipolar spindle formation and its underlying mechanism have not been addressed explicitly in the acentrosomal cells and awaits further investigation.

Towards cancer therapeutics
Kinesin-5 is required for successive cell division. Furthermore, in actively growing cancer cell lines its activity is tightly, and sometimes causally, linked to tumour progression and malignancy [108]. Accordingly, this kinesin has been deemed to be an attractive target of cancer chemotherapeutics. Indeed, several Kinesin-5 inhibitors were developed and their clinical trials conducted [109]. However, drug-resistant cell lines often appeared to hamper the clinical usage of these inhibitors [110][111][112]. To tackle this conundrum, a comprehensive understanding of in vivo Kinesin-5 functions and regulations in addition to structural information on the interaction between Kinesin-5 and specific inhibitors [109,113] are necessary. As Kinesin-12 is essential for the survival of HeLa cells that become resistant to Kinein-5 inhibitors, the development of specific Kinesin-12 inhibitors would be important [95]. Given that destabilisation and/or the reduced dynamics of the microtubule rescues the lethality derived from Kinesin-5 inactivation, the combined treatment of Kinesin-5 inhibitors and microtubule stabilising reagents such as Paclitaxel (Taxol, on its own used widely for chemotherapy) [114,115] might provide a more effective treatment for cancer therapeutics. Acknowledgments: We thank all the laboratory members, past and present, and collaborators for their contributions to our work described in this review. We are grateful to Risa Mori for critical reading of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.