Laser microsurgery in fission yeast; role of the mitotic spindle midzone in anaphase B

INTRODUCTION: During anaphase B in mitosis, polymerization and sliding of overlapping spindle microtubules (MTs) contribute to the outward movement the spindle pole bodies (SPBs). To probe the mechanism of spindle elongation, we combine fluorescence microscopy, photobleaching, and laser microsurgery in the fission yeast Schizosaccharomyces pombe. RESULTS: We demonstrate that a green laser cuts intracellular structures in yeast cells with high spatial specificity. By using laser microsurgery, we cut mitotic spindles labeled with GFP-tubulin at various stages of anaphase B. Although cutting generally caused early anaphase spindles to disassemble, midanaphase spindle fragments continued to elongate. In particular, when the spindle was cut near a SPB, the larger spindle fragment continued to elongate in the direction of the cut. Photobleach marks showed that sliding of overlapping midzone MTs was responsible for the elongation of the spindle fragment. Spindle midzone fragments not connected to either of the two spindle poles also elongated. Equatorial microtubule organizing center (eMTOC) activity was not affected in cells with one detached pole but was delayed or absent in cells with two detached poles. CONCLUSIONS: These studies reveal that the spindle midzone is necessary and sufficient for the stabilization of MT ends and for spindle elongation. By contrast, SPBs are not required for elongation, but they contribute to the attachment of the nuclear envelope and chromosomes to the spindle, and to cell cycle progression. Laser microsurgery provides a means by which to dissect the mechanics of the spindle in yeast.     

Ase1 domains dynamically slow anaphase spindle elongation and recruit Bim1 to the midzone

How cells regulate microtubule cross-linking activity to control the rate and duration of spindle elongation during anaphase is poorly understood. In this study, we test the hypothesis that PRC1/Ase1 proteins use distinct microtubule-binding domains to control the spindle elongation rate. Using the budding yeast Ase1, we identify unique contributions for the spectrin and carboxy-terminal domains during different phases of spindle elongation. We show that the spectrin domain uses conserved basic residues to promote the recruitment of Ase1 to the midzone before anaphase onset and slow spindle elongation during early anaphase. In contrast, a partial Ase1 carboxy-terminal truncation fails to form a stable midzone in late anaphase, produces higher elongation rates after early anaphase, and exhibits frequent spindle collapses. We find that the carboxy-terminal domain interacts with the plus-end tracking protein EB1/Bim1 and recruits Bim1 to the midzone to maintain midzone length. Overall, our results suggest that the Ase1 domains provide cells with a modular system to tune midzone activity and control elongation rates.     

The mechanism of anaphase spindle elongation: uncoupling of tubulin incorporation and microtubule sliding during in vitro spindle reactivation

To study tubulin polymerization and microtubule sliding during spindle elongation in vitro, we developed a method of uncoupling the two processes. When isolated diatom spindles were incubated with biotinylated tubulin (biot-tb) without ATP, biot-tb was incorporated into two regions flanking the zone of microtubule overlap, but the spindles did not elongate. After biot-tb was removed, spindle elongation was initiated by addition of ATP. The incorporated biot-tb was found in the midzone between the original half-spindles. The extent and rate of elongation were increased by preincubation in biot-tb. Serial section reconstruction of spindles elongating in tubulin and ATP showed that the average length of half-spindle microtubules increased due to growth of microtubules from the ends of native microtubules. The characteristic packing pattern between antiparallel microtubules was retained even in the "new" overlap region. Our results suggest that the forces required for spindle elongation are generated by enzymes in the overlap zone that mediate the sliding apart of antiparallel microtubules, and that tubulin polymerization does not contribute to force generation. Changes in the extent of microtubule overlap during spindle elongation were affected by tubulin and ATP concentration in the incubation medium. Spindles continued to elongate even after the overlap zone was composed entirely of newly polymerized microtubules, suggesting that the enzyme responsible for microtubule translocation either is bound to a matrix in the spindle midzone, or else can move on one microtubule toward the spindle midzone and push another microtubule of opposite polarity toward the pole.     

Cdc14-regulated midzone assembly controls anaphase B

Spindle elongation in anaphase of mitosis is a cell cycle-regulated process that requires coordination between polymerization, cross-linking, and sliding of microtubules (MTs). Proteins that assemble at the spindle midzone may be important for this process. In this study, we show that Ase1 and the separase-Slk19 complex drive midzone assembly in yeast. Whereas the conserved MT-bundling protein Ase1 establishes a midzone, separase-Slk19 is required to focus and center midzone components. An important step leading to spindle midzone assembly is the dephosphorylation of Ase1 by the protein phosphatase Cdc14 at the beginning of anaphase. Failure to dephosphorylate Ase1 delocalizes midzone proteins and delays the second, slower phase of anaphase B. In contrast, in cells expressing nonphosphorylated Ase1, anaphase spindle extension is faster, and spindles frequently break. Cdc14 also controls the separase-Slk19 complex indirectly via the Aurora B kinase. Thus, Cdc14 regulates spindle midzone assembly and function directly through Ase1 and indirectly via the separase-Slk19 complex.     

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.     

Stu2 promotes mitotic spindle elongation in anaphase

During anaphase, mitotic spindles elongate up to five times their metaphase length. This process, known as anaphase B, is essential for correct segregation of chromosomes. Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae. We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2. We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3. Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I). We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.     

Mechanisms of the Ase1/PRC1/MAP65 family in central spindle assembly

During cytokinesis, the organization of the spindle midzone and chromosome segregation is controlled by the central spindle, a microtubule cytoskeleton containing kinesin motors and non‐motor microtubule‐associated proteins. The anaphase spindle elongation 1/protein regulator of cytokinesis 1/microtubule associated protein 65 (Ase1/PRC1/MAP65) family of microtubule‐bundling proteins are key regulators of central spindle assembly, mediating microtubule crosslinking and spindle elongation in the midzone. Ase1/PRC1/MAP65 serves as a complex regulatory platform for the recruitment of other midzone proteins at the spindle midzone. Herein, we summarize recent advances in understanding of the structural domains and molecular kinetics of the Ase1/PRC1/MAP65 family. We summarize the regulatory network involved in post‐translational modifications of Ase1/PRC1 by cyclin‐dependent kinase 1 (Cdk1), cell division cycle 14 (Cdc14) and Polo‐like kinase 1 (Plk1) and also highlight multiple functions of Ase1/PRC1 in central spindle organization, spindle elongation and cytokinesis during cell division.     

Chromosome passenger complexes control anaphase duration and spindle elongation via a kinesin-5 brake

During mitosis, chromosome passenger complexes (CPCs) exhibit a well-conserved association with the anaphase spindle and have been implicated in spindle stability. However, their precise effect on the spindle is not clear. In this paper, we show, in budding yeast, that a CPC consisting of CBF3, Bir1, and Sli15, but not Ipl1, is required for normal spindle elongation. CPC mutants slow spindle elongation through the action of the bipolar kinesins Cin8 and Kip1. The same CPC mutants that slow spindle elongation also result in the enrichment of Cin8 and Kip1 at the spindle midzone. Together, these findings argue that CPCs function to organize the spindle midzone and potentially switch motors between force generators and molecular brakes. We also find that slowing spindle elongation delays the mitotic exit network (MEN)-dependent release of Cdc14, thus delaying spindle breakdown until a minimal spindle size is reached. We propose that these CPC- and MEN-dependent mechanisms are important for coordinating chromosome segregation with spindle breakdown and mitotic exit.     

The role of centromere-binding factor 3 (CBF3) in spindle stability, cytokinesis, and kinetochore attachment.

The spindle midzone is critical for spindle stability and cytokinesis. Chromosomal passenger proteins relocalize from chromosomes to the spindle midzone after anaphase onset. The recent localization of the inner-kinetochore, centromere-binding factor 3 (CBF3) complex to the spindle midzone in budding yeast has led to the discovery of novel functions for this complex in addition to its essential role at kinetochores. In G1/S cells, CBF3 components are detected along dynamic microtubules, where they can "search-and-capture" newly replicated centromeres. During anaphase, CBF3 is transported to the microtubule plus-ends of the spindle midzone. Consistent with this localization, cells containing a mutation in the CBF3 subunit Ndc10p show defects in spindle stability during anaphase. In addition, ndc10-1 cells show defects during cytokinesis, resulting in a defect in cell abscission. These results highlight the importance of midzone-targeted proteins in coordinating mitosis with cell division. Here we discuss these findings and explore the significance of CBF3 transport to microtubule plus-ends at the spindle midzone.     

The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. Microtubule-associated proteins

The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B). Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function. Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs. By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer. Ase1p bound and bundled MTs in vitro. By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase. Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase-arrested cells. FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs. We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone. MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix.     

The role of tubulin polymerization during spindle elongation in vitro

We describe the effect of exogenous tubulin on reactivation of anaphase spindle elongation in isolated diatom spindles. In the absence of tubulin, spindle elongation is limited to the equivalent of the microtubule overlap zone, but in the presence of tubulin spindle elongation is several times the length of the overlap zone. Biotinylated neurotubulin is incorporated into the overlap zone and around the poles. Before spindles have elongated by the equivalent of the overlap zone, there are two regions of incorporated tubulin flanking this zone. After further elongation, there is one broad zone of incorporated tubulin in the spindle midzone. Spindle elongation and the pattern of tubulin incorporation into the midzone, but not the poles, are ATP-dependent and vanadate-sensitive. These results suggest that tubulin adds onto the ends of microtubules in the overlap zone, which then slide through the midzone as the spindle elongates.     

Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner

During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.     

The Saccharomyces cerevisiae Kinesin-related Motor Kar3p Acts at Preanaphase Spindle Poles to Limit the Number and Length of Cytoplasmic Microtubules

The Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle–specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential α-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.     

Aurora B and Kif2A control microtubule length for assembly of a functional central spindle during anaphase

The central spindle is built during anaphase by coupling antiparallel microtubules (MTs) at a central overlap zone, which provides a signaling scaffold for the regulation of cytokinesis. The mechanisms underlying central spindle morphogenesis are still poorly understood. In this paper, we show that the MT depolymerase Kif2A controls the length and alignment of central spindle MTs through depolymerization at their minus ends. The distribution of Kif2A was limited to the distal ends of the central spindle through Aurora B–dependent phosphorylation and exclusion from the spindle midzone. Overactivation or inhibition of Kif2A affected interchromosomal MT length and disorganized the central spindle, resulting in uncoordinated cell division. Experimental data and model simulations suggest that the steady-state length of the central spindle and its symmetric position between segregating chromosomes are predominantly determined by the Aurora B activity gradient. On the basis of these results, we propose a robust self-organization mechanism for central spindle formation.     

Reactivation of spindle elongation in vitro is correlated with the phosphorylation of a 205 kd spindle-associated protein

Mitotic spindles isolated from the diatom Stephanopyxis turris consist of two half-spindles of closely interdigitating microtubules that slide relative to one another in the presence of ATP, reinitiating spindle elongation (anaphase B) in vitro. Purified spindles that have been exposed to ATP-gamma-S undergo ATP-dependent reactivation more readily than do control spindles. Thiophosphorylated proteins in such spindles are located in the spindle midzone, kinetochores, and a portion of the pole complex. One major thiophosphorylated peptide of 205 kd is detected in extracts prepared from spindles labeled with [35S]ATP-gamma-S, and is also localized in the spindle midzone by using an antibody that recognizes thiophosphorylated proteins. It is likely that this 205 kd peptide is either a positive regulator or mechanochemical transducer of microtubule sliding when it is in a phosphorylated state.     

Inhibition of spindle elongation by taxol.

During anaphase B spindle elongation, interzonal microtubules lengthen to accomplish pole-pole separation, while at the same time remaining highly dynamic [Shelden and Wadsworth, J. Cell Sci. 97:273-281, 1990]. To further examine the role of microtubule polymerization and dynamics during spindle elongation, cells have been treated with taxol, which induces microtubule polymerization and stabilizes microtubules. Taxol was added to PtK1 cells 3 minutes after initial chromatid separation, so that the effect on anaphase B could be observed with minimal disruption to anaphase A movement. In 20 microM taxol, the rate and extent of pole-pole separation, measured from time-lapse video records, are reduced to 4% and 9.5% of controls, respectively. The organization of microtubules in taxol treated cells was examined using tubulin immunofluorescence and confocal fluorescence microscopy. Taxol induces a dramatic reorganization of interzonal microtubules resulting in a narrow gap, which is nearly completely lacking in MTs, across the center of the interzone. Furthermore, microtubules in taxol treated cells are resistant to nocodazole induced microtubule disassembly. Our results reveal that taxol rapidly inhibits anaphase B spindle elongation; inhibition is accompanied by a depletion of interdigitated interzonal microtubules and a reduction in microtubule dynamic behavior.     

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.     

Mitotic spindle function in Saccharomyces cerevisiae requires a balance between different types of kinesin-related motors.

Two Saccharomyces cerevisiae kinesin-related motors, Cin8p and Kip1p, perform an essential role in the separation of spindle poles during spindle assembly and a major role in spindle elongation. Cin8p and Kip1p are also required to prevent an inward spindle collapse prior to anaphase. A third kinesin-related motor, Kar3p, may act antagonistically to Cin8p and Kip1p since loss of Kar3p partially suppresses the spindle collapse in cin8 kip1 mutants. We have tested the relationship between Cin8p and Kar3p by overexpressing both motors using the inducible GAL1 promoter. Overexpression of KAR3 results in a shrinkage of spindle size and a temperature-dependent inhibition of the growth of wild-type cells. Excess Kar3p has a stronger inhibitory effect on the growth of cin8 kip1 mutants and can completely block anaphase spindle elongation in these cells. In contrast, overexpression of CIN8 leads to premature spindle elongation in all cells tested. This is the first direct demonstration of antagonistic motors acting on the intact spindle and suggests that spindle length is determined by the relative activity of Kar3p-like and Cin8p/Kip1p-like motors.     

PLK1 phosphorylates mitotic centromere-associated kinesin and promotes its depolymerase activity

During cell division, interaction between kinetochores and dynamic spindle microtubules governs chromosome movements. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator of mitotic spindle assembly and dynamics. However, the regulatory mechanisms underlying its depolymerase activity during the cell cycle remain elusive. Here, we showed that PLK1 is a novel regulator of MCAK in mammalian cells. MCAK interacts with PLK1 in vitro and in vivo. The neck and motor domain of MCAK associates with the kinase domain of PLK1. MCAK is a novel substrate of PLK1, and the phosphorylation stimulates its microtubule depolymerization activity of MCAK in vivo. Overexpression of a polo-like kinase 1 phosphomimetic mutant MCAK causes a dramatic increase in misaligned chromosomes and in multipolar spindles in mitotic cells, whereas overexpression of a nonphosphorylatable MCAK mutant results in aberrant anaphase with sister chromatid bridges, suggesting that precise regulation of the MCAK activity by PLK1 phosphorylation is critical for proper microtubule dynamics and essential for the faithful chromosome segregation. We reasoned that dynamic regulation of MCAK phosphorylation by PLK1 is required to orchestrate faithful cell division, whereas the high levels of PLK1 and MCAK activities seen in cancer cells may account for a mechanism underlying the pathogenesis of genomic instability.     

Directionality of individual kinesin-5 Cin8 motors is modulated by loop 8, ionic strength and microtubule geometry

Kinesin-5 motors fulfil essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT) plus-end directed motors. The Saccharomyces cerevisiae kinesin-5 Cin8 was found, surprisingly, to switch directionality. Here, we have examined directionality using single-molecule fluorescence motility assays and live-cell microscopy. On spindles, Cin8 motors mostly moved slowly (～25 nm/s) towards the midzone, but occasionally also faster (～55 nm/s) towards the spindle poles. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid (380 nm/s) and processive minus-end to slow plus-end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel MTs, while driving steady plus-end relative sliding. Between parallel MTs, plus-end motion was only occasionally observed. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus-end motility and affected the ionic strength-dependent directional switching of Cin8 in vitro. The deletion mutant cells exhibited reduced midzone-directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8.