Mechanism for Anaphase B: Evaluation of “Slide-and-Cluster” versus “Slide-and-Flux-or-Elongate” Models

Elongation of the mitotic spindle during anaphase B contributes to chromosome segregation in many cells. Here, we quantitatively test the ability of two models for spindle length control to describe the dynamics of anaphase B spindle elongation using experimental data from Drosophila embryos. In the slide-and-flux-or-elongate (SAFE) model, kinesin-5 motors persistently slide apart antiparallel interpolar microtubules (ipMTs). During pre-anaphase B, this outward sliding of ipMTs is balanced by depolymerization of their minus ends at the poles, producing poleward flux, while the spindle maintains a constant length. Following cyclin B degradation, ipMT depolymerization ceases so the sliding ipMTs can push the poles apart. The competing slide-and-cluster (SAC) model proposes that MTs nucleated at the equator are slid outward by the cooperative actions of the bipolar kinesin-5 and a minus-end-directed motor, which then pulls the sliding MTs inward and clusters them at the poles. In assessing both models, we assume that kinesin-5 preferentially cross-links and slides apart antiparallel MTs while the MT plus ends exhibit dynamic instability. However, in the SAC model, minus-end-directed motors bind the minus ends of MTs as cargo and transport them poleward along adjacent, parallel MT tracks, whereas in the SAFE model, all MT minus ends that reach the pole are depolymerized by kinesin-13. Remarkably, the results show that within a narrow range of MT dynamic instability parameters, both models can reproduce the steady-state length and dynamics of pre-anaphase B spindles and the rate of anaphase B spindle elongation. However, only the SAFE model reproduces the change in MT dynamics observed experimentally at anaphase B onset. Thus, although both models explain many features of anaphase B in this system, our quantitative evaluation of experimental data regarding several different aspects of spindle dynamics suggests that the SAFE model provides a better fit.     

KLP10A and KLP59C: The Dynamic Duo of Microtubule Depolymerization

Kinesin-13s are important effectors of microtubule depolymerization in cells. In a recent series of studies, we examined the roles played by kinesin-13s throughout the cell cycle in Drosophila. Our findings have revealed remarkable coordination between two family members, KLP10A and KLP59C, in which alterations in the relative targeting of these proteins allows them to participate in markedly different tasks at distinct points in the cell cycle. During mitosis, KLP10A and KLP59C function in parallel by targeting to and depolymerizing the opposite ends of kinetochore-associated microtubules, thereby driving poleward chromatid motility by a Pacman-Flux mechanism. Alternatively, during interphase, both proteins target to the same end of the microtubule but act in series to divide the labor of microtubule depolymerization. KLP10A initiates depolymerization while KLP59C perpetuates depolymerization after its initiation. Below, we detail these findings and examine some of their implications.     

A Kin I-Independent Pacman: Flux Mechanism for Anaphase A

Anaphase A chromatid-to-pole motion is fundamental for proper chromosome segregation in most systems. During the past several decades, two models for the mechanism of anaphase A have come to prominence. The Pacman model posits that chromatids induce the depolymerization of microtubule plus-ends embedded in kinetochores, thereby ‘chewing’ their way poleward. Alternatively, the Poleward-flux model posits that chromatids are ‘reeled-in’ to poles by the continual depolymerization of the minus-ends of kinetochore-associated microtubules, which are focused at spindle poles. In a recent study, we reported that anaphase A in Drosophila requires the depolymerization of both ends of kinetochore-associated microtubules, simultaneously. This is driven by two members of the Kin I subfamily of kinesins, termed KLP59C and KLP10A, which target specifically to chromatids and spindle poles, respectively. We have termed this hybrid of Pacman and Poleward flux the Kin I-dependent Pacman-flux mechanism for anaphase A. Here, we discuss the implications of these findings and explore potential additional components required to drive chromatid-to-pole motion by such a mechanism.     

Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Here, we report that two proteins of the Drosophila melanogaster kinesin-13 family, KLP10A and KLP59C, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; KLP10A stimulates catastrophe (a switch from growth to shrinkage) whereas KLP59C suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live analyses of cells expressing tagged kinesins reveal that KLP10A and KLP59C target to polymerizing and depolymerizing microtubule plus ends, respectively. Our data also suggest that KLP10A is deposited on microtubules by the plus-end tracking protein, EB1. Our findings support a model in which these two members of the kinesin-13 family divide the labour of microtubule depolymerization.     

Mitotic motors and chromosome segregation: the mechanism of anaphase B

Anaphase B spindle elongation plays an important role in chromosome segregation. In the present paper, we discuss our model for anaphase B in Drosophila syncytial embryos, in which spindle elongation depends on an ip (interpolar) MT (microtubule) sliding filament mechanism generated by homotetrameric kinesin-5 motors acting in concert with poleward ipMT flux, which acts as an 'on/off' switch. Specifically, the pre-anaphase B spindle is maintained at a steady-state length by the balance between ipMT sliding and ipMT depolymerization at spindle poles, producing poleward flux. Cyclin B degradation at anaphase B onset triggers: (i) an MT catastrophe gradient causing ipMT plus ends to invade the overlap zone where ipMT sliding forces are generated; and (ii) the inhibition of ipMT minus-end depolymerization so flux is turned 'off', tipping the balance of forces to allow outward ipMT sliding to push apart the spindle poles. We briefly comment on the relationship of this model to anaphase B in other systems.     

A Second Tubulin Binding Site on the Kinesin-13 Motor Head Domain Is Important during Mitosis

Kinesin-13s are microtubule (MT) depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD) containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2) on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.     

A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes

The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.     

A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays

BACKGROUND: Mitosis depends upon the cooperative action of multiple microtubule (MT)-based motors. Among these, a kinesin-5, KLP61F, and the kinesin-14, Ncd, are proposed to generate antagonistic-sliding forces that control the spacing of the spindle poles. We tested whether purified KLP61F homotetramers and Ncd homodimers can generate a force balance capable of maintaining a constant spindle length in Drosophila embryos. RESULTS: Using fluorescence microscopy and cryo-EM, we observed that purified full-length, motorless, and tailless KLP61F tetramers (containing a tetramerization domain) and Ncd dimers can all cross-link MTs into bundles in MgATP. In multiple-motor motility assays, KLP61F and Ncd drive plus-end and minus-end MT sliding at 0.04 and 0.1 microm/s, respectively, but the motility of either motor is decreased by increasing the mole fraction of the other. At the "balance point," the mean velocity was zero and MTs paused briefly and then oscillated, taking approximately 0.3 microm excursions at approximately 0.02 microm/s toward the MT plus end and then the minus end. CONCLUSIONS: The results, combined with quantitative analysis, suggest that these motors could act as mutual brakes to modulate the rate of pole-pole separation and could maintain a prometaphase spindle displaying small fluctuations in its steady-state length.     

The microtubule cross-linker Feo controls the midzone stability,motor composition,and elongation of the anaphase B spindle in Drosophila embryos

Chromosome segregation during anaphase depends on chromosome-to-pole motility and pole-to-pole separation. We propose that in Drosophila embryos, the latter process (anaphase B) depends on a persistent kinesin-5–generated interpolar (ip) microtubule (MT) sliding filament mechanism that “engages” to push apart the spindle poles when poleward flux is turned off. Here we investigated the contribution of the midzonal, antiparallel MT-cross-linking nonmotor MAP, Feo, to this “slide-and-flux-or-elongate” mechanism. Whereas Feo homologues in other systems enhance the midzone localization of the MT-MT cross-linking motors kinesin-4, -5 and -6, the midzone localization of these motors is respectively enhanced, reduced, and unaffected by Feo. Strikingly, kinesin-5 localizes all along ipMTs of the anaphase B spindle in the presence of Feo, including at the midzone, but the antibody-induced dissociation of Feo increases kinesin-5 association with the midzone, which becomes abnormally narrow, leading to impaired anaphase B and incomplete chromosome segregation. Thus, although Feo and kinesin-5 both preferentially cross-link MTs into antiparallel polarity patterns, kinesin-5 cannot substitute for loss of Feo function. We propose that Feo controls the organization, stability, and motor composition of antiparallel ipMTs at the midzone, thereby facilitating the kinesin-5–driven sliding filament mechanism underlying proper anaphase B spindle elongation and chromosome segregation.     

A computational model predicts Xenopus meiotic spindle organization

The metaphase spindle is a dynamic bipolar structure crucial for proper chromosome segregation, but how microtubules (MTs) are organized within the bipolar architecture remains controversial. To explore MT organization along the pole-to-pole axis, we simulated meiotic spindle assembly in two dimensions using dynamic MTs, a MT cross-linking force, and a kinesin-5-like motor. The bipolar structures that form consist of antiparallel fluxing MTs, but spindle pole formation requires the addition of a NuMA-like minus-end cross-linker and directed transport of MT depolymerization activity toward minus ends. Dynamic instability and minus-end depolymerization generate realistic MT lifetimes and a truncated exponential MT length distribution. Keeping the number of MTs in the simulation constant, we explored the influence of two different MT nucleation pathways on spindle organization. When nucleation occurs throughout the spindle, the simulation quantitatively reproduces features of meiotic spindles assembled in Xenopus egg extracts.     

Model of chromosome motility in Drosophila embryos: adaptation of a general mechanism for rapid mitosis

During mitosis, ensembles of dynamic MTs and motors exert forces that coordinate chromosome segregation. Typically, chromosomes align at the metaphase spindle equator where they oscillate along the pole-pole axis before disjoining and moving poleward during anaphase A, but spindles in different cell types display differences in MT dynamicity, in the amplitude of chromosome oscillations and in rates of chromatid-to-pole motion. Drosophila embryonic mitotic spindles, for example, display remarkably dynamic MTs, barely detectable metaphase chromosome oscillations, and a rapid rate of "flux-pacman-dependent" anaphase chromatid-to-pole motility. Here we develop a force-balance model that describes Drosophila embryo chromosome motility in terms of a balance of forces acting on kinetochores and kMTs that is generated by multiple polymer ratchets and mitotic motors coupled to tension-dependent kMT dynamics. The model shows that i), multiple MTs displaying high dynamic instability can drive steady and rapid chromosome motion; ii), chromosome motility during metaphase and anaphase A can be described by a single mechanism; iii), high kinetochore dynein activity is deployed to dampen metaphase oscillations, to augment the basic flux-pacman mechanism, and to drive rapid anaphase A; iv), modulation of the MT rescue frequency by the kinetochore-associated kinesin-13 depolymerase promotes metaphase chromosome oscillations; and v), this basic mechanism can be adapted to a broad range of spindles.     

Kinesin-8 from Fission Yeast: A Heterodimeric, Plus-End–directed Motor that Can Couple Microtubule Depolymerization to Cargo Movement

Fission yeast expresses two kinesin-8s, previously identified and characterized as products of the klp5⁺ and klp6⁺ genes. These polypeptides colocalize throughout the vegetative cell cycle as they bind cytoplasmic microtubules during interphase, spindle microtubules, and/or kinetochores during early mitosis, and the interpolar spindle as it elongates in anaphase B. Here, we describe in vitro properties of these motor proteins and some truncated versions expressed in either bacteria or Sf9 cells. The motor-plus-neck domain of Klp6p formed soluble dimers that cross-linked microtubules and showed both microtubule-activated ATPase and plus-end–directed motor activities. Full-length Klp5p and Klp6p, coexpressed in Sf9 cells, formed soluble heterodimers with the same activities. The latter recombinant protein could also couple microbeads to the ends of shortening microtubules and use energy from tubulin depolymerization to pull a load in the minus end direction. These results, together with the spindle localizations of these proteins in vivo and their requirement for cell viability in the absence of the Dam1/DASH kinetochore complex, support the hypothesis that fission yeast kinesin-8 contributes both to chromosome congression to the metaphase plate and to the coupling of spindle microtubules to kinetochores during anaphase A.     

Microtubule dynamics in the spindle. Theoretical aspects of assembly/disassembly reactions in vivo

The mitotic spindle contains several classes of microtubules (MTs) whose lengths change independently during mitosis. Precise control over MT polymerization and depolymerization during spindle formation, anaphase chromosome movements, and spindle breakdown is necessary for successful cell division. This model proposes the site of addition and removal of MT subunits in each of four classes of spindle MTs at different stages of mitosis, and suggests how this addition and removal is controlled. We propose that spindle poles and kinetochores significantly alter the assembly-disassembly kinetics of associated MT ends. Control of MT length is further modulated by localized forces affecting assembly and disassembly kinetics of individual sets of MTs.     

Efficient mitosis in human cells lacking poleward microtubule flux

Chromosome segregation relies on the dynamic properties of spindle microtubules (MTs). Poleward MT flux contributes to spindle dynamics through the disassembly of MT minus ends at spindle poles coupled to the continuous poleward transport of spindle MTs. Despite being conserved in metazoan cells, the function of flux remains controversial because flux rates differ widely in different cell types. In meiotic systems, the rate of flux nearly matches that of chromosome movement, but in mitotic systems, flux is significantly slower than chromosome movement. Here, we show that spindles in human mitotic cells depleted of the kinesin-13 proteins Kif2a and MCAK lack detectable flux and that such cells frequently fail to segregate all chromosomes appropriately at anaphase. Elimination of flux reduces poleward chromosome velocity approximately 20%, but does not hinder bipolar spindle assembly, chromosome alignment, or mitotic progression. Thus, mitosis proceeds efficiently in human cells lacking detectable poleward MT flux. These data demonstrate that in human cultured cells, kinetochores are sufficient to effectively power chromosome movement, leading us to speculate that flux is maintained in these cells to fulfill other functional roles such as error correction or kinetochore regulation.     

The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility

Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic spindle morphogenesis and chromosome movements in Drosophila embryos and cultured S2 cells. By immunofluorescence, KLP3A associates with nonfibrous punctae that concentrate in nuclei and display MT-dependent associations with spindles. These punctae concentrate in indistinct domains associated with chromosomes and central spindles and form distinct bands associated with telophase midbodies. The functional disruption of KLP3A by antibodies or dominant negative proteins in embryos, or by RNA interference (RNAi) in S2 cells, does not block mitosis but produces defects in mitotic spindles. Time-lapse confocal observations of mitosis in living embryos reveal that KLP3A inhibition disrupts the organization of interpolar (ip) MTs and produces short spindles. Kinetic analysis suggests that KLP3A contributes to spindle pole separation during the prometaphase-to-metaphase transition (when it antagonizes Ncd) and anaphase B, to normal rates of chromatid motility during anaphase A, and to the proper spacing of daughter nuclei during telophase. We propose that KLP3A acts on MTs associated with chromosome arms and the central spindle to organize ipMT bundles, to drive spindle pole separation and to facilitate chromatid motility.     

Dynamic partitioning of mitotic kinesin-5 cross-linkers between microtubule-bound and freely diffusing states

The dynamic behavior of homotetrameric kinesin-5 during mitosis is poorly understood. Kinesin-5 may function only by binding, cross-linking, and sliding adjacent spindle microtubules (MTs), or, alternatively, it may bind to a stable "spindle matrix" to generate mitotic movements. We created transgenic Drosophila melanogaster expressing fluorescent kinesin-5, KLP61F-GFP, in a klp61f mutant background, where it rescues mitosis and viability. KLP61F-GFP localizes to interpolar MT bundles, half spindles, and asters, and is enriched around spindle poles. In fluorescence recovery after photobleaching experiments, KLP61F-GFP displays dynamic mobility similar to tubulin, which is inconsistent with a substantial static pool of kinesin-5. The data conform to a reaction-diffusion model in which most KLP61F is bound to spindle MTs, with the remainder diffusing freely. KLP61F appears to transiently bind MTs, moving short distances along them before detaching. Thus, kinesin-5 motors can function by cross-linking and sliding adjacent spindle MTs without the need for a static spindle matrix.     

Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis

Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.     

Xorbit/CLASP links dynamic microtubules to chromosomes in the Xenopus meiotic spindle

A family of microtubule (MT)-binding proteins, Orbit/multiple asters/cytoplasmic linker protein-associated protein, has emerged as an important player during mitosis, but their functional mechanisms are poorly understood. In this study, we used meiotic egg extracts to gain insight into the role of the Xenopus laevis homologue Xorbit in spindle assembly and function. Xorbit immunodepletion or its inhibition by a dominant-negative fragment resulted in chromosome alignment defects and aberrant MT structures, including monopolar and small spindles. Xorbit-depleted extracts failed to nucleate MTs around chromatin-coated beads, indicating its essential requirement for spindle assembly in the absence of centrosomes and kinetochores. Xorbit's MT stabilizing effect was most apparent during anaphase, when spindle MTs depolymerized rapidly upon Xorbit inhibition. Biochemical interaction between a COOH-terminal Xorbit fragment and the kinetochore-associated kinesin centromeric protein E may contribute to Xorbit's role in chromosome congression. We propose that Xorbit tethers dynamic MT plus ends to kinetochores and chromatin, providing a stabilizing activity that is crucial for spindle assembly and chromosome segregation.     

Microcephaly protein Asp focuses the minus ends of spindle microtubules at the pole and within the spindle

Depletion of Drosophila melanogaster Asp, an orthologue of microcephaly protein ASPM, causes spindle pole unfocusing during mitosis. However, it remains unclear how Asp contributes to pole focusing, a process that also requires the kinesin-14 motor Ncd. We show that Asp localizes to the minus ends of spindle microtubule (MT) bundles and focuses them to make the pole independent of Ncd. We identified a critical domain in Asp exhibiting MT cross-linking activity in vitro. Asp was also localized to, and focuses the minus ends of, intraspindle MTs that were nucleated in an augmin-dependent manner and translocated toward the poles by spindle MT flux. Ncd, in contrast, functioned as a global spindle coalescence factor not limited to MT ends. We propose a revised molecular model for spindle pole focusing in which Asp at the minus ends cross-links MTs at the pole and within the spindle. Additionally, this study provides new insight into the dynamics of intraspindle MTs by using Asp as a minus end marker.     

Microtubule Cytoskeleton Remodeling by Acentriolar Microtubule-organizing Centers at the Entry and Exit from Mitosis in Drosophila Somatic Cells

Cytoskeleton microtubules undergo a reversible metamorphosis as cells enter and exit mitosis to build a transient mitotic spindle required for chromosome segregation. Centrosomes play a dominant but dispensable role in microtubule (MT) organization throughout the animal cell cycle, supporting the existence of concurrent mechanisms that remain unclear. Here we investigated MT organization at the entry and exit from mitosis, after perturbation of centriole function in Drosophila S2 cells. We found that several MTs originate from acentriolar microtubule-organizing centers (aMTOCs) that contain γ-tubulin and require Centrosomin (Cnn) for normal architecture and function. During spindle assembly, aMTOCs associated with peripheral MTs are recruited to acentriolar spindle poles by an Ncd/dynein-dependent clustering mechanism to form rudimentary aster-like structures. At anaphase onset, down-regulation of CDK1 triggers massive formation of cytoplasmic MTs de novo, many of which nucleated directly from aMTOCs. CDK1 down-regulation at anaphase coordinates the activity of Msps/XMAP215 and the kinesin-13 KLP10A to favor net MT growth and stability from aMTOCs. Finally, we show that microtubule nucleation from aMTOCs also occurs in cells containing centrosomes. Our data reveal a new form of cell cycle–regulated MTOCs that contribute for MT cytoskeleton remodeling during mitotic spindle assembly/disassembly in animal somatic cells, independently of centrioles.