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.     

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.     

The mechanism of anaphase spindle elongation

At anaphase chromosomes move to the spindle poles (anaphase A) and the spindle poles move apart (anaphase B). In vitro studies using isolated diatom spindles demonstrate that the primary mechanochemical event responsible for spindle elongation is the sliding apart of half-spindle microtubules. Further, these forces are generated within the zone of microtubule overlap in the spindle mid-zone.     

Physiological and ultrastructural analysis of elongating mitotic spindles reactivated in vitro

We have developed a simple procedure for isolating mitotic spindles from the diatom Stephanopyxis turris and have shown that they undergo anaphase spindle elongation in vitro upon addition of ATP. The isolated central spindle is a barrel-shaped structure with a prominent zone of microtubule overlap. After ATP addition greater than 75% of the spindle population undergoes distinct structural rearrangements: the spindles on average are longer and the two half-spindles are separated by a distinct gap traversed by only a small number of microtubules, the phase-dense material in the overlap zone is gone, and the peripheral microtubule arrays have depolymerized. At the ultrastructural level, we examined serial cross-sections of spindles after 1-, 5-, and 10-min incubations in reactivation medium. Microtubule depolymerization distal to the poles is confirmed by the increased number of incomplete, i.e., c-microtubule profiles specifically located in the region of overlap. After 10 min we see areas of reduced microtubule number which correspond to the gaps seen in the light microscope and an overall reduction in the number of half-spindle microtubules to about one-third the original number. The changes in spindle structure are highly specific for ATP, are dose-dependent, and do not occur with nonhydrolyzable nucleotide analogues. Spindle elongation and gap formation are blocked by 10 microM vanadate, equimolar mixtures of ATP and AMPPNP, and by sulfhydryl reagents. This process is not affected by nocodazole, erythro-9-[3-(2-hydroxynonyl)]adenine, cytochalasin D, and phalloidin. In the presence of taxol, the extent of spindle elongation is increased; however, distinct gaps still form between the two half-spindles. These results show that the response of isolated spindles to ATP is a complex process consisting of several discrete steps including initiation events, spindle elongation mechanochemistry, controlled central spindle microtubule plus-end depolymerization, and loss of peripheral microtubules. They also show that the microtubule overlap zone is an important site of ATP action and suggest that spindle elongation in vitro is best explained by a mechanism of microtubule-microtubule sliding. Spindle elongation in vitro cannot be accounted for by cytoplasmic forces pulling on the poles or by microtubule polymerization.     

Three-dimensional reconstruction and analysis of mitotic spindles from the yeast, Schizosaccharomyces pombe

Mitotic spindles of Schizosaccharomyces pombe have been studied by EM, using serial cross sections to reconstruct 12 spindles from cells that were ultrarapidly frozen and fixed by freeze substitution. The resulting distributions of microtubules (MTs) have been analyzed by computer. Short spindles contain two kinds of MTs: continuous ones that run from pole to pole and MTs that originate at one pole and end in the body of the spindle. Among the latter there are three pairs of MT bundles that end on fibrous, darkly staining structures that we interpret as kinetochores. The number of MTs ending at each putative kinetochore ranges from two to four; all kinetochore-associated MTs disappear as the spindle elongates from 3-6 microns. At this and greater spindle lengths, there are no continuous MTs, only polar MTs that interdigitate at the spindle midzone, but the spindle continues to elongate. An analysis of the density of neighboring MTs at the midzone of long spindles shows that their most common spacing is approximately 40 nm, center to center, and that there is a preferred angular separation of 90 degrees. Only hints of such square-packing are found at the midzone of short spindles, and near the poles there is no apparent order at any mitotic stage. Our data suggest that the kinetochore MTs (KMTs) do not interact directly with nonkinetochore MTs, but that interdigitating MTs from the two spindle poles do interact to form a mechanically stable bundle that connects the poles. As the spindle elongates, the number of MTs decreases while the mean length of the MTs that remain increases. We conclude that the chromosomes of S. pombe become attached to the spindle by kinetochore MTs, that these MTs disappear as the chromosomes segregate, that increased separation of daughter nuclei is accompanied by a sliding apart of anti-parallel MTs, and that the mitotic processes of S. pombe are much like those in other eukaryotic cells.     

Reactivation of isolated mitotic apparatus: metaphase versus anaphase spindles

Mitotic spindles isolated from sea urchin eggs can be reactivated to undergo mitotic processes in vitro. Spindles incubated in reactivation media containing sea urchin tubulin and nucleotides undergo pole-pole elongation similar to that observed in living cells during anaphase-B. The in vitro behavior of spindles isolated during metaphase and anaphase are compared. Both metaphase and anaphase spindles undergo pole-pole elongation with similar rates, but only in the presence of added tubulin. In contrast, metaphase but not anaphase spindles increase chromosome-pole distance in the presence of exogenous tubulin, suggesting that in vitro, tubulin can be incorporated at the kinetochores of metaphase but not anaphase chromosomes. The rate of spindle elongation, ultimate length achieved, and the increase in chromosome-pole distance for isolated metaphase spindles is related to the concentration of available tubulin. Pole-pole elongation and chromosome-pole elongation does not require added adenosine triphosphate (ATP). Guanosine triphosphate (GTP) will support all activities observed. Thus, the force generation mechanism for anaphase-B in isolated sea urchin spindles is independent of added ATP, but dependent on the availability of tubulin. These results support the hypothesis that the mechanism of force generation for anaphase-B is linked to the incorporation of tubulin into the mitotic apparatus. (If, in addition, a microtubule-dependent motor-protein(s) is acting to generate force, it does not appear to be dependent on ATP as the exclusive energy source.     

The kinesin ATK5 functions in early spindle assembly in Arabidopsis

During cell division, the mitotic spindle partitions chromosomes into daughter nuclei. In higher plants, the molecular mechanisms governing spindle assembly and function remain largely unexplored. Here, live cell imaging of mitosis in Arabidopsis thaliana plants lacking a kinesin-14 (ATK5) reveals defects during early spindle formation. Beginning during prophase and lasting until late prometaphase, spindles of atk5-1 plants become abnormally elongated, are frequently bent, and have splayed poles by prometaphase. The period of spindle elongation during prophase and prometaphase is prolonged in atk5-1 cells. Time-lapse imaging of yellow fluorescent protein:ATK5 reveals colocalization with perinuclear microtubules before nuclear envelope breakdown, after which it congresses inward from the poles to the midzone, where it becomes progressively enriched at regions of overlap between antiparallel microtubules. In vitro microtubule motility assays demonstrate that in the presence of ATK5, two microtubules encountering one another at an angle can interact and coalign, forming a linear bundle. These data indicate that ATK5 participates in the search and capture of antiparallel interpolar microtubules, where it aids in generating force to coalign microtubules, thereby affecting spindle length, width, and integrity.     

On the mechanism of anaphase spindle elongation in Diatoma vulgare

Central spindles from five dividing cells (one metaphase, three anaphase, and one telophase) of Diatoma vulgare were reconstructed from serial sections. Each spindle is made up of two half-spindles that are composed almost entirely of polar microtubules. A small percentage of continuous microtubules and free microtubules were present in every stage except telophase. The half-spindles interdigitate at the midregion of the central spindle, forming a zone of overlap where the microtubules from one pole intermingle with those of the other. At metaphase the overlap zone is fairly extensive, but as elongation proceeds, the spindle poles move apart and the length of the overlap decreases because fewer microtubules are sufficiently long to reach from the pole to the zone of interdigitation. At telophase, only a few tubules are long enough to overlap at the midregion. Concurrent with the decrease in the length of the overlap zone is an increase in the staining density of the intermicrotubule matrix at the same region. These changes in morphology can most easily be explained by assuming zone mechanochemical interaction between microtubules in the overlap zone which results in a sliding apart of the two half-spindles.     

Eg5 causes elongation of meiotic spindles when flux-associated microtubule depolymerization is blocked

In higher eukaryotes, microtubules (MT) in both halves of the mitotic spindle translocate continuously away from the midzone in a phenomenon called poleward microtubule flux. Because the spindle maintains constant length and microtubule density, this microtubule translocation must somehow be coupled to net MT depolymerization at spindle poles. The molecular mechanisms underlying both flux-associated translocation and flux-associated depolymerization are not well understood, but it can be predicted that blocking pole-based destabilization will increase spindle length, an idea that has not been tested in meiotic spindles. Here, we show that simultaneous addition of two pole-disrupting reagents p50/dynamitin and a truncated version of Xklp2 results in continuous spindle elongation in Xenopus egg extracts, and we quantitatively correlate this elongation rate with the poleward translocation of stabilized microtubules. We further use this system to demonstrate that this poleward translocation requires the activity of the kinesin-related protein Eg5. These results suggest that Eg5 is responsible for flux-associated MT translocation and that dynein and Xklp2 regulate flux-associated microtubule depolymerization at spindle poles.     

Positioning and elongation of the fission yeast spindle by microtubule-based pushing

In eukaryotic cells, proper position of the mitotic spindle is necessary for successful cell division and development. We explored the nature of forces governing the positioning and elongation of the mitotic spindle in Schizosaccharomyces pombe. We hypothesized that astral microtubules exert mechanical force on the S. pombe spindle and thus help align the spindle with the major axis of the cell. Microtubules were tagged with green fluorescent protein (GFP) and visualized by two-photon microscopy. Forces were inferred both from time-lapse imaging of mitotic cells and, more directly, from mechanical perturbations induced by laser dissection of the spindle and astral microtubules. We found that astral microtubules push on the spindle poles in S. pombe, in contrast to the pulling forces observed in a number of other cell types. Further, laser dissection of the spindle midzone induced spindle collapse inward. This offers direct evidence in support of the hypothesis that spindle elongation is driven by the sliding apart of antiparallel microtubules in the spindle midzone. Broken spindles recovered and mitosis completed as usual. We propose a model of spindle centering and elongation by microtubule-based pushing forces.     

Distribution of phosphorylated spindle-associated proteins in the diatom Stephanopyxis turris

Mitotic spindles isolated from the diatom Stephanopyxis turris become thiophosphorylated in the presence of ATP gamma S at specific locations within the mitotic apparatus, resulting in a stimulation of ATP-dependent spindle elongation in vitro. Here, using indirect immunofluorescence, we compare the staining pattern of an antibody against thiophosphorylated proteins to that of MPM-2, an antibody against mitosis-specific phosphoproteins, in isolated spindles. Both antibodies label spindle poles, kinetochores, and the midzone. Neither antibody exhibits reduced labeling in salt-extracted spindles, although prior salt extraction inhibits thiophosphorylation in ATP gamma S. Furthermore, both antibodies recognize a 205 kd band on immunoblots of spindle extracts. Microtubule-organizing centers and mitotic spindles label brightly with the MPM-2 antibody in intact cells. These results show that functional mitotic spindles isolated from S. turris are phosphorylated both in vivo and in vitro. We discuss the possible role of phosphorylated cytoskeletal proteins in the control of mitotic spindle function.     

Feo, the Drosophila homolog of PRC1, is required for central-spindle formation and cytokinesis

We performed a functional analysis of fascetto (feo), a Drosophila gene that encodes a protein homologous to the Ase1p/PRC1/MAP65 conserved family of microtubule-associated proteins (MAPs). These MAPs are enriched at the spindle midzone in yeast and mammals and at the fragmoplast in plants, and are essential for the organization and function of these microtubule arrays. Here we show that the Feo protein is specifically enriched at the central-spindle midzone and that its depletion either by mutation or by RNAi results in aberrant central spindles. In Feo-depleted cells, late anaphases showed normal overlap of the antiparallel MTs at the cell equator, but telophases displayed thin MT bundles of uniform width instead of robust hourglass-shaped central spindles. These thin central spindles exhibited diffuse localizations of both the Pav and Asp proteins, suggesting that these spindles comprise improperly oriented MTs. Feo-depleted cells also displayed defects in the contractile apparatus that correlated with those in the central spindle; late anaphase cells formed regular contractile structures, but these structures did not constrict during telophase, leading to failures in cytokinesis. The phenotype of Feo-depleted telophases suggests that Feo interacts with the plus ends of central spindle MTs so as to maintain their precise interdigitation during anaphase-telophase MT elongation and antiparallel sliding.     

Ultraviolet microbeam irradiations of mitotic diatoms: investigation of spindle elongation

Our simple instrumentation for generating a UV-microbeam is described UV microbeam irradiations of the central spindle in the pennate diatom Hantzschia amphioxys have been examined through correlated birefringence light microscopy and TEM. A precise correlation between the region of reduced birefringence and the UV-induced lesion in the microtubules (MTs) of the central spindle is demonstrated. The UV beam appears to dissociate MTs, as MT fragments were rarely encountered. The forces associated with metaphase and anaphase spindles have been studied via localized UV-microbeam irradiation of the central spindle. These spindles were found to be subjected to compressional forces, presumably exerted by stretched or contracting chromosomes. Comparisons are made with the results of other writers. These compressional forces caused the poles of a severed anaphase spindle to move toward each other and the center of the cell. As these poles moved centrally, the larger of the two postirradiational central spindle remnants elongated with a concomitant decrease in the length of the overlap. Metaphase spindles, in contrast, did not elongate nor lose their overlap region. Our interpretation is that the force for anaphase spindle elongation in Hantzschia is generated between half-spindles in the region of MT overlap.     

Three-dimensional structure of the central mitotic spindle of Diatoma vulgare

Central mitotic spindles in Diatoma vulgare have been investigated using serial sections and electron microscopy. Spindles at both early stages (before metaphase) and later stages of mitosis (metaphase to telophase) have been analyzed. We have used computer graphics technology to facilitate the analysis and to produce stereo images of the central spindle reconstructed in three dimensions. We find that at prometaphase, when the nuclear envelope is dissassembling, the spindle is constructed from two sets of polar microtubules (MTs) that interdigitate to form a zone of overlap. As the chromosomes become organized into the metaphase configuration, the polar MTs, the spindle, and the zone of overlap all elongate, while the number of MTs in the central spindle decreases from greater than 700 to approximately 250. Most of the tubules lost are short ones that reside near the spindle poles. The previously described decrease in the length of the zone of overlap during anaphase central spindle elongation is clearly demonstrated in stereo images. In addition, we have used our three- dimensional data to determine the lengths of the spindle MTs at various times during mitotis. The distribution of lengths is bimodal during prometaphase, but the short tubules disappear and the long tubules elongate as mitosis proceeds. The distributions of MT lengths are compared to the length distributions of MTs polymerized in vitro, and a model is presented to account for our findings about both MT length changes and microtubule movements.     

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.     

Contractile ring-independent localization of DdINCENP, a protein important for spindle stability and cytokinesis

Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone, and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule-depolymerizing drugs, suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. Although not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. Finally, we show that the localization of DdINCENP at the cleavage furrow is modulated by myosin II but it occurs by a mechanism different from that controlling the formation of the contractile ring.     

Live-cell analysis of mitotic spindle formation in taxol-treated cells

Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.     

The microtubule-based motor Kar3 and plus end-binding protein Bim1 provide structural support for the anaphase spindle

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ~8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs.     

Male meiotic spindle lengths in normal and mutant arabidopsis cells

Spindle elongation is crucial to normal chromosome separation in eukaryotes; in particular, it is required for or associated with the extension of distance between spindle poles and the further moving apart of the already separated chromosomes. However, little is known about the relationship between spindle elongation and the status of chromosome separation, and it is unknown whether spindle elongation in different organisms shares any quantitative feature. The Arabidopsis ask1-1 mutant might be a unique material for addressing these questions because it appears to have functional spindles, but a severe defect in homolog separation at male anaphase I (M. Yang, Y. Hu, M. Lodhi, W.R. McCombie, H Ma [1999] Proc Natl Acad Sci USA 96: 11416-11421). We have characterized male meiotic spindle lengths in wild-type and the ask1-1 mutant plants. We observed that during meiosis I some ask1-1 cells had spindles that were similar in length to fully elongated normal spindles, but the chromosomes in these cells did not show appreciable movement from the equator. Furthermore, greater movement of chromosomes from the equator was usually found in the ask1-1 cells that had longer than normal spindles. These results suggest that additional elongation of ask1-1 spindles occurred; one possible reason for the extra-long spindles may be that it is a consequence of chromosome non-separation. We also found that normal and ask1-1 spindle lengths are clustered at discrete values, and their differences are of multiples of 0.7 microm. A search of the literature revealed that in each of several organisms, spindle lengths also differ by multiples of 0.7 microm. These findings strongly suggest that the spindle elongates in response to status of chromosome separation, and perhaps there are conserved mechanisms controlling the extent of spindle elongation.     

The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization

Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.