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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., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2015 Wang et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Mitosis depends upon the action of the mitotic spindle, a subcellular machine that uses microtubules (MTs) and motors to assemble itself and to coordinate chromosome segregation. Recent work illuminates how the motor-driven poleward sliding of MTs - nucleated at centrosomes, chromosomes and on pre-existing MTs - contributes to spindle assembly and length control.

Anaphase B spindle elongation contributes to chromosome segregation during Drosophila melanogaster embryo mitosis. We propose that this process is driven by a kinesin-5-generated interpolar microtubule (MT; ipMT) sliding filament mechanism that engages when poleward flux is turned off. In this paper, we present evidence that anaphase B is induced by the minus end-stabilizing protein patronin, which antagonizes the kinesin-13 depolymerase KLP10A at spindle poles, thereby switching off the depolymerization of the minus ends of outwardly sliding ipMTs to suppress flux. Although intact cortices, kinetochore MTs, and midzone augmentation are dispensable, this patronin-based change in ipMT minus-end dynamics is sufficient to induce the elongation of spindles capable of separating chromosomes.

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.

Mitotic spindle length control requires coordination between microtubule (MT) dynamics and motor-generated forces. To investigate how MT plus-end polymerization contributes to spindle length in Drosophila embryos, we studied the dynamics of the MT plus-end depolymerase, kinesin-8, and the effects of kinesin-8 inhibition using mutants and antibody microinjection. As expected, kinesin-8 was found to contribute to anaphase A. Furthermore, kinesin-8 depletion caused: (i) excessive polymerization of interpolar (ip) MT plus ends, which "overgrow" to penetrate distal half spindles; (ii) an increase in the poleward ipMT sliding rate that is coupled to MT plus-end polymerization; (iii) premature spindle elongation during metaphase/anaphase A; and (iv) an increase in the anaphase B spindle elongation rate which correlates linearly with the MT sliding rate. This is best explained by a revised "ipMT sliding/minus-end depolymerization" model for spindle length control which incorporates a coupling between ipMT plus end dynamics and the outward ipMT sliding that drives poleward flux and spindle elongation., (© 2010 Wiley-Liss, Inc.)

To understand how mitotic kinesins contribute to the assembly and function of the mitotic spindle, we need to purify these motors and analyze their biochemical and ultrastructural properties. Here we briefly review our use of microtubule (MT) affinity and biochemical fractionation to obtain information about the oligomeric state of native mitotic kinesin holoenzymes from eggs and early embryos. We then detail the methods we use to purify full length recombinant Drosophila embryo mitotic kinesins, using the baculovirus expression system, in sufficient yields for detailed in vitro assays. These two approaches provide complementary biochemical information on the basic properties of these key mitotic proteins, and permit assays of critical motor activities, such as MT-MT crosslinking and sliding, that are not revealed by assaying motor domain subfragments.

We tested the classical hypothesis that astral, prometaphase bipolar mitotic spindles are maintained by balanced outward and inward forces exerted on spindle poles by kinesin-5 and -14 using modeling of in vitro and in vivo data from Drosophila melanogaster embryos. Throughout prometaphase, puncta of both motors aligned on interpolar microtubules (MTs [ipMTs]), and motor perturbation changed spindle length, as predicted. Competitive motility of purified kinesin-5 and -14 was well described by a stochastic, opposing power stroke model incorporating motor kinetics and load-dependent detachment. Motor parameters from this model were applied to a new stochastic force-balance model for prometaphase spindles, providing a good fit to data from embryos. Maintenance of virtual spindles required dynamic ipMTs and a narrow range of kinesin-5 to kinesin-14 ratios matching that found in embryos. Functional perturbation and modeling suggest that this range can be extended significantly by a disassembling lamin-B envelope that surrounds the prometaphase spindle and augments the finely tuned, antagonistic kinesin force balance to maintain robust prometaphase spindles as MTs assemble and chromosomes are pushed to the equator.

The mitotic spindle accurately segregates genetic instructions by moving chromosomes to spindle poles (anaphase A) and separating the poles (anaphase B) so that, in general, the chromosomes and poles are positioned near the centers of the nascent daughter cell products of each cell division. Because the size of different types of dividing cells, and thus the spacing of their daughter cell centers, can vary significantly, the length of the metaphase or postanaphase B spindle often scales with cell size. However, significant exceptions to this scaling rule occur, revealing the existence of cell size–independent, spindle-associated mechanisms of spindle length control. The control of spindle length reflects the action of mitotic force-generating mechanisms, and its study may illuminate general principles by which cells regulate the size of internal structures. Here we review molecules and mechanisms that control spindle length, how these mechanisms are deployed in different systems, and some quantitative models that describe the control of spindle length.

The Drosophila syncytial embryo uses multiple astral mitotic spindles that are specialized for rapid mitosis. The homotetrameric kinesin-5, KLP61F contributes to various aspects of mitosis in this system, all of which are consistent with it exerting outward forces on spindle poles. In principle, kinesin-5 could accomplish this by (i) sliding microtubules (MTs), minus end leading, relative to a static spindle matrix or (ii) crosslinking and sliding apart adjacent pairs of antiparallel interpolar (ip) MTs. Here, I critically review data on the biochemistry of purified KLP61F, its localization and dynamic properties within spindles, and quantitative modeling of KLP61F function. While a matrix-based mechanism may operate in some systems, the work tends to support the latter "sliding filament" mechanism for KLP61F action in Drosophila embryo spindles. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

We used antibody microinjection and genetic manipulations to dissect the various roles of the homotetrameric kinesin-5, KLP61F, in astral, centrosome-controlled Drosophila embryo spindles and to test the hypothesis that it slides apart interpolar (ip) microtubules (MT), thereby controlling poleward flux and spindle length. In wild-type and Ncd null mutant embryos, anti-KLP61F dissociated the motor from spindles, producing a spatial gradient in the KLP61F content of different spindles, which was visible in KLP61F-GFP transgenic embryos. The resulting mitotic defects, supported by gene dosage experiments and time-lapse microscopy of living klp61f mutants, reveal that, after NEB, KLP61F drives persistent MT bundling and the outward sliding of antiparallel MTs, thereby contributing to several processes that all appear insensitive to cortical disruption. KLP61F activity contributes to the poleward flux of both ipMTs and kinetochore MTs and to the length of the metaphase spindle. KLP61F activity maintains the prometaphase spindle by antagonizing Ncd and another unknown force-generator and drives anaphase B, although the rate of spindle elongation is relatively insensitive to the motor's concentration. Finally, KLP61F activity contributes to normal chromosome congression, kinetochore spacing, and anaphase A rates. Thus, a KLP61F-driven sliding filament mechanism contributes to multiple aspects of mitosis in this system.

A kinesin-5-dependent 'sliding filament' mechanism is commonly used to actively push apart the poles during mitotic spindle assembly and elongation, but a recent study now shows that, in C. elegans, kinesin-5 is deployed as a brake to slow down spindle-pole separation.

The formation of a metaphase spindle, a bipolar microtubule array with centrally aligned chromosomes, is a prerequisite for the faithful segregation of a cell's genetic material. Using a full-genome RNA interference screen of Drosophila S2 cells, we identified about 200 genes that contribute to spindle assembly, more than half of which were unexpected. The screen, in combination with a variety of secondary assays, led to new insights into how spindle microtubules are generated; how centrosomes are positioned; and how centrioles, centrosomes, and kinetochores are assembled.

Mitosis, the process by which the replicated chromosomes are segregated equally into daughter cells, has been studied for over a century. Drosophila melanogaster is an ideal organism for this research. Drosophila embryos are well suited to image mitosis, because during cycles 10-13 nuclei divide rapidly at the surface of the embryo, but mitotic cells during larval stages and spermatocytes are also used for the study of mitosis. Drosophila can be easily maintained, many mutant stocks exist, and transgenic flies expressing mutated or fluorescently labeled proteins can be made. In addition, the genome has been completed and RNA interference can be used in Drosophila tissue culture cells. Here, we review our current understanding of spindle dynamics, looking at the experiments and quantitative modeling on which it is based. Many molecular players in the Drosophila mitotic spindle are similar to those in mammalian spindles, so findings in Drosophila can be extended to other organisms.

Background: The pole-to-pole distance of the metaphase spindle is reasonably constant in a given cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved., Results: Using the Drosophila S2 cell line, we depleted or overexpressed proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, we found a reduction in spindle size after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces., Conclusions: By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, we reach to a conclusion that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force.

Dynein is a critical mitotic motor whose inhibition causes defects in spindle pole organization and separation, chromosome congression or segregation, and anaphase spindle elongation, but results differ in different systems. We evaluated the functions of the dynein-dynactin complex by using RNA interference (RNAi)-mediated depletion of distinct subunits in Drosophila S2 cells. We observed a striking detachment of centrosomes from spindles, an increase in spindle length, and a loss of spindle pole focus. RNAi depletion of Ncd, another minus-end motor, produced disorganized spindles consisting of multiple disconnected mini-spindles, a different phenotype consistent with distinct pathways of spindle pole organization. Two candidate dynein-dependent spindle pole organizers also were investigated. RNAi depletion of the abnormal spindle protein, Asp, which localizes to focused poles of control spindles, produced a severe loss of spindle pole focus, whereas depletion of the pole-associated microtubule depolymerase KLP10A increased spindle microtubule density. Depletion of either protein produced long spindles. After RNAi depletion of dynein-dynactin, we observed subtle but significant mislocalization of KLP10A and Asp, suggesting that dynein-dynactin, Asp, and KLP10A have complex interdependent functions in spindle pole focusing and centrosome attachment. These results extend recent findings from Xenopus extracts to Drosophila cultured cells and suggest that common pathways contribute to spindle pole organization and length determination.

Drosophila melanogaster is an excellent model for studying mitosis. Syncytial embryos are amenable to time-lapse imaging of hundreds of synchronously dividing spindles, allowing the quantitation of spindle and chromosome dynamics with unprecedented fidelity. Other Drosophila cell types, including neuroblasts, cultured cells, spermatocytes and oocytes, contain spindles that differ in their design, providing cells amenable to different types of experiments and allowing identification of common core mechanisms. The function of mitotic proteins can be studied using mutants, inhibitor microinjection and RNA interference (RNAi) to identify the full inventory of mitotic proteins encoded by the genome. Here, we review recent advances in understanding how ensembles of mitotic proteins coordinate spindle assembly and chromosome motion in this system.

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.

In creating the mitotic spindle and the contractile ring, natural selection has engineered fascinating precision machines whose movements depend upon forces generated by ensembles of cytoskeletal proteins. These machines segregate chromosomes and divide the cell with high fidelity. Current research on the mechanisms and regulation of spindle morphogenesis, chromosome motility and cytokinesis emphasizes how ensembles of dynamic cytoskeletal polymers and multiple motors cooperate to generate the forces that guide the cell through mitosis and cytokinesis.

We proposed that spindle morphogenesis in Drosophila embryos involves progression through four transient isometric structures in which a constant spacing of the spindle poles is maintained by a balance of forces generated by multiple microtubule (MT) motors and that tipping this balance drives pole-pole separation. Here we used fluorescent speckle microscopy to evaluate the influence of MT dynamics on the isometric state that persists through metaphase and anaphase A and on pole-pole separation in anaphase B. During metaphase and anaphase A, fluorescent punctae on kinetochore and interpolar MTs flux toward the poles at 0.03 microm/s, too slow to drive chromatid-to-pole motion at 0.11 microm/s, and during anaphase B, fluorescent punctae on interpolar MTs move away from the spindle equator at the same rate as the poles, consistent with MT-MT sliding. Loss of Ncd, a candidate flux motor or brake, did not affect flux in the metaphase/anaphase A isometric state or MT sliding in anaphase B but decreased the duration of the isometric state. Our results suggest that, throughout this isometric state, an outward force exerted on the spindle poles by MT sliding motors is balanced by flux, and that suppression of flux could tip the balance of forces at the onset of anaphase B, allowing MT sliding and polymerization to push the poles apart.

The mitotic spindle is a complex macromolecular machine that coordinates accurate chromosome segregation. The spindle accomplishes its function using forces generated by microtubules (MTs) and multiple molecular motors, but how these forces are integrated remains unclear, since the temporal activation profiles and the mechanical characteristics of the relevant motors are largely unknown. Here, we developed a computational search algorithm that uses experimental measurements to 'reverse engineer' molecular mechanical machines. Our algorithm uses measurements of length time series for wild-type and experimentally perturbed spindles to identify mechanistic models for coordination of the mitotic force generators in Drosophila embryo spindles. The search eliminated thousands of possible models and identified six distinct strategies for MT-motor integration that agree with available data. Many features of these six predicted strategies are conserved, including a persistent kinesin-5-driven sliding filament mechanism combined with the anaphase B-specific inhibition of a kinesin-13 MT depolymerase on spindle poles. Such conserved features allow predictions of force-velocity characteristics and activation-deactivation profiles of key mitotic motors. Identified differences among the six predicted strategies regarding the mechanisms of prometaphase and anaphase spindle elongation suggest future experiments.

The mechanical events of mitosis depend on the action of microtubules and mitotic motors, but whether these spindle components act alone or in concert with a spindle matrix is an important question.

Anaphase B spindle elongation is characterized by the sliding apart of overlapping antiparallel interpolar (ip) microtubules (MTs) as the two opposite spindle poles separate, pulling along disjoined sister chromatids, thereby contributing to chromosome segregation and the propagation of all cellular life. The major biochemical “modules” that cooperate to mediate pole–pole separation include: (i) midzone pushing or (ii) braking by MT crosslinkers, such as kinesin-5 motors, which facilitate or restrict the outward sliding of antiparallel interpolar MTs (ipMTs); (iii) cortical pulling by disassembling astral MTs (aMTs) and/or dynein motors that pull aMTs outwards; (iv) ipMT plus end dynamics, notably net polymerization; and (v) ipMT minus end depolymerization manifest as poleward flux. The differential combination of these modules in different cell types produces diversity in the anaphase B mechanism. Combinations of antagonist modules can create a force balance that maintains the dynamic pre-anaphase B spindle at constant length. Tipping such a force balance at anaphase B onset can initiate and control the rate of spindle elongation. The activities of the basic motor filament components of the anaphase B machinery are controlled by a network of non-motor MT-associated proteins (MAPs), for example the key MT cross-linker, Ase1p/PRC1, and various cell-cycle kinases, phosphatases, and proteases. This review focuses on the molecular mechanisms of anaphase B spindle elongation in eukaryotic cells and briefly mentions bacterial DNA segregation systems that operate by spindle elongation. [ABSTRACT FROM AUTHOR]

The mitotic spindle uses dynamic microtubules and mitotic motors to generate the pico-Newton scale forces that are needed to drive the mitotic movements that underlie chromosome capture, alignment and segregation. Here, we consider the biophysical and molecular basis of force-generation for chromosome movements in the spindle, and, with reference to the Drosophila embryo mitotic spindle, we briefly discuss how mathematical modeling can complement experimental analysis to illuminate the mechanisms of chromosome-to-pole motility during anaphase A and spindle elongation during anaphase B. [ABSTRACT FROM AUTHOR]

Focuses on the mitotic spindle, which uses microtubule-based motor proteins to assemble itself and to segregate its sister chromatids. Research which suggests that motors invoke several distinct mechanisms involved in mitosis; Series of transient steady-state structures through which the spindle passes, in which motors appear to be involved.

Mitosis depends on the mitotic spindle, a subcellular protein machine that uses dynamic microtubules and mitotic motors to assemble itself and to coordinate chromosome movements. Spindle function depends critically on the interplay of microtubule polymer dynamics and the motor proteins and non-motor microtubule-associated proteins (MAPs) that crosslink adjacent microtubules. These microtubule crosslinkers can organize microtubules into bundles with specific polarity patterns and some of them can slide adjacent microtubules in relation to one another. Here, we discuss the functions and mechanisms of action of three such crosslinkers: the motors kinesin-5 and kinesin-14, and the non-motor MAPs of the Ase1p family. [Copyright &y& Elsevier]

Summary: The segregation of genetic material during mitosis is coordinated by the mitotic spindle, whose action depends upon the polarity patterns of its microtubules (MTs) . Homotetrameric mitotic kinesin-5 motors can crosslink and slide adjacent spindle MTs , but it is unknown whether they or other motors contribute to establishing these MT polarity patterns. Here, we explored whether the Drosophila embryo kinesin-5 KLP61F, which plausibly crosslinks both parallel and antiparallel MTs , displays a preference for parallel or antiparallel MT orientation. In motility assays, KLP61F was observed to crosslink and slide adjacent MTs, as predicted. Remarkably, KLP61F displayed a 3-fold higher preference for crosslinking MTs in the antiparallel orientation. This polarity preference was observed in the presence of ADP or ATP plus AMPPNP, but not AMPPNP alone, which induces instantaneous rigor binding. Also, a purified motorless tetramer containing the C-terminal tail domains displayed an antiparallel orientation preference, confirming that motor activity is not required. The results suggest that, during morphogenesis of the Drosophila embryo mitotic spindle, KLP61F''s crosslinking and sliding activities could facilitate the gradual accumulation of KLP61F within antiparallel interpolar MTs at the equator, where the motor could generate force to drive poleward flux and pole-pole separation. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Anaphase B in Drosophila embryos is initiated by the inhibition of microtubule (MT) depolymerization at spindle poles, which allows outwardly sliding interpolar (ip) MTs to drive pole-pole separation. Using fluorescence recovery after photobleaching, we observed that MTs throughout the preanaphase B spindle are very dynamic and display complete recovery of fluorescence, but during anaphase B, MTs proximal to the poles stabilize and therefore display lower recovery than those elsewhere. Fluorescence microscopy of the MT tip tracker EB1 revealed that growing MT plus ends localize throughout the preanaphase B spindle but concentrate in the overlap region of interpolar MTs (ipMTs) at anaphase B onset. None of these changes occurred in the presence of nondegradable cyclin B. Modeling suggests that they depend on the establishment of a spatial gradient of MT plus-end catastrophe frequencies, decreasing toward the equator. The resulting redistribution of ipMT plus ends to the overlap zone, together with the suppression of minus-end depolymerization at the poles, could constitute a mechanical switch that initiates spindle elongation. [ABSTRACT FROM AUTHOR]