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Mitosis depends upon the mitotic spindle, a dynamic protein machine that uses ensembles of dynamic microtubules (MTs) and MT-based motor proteins to assemble itself, control its own length (pole-pole spacing), and segregate chromosomes during anaphase A (chromosome-to-pole motility) and anaphase B (spindle elongation). In this review, we describe how the molecular and biophysical mechanisms of these processes can be analyzed in the syncytial Drosophila embryo by combining (1) time-lapse imaging and other fluorescence light microscopy techniques to study the dynamics of mitotic proteins such as tubulins, mitotic motors, and chromosome or centrosome proteins; (2) the perturbation of specific mitotic protein function using microinjected inhibitors (e.g., antibodies) or mutants to infer protein function; and (3) mathematical modeling of the qualitative models derived from these experiments, which can then be used to make predictions which are in turn tested experimentally. We provide details of the methods we use for embryo preparation, fluorescence imaging, and mathematical modeling.

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.

An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitotic motors to coordinate accurate chromosome segregation during mitosis? This process depends upon the mitotic motor, kinesin-5, whose unique bipolar architecture, with pairs of motor domains lying at opposite ends of a central rod, allows it to crosslink microtubules within the mitotic spindle and to coordinate their relative sliding during spindle assembly, maintenance and elongation. The structural basis of kinesin-5's bipolarity is, however, unknown, as protein asymmetry has so far precluded its crystallization. Here we use electron microscopy of single molecules of kinesin-5 and its subfragments, combined with hydrodynamic analysis plus mass spectrometry, circular dichroism and site-directed spin label electron paramagnetic resonance spectroscopy, to show how a staggered antiparallel coiled-coil 'BASS' (bipolar assembly) domain directs the assembly of four kinesin-5 polypeptides into bipolar minifilaments.

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.

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.

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.

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system. The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, asymptotically equal to 10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

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.

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.

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.

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.

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.

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.

During anaphase identical sister chromatids separate and move towards opposite poles of the mitotic spindle. In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochore and their minus ends at the spindle pole. Two models have been proposed to account for the movement of chromatids during anaphase. In the 'Pac-Man' model, kinetochores induce the depolymerization of kinetochore microtubules at their plus ends, which allows chromatids to move towards the pole by 'chewing up' microtubule tracks. In the 'poleward flux' model, kinetochores anchor kinetochore microtubules and chromatids are pulled towards the poles through the depolymerization of kinetochore microtubules at the minus ends. Here, we show that two functionally distinct microtubule-destabilizing KinI kinesin enzymes (so named because they possess a kinesin-like ATPase domain positioned internally within the polypeptide) are responsible for normal chromatid-to-pole motion in Drosophila. One of them, KLP59C, is required to depolymerize kinetochore microtubules at their kinetochore-associated plus ends, thereby contributing to chromatid motility through a Pac-Man-based mechanism. The other, KLP10A, is required to depolymerize microtubules at their pole-associated minus ends, thereby moving chromatids by means of poleward flux.

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.

Chromosome segregation during mitosis depends upon Kinesin-5 motors, which display a conserved, bipolar homotetrameric organization consisting of two motor dimers at opposite ends of a central rod. Kinesin-5 motors crosslink adjacent microtubules to drive or constrain their sliding apart, but the structural basis of their organization is unknown. In this study, we report the atomic structure of the bipolar assembly (BASS) domain that directs four Kinesin-5 subunits to form a bipolar minifilament. BASS is a novel 26-nm four-helix bundle, consisting of two anti-parallel coiled-coils at its center, stabilized by alternating hydrophobic and ionic four-helical interfaces, which based on mutagenesis experiments, are critical for tetramerization. Strikingly, N-terminal BASS helices bend as they emerge from the central bundle, swapping partner helices, to form dimeric parallel coiled-coils at both ends, which are offset by 90°. We propose that BASS is a mechanically stable, plectonemically-coiled junction, transmitting forces between Kinesin-5 motor dimers during microtubule sliding. DOI: http://dx.doi.org/10.7554/eLife.02217.001.

Cilia and mitotic spindles are microtubule (MT)-based, macromolecular machines that consecutively assemble and disassemble during interphase and M phase of the cell cycle, respectively, and play fundamental roles in how eukaryotic cells swim through a fluid, sense their environment, and divide to reproduce themselves. The formation and function of these structures depend on several types of cytoskeletal motors, notably MT-based kinesins and dyneins, supplemented by actin-based myosins, which may function independently or collaboratively during specific steps in the pathway of mitosis or ciliogenesis. System-specific differences in these pathways occur because, instead of conforming to a simple one motor–one function rule, ciliary and mitotic motors can be deployed differently by different cell types. This reflects the well-known influence of natural selection on basic molecular processes, creating diversity at subcellular scales. Here we review our current understanding of motor function and cooperation during the assembly–disassembly, maintenance, and functions of cilia and mitotic spindles. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

The movement of chromosomes during mitosis occurs on a bipolar, microtubule-based protein machine, the mitotic spindle. It has long been proposed that poleward chromosome movements that occur during prometaphase and anaphase A are driven by the microtubule motor cytoplasmic dynein, which binds to kinetochores and transports them toward the minus ends of spindle microtubules. Here we evaluate this hypothesis using time-lapse confocal microscopy to visualize, in real time, kinetochore and chromatid movements in living Drosophila embryos in the presence and absence of specific inhibitors of cytoplasmic dynein. Our results show that dynein inhibitors disrupt the alignment of kinetochores on the metaphase spindle equator and also interfere with kinetochore- and chromatidto-pole movements during anaphase A. Thus, dynein is essential for poleward chromosome motility throughout mitosis in Drosophila embryos. [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 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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]