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Address correspondence to David Pellman, Dana-Farber Cancer Institute, 44 Binney St., Mayer 621A, Boston, MA 02115. Tel.: 617-632-4918. Fax: 617-632-5757. E-mail: david_pellman{at}dfci.harvard.edu
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Abstract |
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Key Words: mitosis; microtubule-associated protein; spindle midzone; anaphase; budding yeast
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Introduction |
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A striking feature of spindle midzones from diverse organisms is the organization of antiparallel MTs into highly ordered geometrical arrays. From EM studies it has been inferred that this organization is established either by proteinacious cross-bridges or by a more diffuse electron-dense matrix substance (McIntosh et al., 1969; McDonald et al., 1977). However, there has been debate over whether or not the electron micrographs of cross-bridges or the matrix substance represent genuine in vivo structures. Recent studies using ultra-rapid freeze substitution methods have independently confirmed the presence of spindle cross-bridges (Ding et al., 1993; Mastronarde et al., 1993; Winey et al., 1995). Little is known about the molecular composition of these spindle cross-bridges, however there is compelling evidence that at least some contain kinesin motor proteins (Sharp et al., 1999).
The highly conserved BimC homotetrameric kinesin motors have been shown to form cross-bridges between MTs in vitro and to associate with cross-bridge structures in vivo (Kashina et al., 1996; Sharp et al., 1999). These motor proteins are thought to bind and bundle spindle MTs and to make a direct contribution to force generation during spindle elongation (Hoyt et al., 1992; Saunders and Hoyt, 1992; Sharp et al., 2000). In addition, the MKLP1 motor protein complex localizes to the spindle midzone in anaphase and is necessary for spindle integrity (Sharp et al., 2000; Adams et al., 2001). This complex may contain the microtubule-associated protein (MAP) INCENP and the signaling molecule aurora B kinase and, in Caenorhabditis elegans, appears to be part of the "central spindlin" complex (Mishima et al., 2002). The purified MKLP1 motor protein alone binds to and bundles MTs and slides antiparallel MTs past each other in vitro (Nislow et al., 1992). However, the precise mechanism by which the BimC and MKLP1 motor proteins contribute to spindle midzone organization remains unknown. Specifically, it is not known if these motor proteins are sufficient to create proper spindle midzone organization or whether other nonmotor elements make a contribution.
The major class of nonmotor elements identified in the spindle midzone is MAPs. This class includes the highly conserved "chromosomal passenger" INCENP (Kim et al., 1999; Adams et al., 2001; Morishita et al., 2001; Petersen et al., 2001; Rajagopalan and Balasubramanian, 2002). In budding yeast, the best candidates for midzone MAPs are Ase1p and Stu1p (Pellman et al., 1995; Yin et al., 2002). Ase1p was originally identified as a protein that is essential in cells lacking the MT plus end tracking protein Bik1p (Pellman et al., 1995). Because ase1-1 bik1-S419 cells fail to elongate anaphase spindles, but do not undergo cell cycle arrest, these cells accumulate multiple spindles and spindle pole bodies within a single nucleus. Ase1p is a substrate for the anaphase-promoting complex (APC), and its degradation appears to be required for the normal timing of spindle disassembly (Juang et al., 1997; Huang et al., 2001). It was also shown that telophase spindles in cdc15-2 arrested cells are unstable in the absence of Ase1p (Juang et al., 1997). The analysis of specific double mutants (ase1-1 bik1-S419 and ase1 cdc15-2) previously suggested that Ase1p is important for anaphase spindle stability. However, this hypothesis has not yet been tested directly by live cell microscopy of ase1
cells. Furthermore, how Ase1p contributes at the molecular level to spindle bipolarity and elongation is unknown.
To probe the role of a nonmotor protein in midzone function, we have studied Ase1p function in vitro and in vivo. First, we note that Ase1p is a member of a conserved family of spindle midzone proteins. Next, we found that Ase1p acts as a homodimer that binds to and bundles MTs. Ase1p is required for anaphase spindle elongation, and overexpression of Ase1p is sufficient to induce spindle elongation in S phasearrested cells. In addition, FRAP analysis has revealed that Ase1p is relatively immobile within the midzone during spindle elongation. We propose that Ase1p functions as a spindle cross-bridge that imparts matrix-like characteristics to the spindle midzone, maintaining anaphase spindle integrity.
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Results |
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Ase1p is a homodimer with an extended rod shape
The molecular function of Ase1p is not known. The cellular localization of Ase1p and the phenotypes of cells lacking Ase1p are consistent with it being a structural component of the spindle. However, it could also be a component of a regulatory complex, for example, like the INCENP subunit of the aurora B kinase complex (Adams et al., 2001). If Ase1p is a structural component of the spindle, it could either be a homomeric MAP or a component of a macromolecular complex, for example, a motor protein complex. As a first step to distinguish among these possibilities, the size and shape of an epitope-tagged Ase1p (Ase1pMYC) in native yeast extracts was measured by velocity sedimentation and size exclusion chromatography. For these experiments, the lysis buffer used for extraction contained 150 mM salt and no detergent. The Stokes' radius of the native yeast molecule was determined to be 8.8 nm (Fig. 2 A), and the Svedberg coefficient was 5.9S (Fig. 2 B). From these values, we calculated that the native form of Ase1p has a molecular weight of 225 ± 45 kD (Siegel and Monty, 1966; Schuyler and Pellman, 2002). The predicted molecular weight of Ase1pMYC is 107 kD. The ratio between the measured and calculated value is 2:1, suggesting that Ase1p may form a homodimer in vivo. The axial ratio, if a prolate ellipsoid is assumed, is 17:1 (Schuyler and Pellman, 2002). This suggests that Ase1p in native extracts has an extended rod shape, which is consistent with the high content of coiled-coil structure predicted from its primary sequence.
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Ase1p is an MT binding and bundling protein
Next, we determined if Ase1p interacts with MTs. Ase1p expressed in a rabbit reticulocyte lysate copelleted with bovine Taxol-stabilized MTs (Fig. 3 A). Thus, Ase1p can interact with MTs in the absence of other yeast proteins. Furthermore, Ase1p bound to MTs with an apparent Kd of 0.3 ± 0.13 µM (n = 2; Fig. 3 B), which is similar to the binding constants of other known MAPs.
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Finally, we determined whether in the absence of Ase1p spindle midzone MTs depolymerize, in addition to losing bipolar interactions. Using rapid single focal plane imaging (streaming) of ase1 cells, we found that MTs abruptly depolymerize during spindle collapse (Fig. 5 D). The rate of MT depolymerization was similar to the rate observed for MT depolymerization upon spindle disassembly in wild-type cells at the end of mitosis (
0.18 µm/sec; Maddox et al., 2000). Taken together, our data suggest that loss of Ase1p results in little or no defect in the mitotic fast phase of spindle elongation, but rather a highly penetrant spindle collapse at the beginning of the slow phase.
However, Ase1p may play additional roles at other stages in mitosis. For example, in time-lapse movies of ase1 cells, we noticed a delay in the initiation of anaphase in many cells. This led us to suspect that Ase1p may play a role within the metaphase spindle. One possible explanation for the observed delay is that loss of Ase1p function leads to the activation of the spindle checkpoint (Hoyt, 2001). We have observed that ase1
shows synthetic lethality with a mad1
mutant, and that in ase1
mutant cells released from
-factor, there is about a 20-min delay in the metaphase to anaphase transition (unpublished data). The simplest explanation for this observation is that loss of Ase1p decreases the stability of the polar MTs in preanaphase spindles and therefore indirectly affects the function of kinetochore MTs. However, we cannot exclude the possibility that during preanaphase, Ase1p has a more direct role in regulating kinetochore MTs.
Ase1p overexpression is sufficient to induce premature spindle elongation
Having found that Ase1p was necessary for anaphase spindle stability, we next determined if Ase1p overexpression might be sufficient to trigger spindle elongation. Cells carrying a GAL::ASE1 centromeric plasmid were arrested by hydroxyurea (HU) treatment, which arrests cells in late S phase with short (1.52 µm) preanaphase mitotic spindles. High levels of Ase1p expression were induced by addition of galactose to the medium. Images of cells were acquired at 1-h intervals after induction, and spindle lengths were measured. Ase1p overexpression was sufficient to induce spindle elongation. Within the first hour after induction, the spindles grew to twice the normal length and then displayed a slow increase to approximately three times the normal length by 6 h (Fig. 6 A). At the later time points, we also observed that this spindle elongation was sufficient to deform the shape of the nucleus and, in rare cases, appeared to actually separate DNA masses (Fig. 6 B, bottom panel). The spindle elongation induced by Ase1p overexpression could be due to Ase1p-promoted MT polymerization or to Ase1p-induced advancement of the cell cycle. To distinguish between these two possibilities, the steady-state levels of Pds1p and Clb2p were monitored during the course of the experiment. The levels of both Pds1p and Clb2p remained constant throughout the time course of Ase1p induction, suggesting that the cells neither initiate anaphase (i.e., degrade Pds1p) nor exit mitosis (i.e., degrade Clb2p; Fig. 6 C). Additionally, we have found that Ase1p overexpression also induced spindle elongation in cells lacking CDC23 function, demonstrating that the ability of Ase1p to promote spindle elongation was not restricted to S phase (unpublished data). This demonstrates that Ase1p is sufficient to promote premature spindle elongation, probably by cross-linking and stabilizing spindle MTs.
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Discussion |
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Our experiments suggest that Ase1p is not a component of a large macromolecular complex and likely functions as a homodimer. Importantly, our study excludes the possibility that Ase1p is a kinesin motor light chain or a stoichiometric subunit of a mitotic regulator (for example, INCENP in the aurora B complex). Whether or not other family members exist as dimers remains to be determined, but dimerization might be expected because of coiled-coil motifs found in all family members. Although our findings demonstrate that Ase1p is not in a stable complex with other proteins, Ase1p may participate in important transient or low-affinity interactions.
The central role of Ase1p in anaphase spindle elongation
As a homodimeric spindle midzone MT-bundling protein, Ase1p is ideally situated to regulate spindle elongation. We found that Ase1p was essential for the mitotic slow phase, a mitotic phase that is thought to be the result of midzone MT polymerization coupled with antiparallel MT sliding (Oppenheim et al., 1973; McIntosh, 1994). This role within the spindle midzone and mitotic slow phase appears to be conserved. In C. elegans, a mutant in SPD-1, the homologue of Ase1p, was found to have defects in spindle integrity in anaphase that ultimately gave rise to defects in cytokinesis (O'Connell et al., 1998; Verbrugghe, K., and J. White, personal communication). In cultured cells, RNAi treatments against PRC1 also led to defects of premature spindle collapse in mitosis (Mollinari et al., 2002). Thus, the Ase1p-related family of MAPs appears to be essential to maintain anaphase spindle bipolarity in evolutionarily distant organisms.
Strikingly, we found that overexpression of Ase1p is sufficient to trigger premature spindle elongation. This effect is not due to advancement of the cell cycle and is therefore most likely due to promoting polymerization of interdigitated MTs. We propose that the Ase1p-related family of MAPs plays a central and conserved role in spindle elongation, likely by bundling antiparallel MTs and by promoting their polymerization and/or stabilization. This hypothesis is supported by the previous observation that nt-MAP65 stabilizes MTs directly in vitro (Smertenko et al., 2000).
Finally, the cell cycle control of Ase1p is consistent with its molecular function described here. Ase1p expression is restricted to mitosis in a pattern similar to that of the mitotic cyclin Clb2p (Pellman et al., 1995). Like Clb2p, Ase1p is a substrate of the APC. Proteolysis of Ase1p destabilizes telophase spindles and contributes to the timely disassembly of the mitotic spindle. Nondegradable Ase1p delayed, but did not block, spindle disassembly, leading to the supposition that the APC might have other important substrates that control anaphase spindle stability (Juang et al., 1997). More recent work has indeed identified a set of spindle proteins regulated by APC-dependent proteolysis, such as two budding yeast BimC motors, Cin8p and Kip1p (Gordon and Roof, 2001; Hildebrandt and Hoyt, 2001). Furthermore, the APC-mediated activation of separase and destruction of Pds1p are also required for normal anaphase spindle function (Uhlmann et al., 2000; Severin et al., 2001; Sullivan et al., 2001). The mechanisms underlying these regulatory events are just starting to be understood. However, defining the function of the spindle-associated targets is an important first step.
Ase1p is a static element of the spindle midzone
Ase1p shows a discrete localization to the spindle midzone that appears to mirror the extent of overlap of antiparallel MTs during anaphase. We have observed that the length of the GFPAse1p bar shrinks during late anaphase. This suggests that the GFPAse1p signal corresponds to the region of overlap between antiparallel MTs. This supports the idea that Ase1p, and perhaps other Ase1p-related proteins, preferentially binds antiparallel MTs.
In vivo, we observed that Ase1p is immobile relative to the kinesin motor protein Cin8p or in comparison to previous experiments on -tubulin (Maddox et al., 2000). By FRAP analysis, photobleached GFPAse1p shows some recovery, albeit at a very slow rate. This recovery could be due to the very slow lateral diffusion of Ase1p within the spindle midzone MT lattice. Alternatively, it may be that Ase1p is completely immobile within the midzone and the appearance of recovery is the result of the addition of newly synthesized GFPAse1p to the spindle. Either way, the length of time it takes for GFPAse1p to recover is several orders of magnitude greater than would be expected from the diffusion constant that is predicted from our hydrodynamic data (2.5 x 10-7 cm2/s). It is important to note that our experimental design left a large pool of unbleached GFPAse1p adjacent to the bleached region that could diffuse into the bleached region if it were mobile.
There are several possible explanations for the limited diffusion of Ase1p along midzone MTs. First, the organization of the MT lattice in the spindle midzone is itself predicted to form a geometrical barrier to diffusion (Jacobson and Wojcieszyn, 1984; Blum et al., 1989). Second, the affinity of Ase1p for MTs, and perhaps the even higher affinity of Ase1p for antiparallel MTs, would further limit lateral mobility (Gershon et al., 1985; Blum et al., 1989). Combined, these effects could lower the effective diffusion constant for Ase1p by several orders of magnitude, explaining our FRAP results (Blum et al., 1989). One predicted consequence of the presence of an immobile midzone MT cross-bridge is that the midzone may act to decrease the rate of spindle elongation. Immobile spindle cross-linking MAPs could act to resist the work of kinesin motor proteins sliding apart antiparallel MTs during elongation. Indeed, laser ablation experiments in several organisms suggest that an intact spindle midzone slows the rate of spindle elongation (Aist and Berns, 1981; Aist et al., 1991, 1993).
Finally, in mitotic spindles assembled in frog egg extracts, although Eg5 is dynamic, it exhibits unexpectedly low lateral mobility (Kapoor and Mitchison, 2001). This restricted lateral mobility of Eg5 provided evidence for an immobile spindle matrix. As expected, we found that loss of Ase1p does not affect the turnover of Cin8p on early anaphase spindles, the rate of which is determined by the ability of spindle-associated Cin8p to exchange with a soluble pool. However, the small compact size of early anaphase yeast spindles prevented us from measuring the lateral mobility of Cin8p.
Ase1p provides the molecular functions proposed for a spindle matrix to the spindle midzone
In general, there are three functional characteristics that have been proposed for a spindle matrix: promote the organization of midzone MTs into highly ordered arrays, participate in the execution of spindle elongation in anaphase B, and provide a static or relatively immobile structure that limits the lateral mobility of kinesin motor proteins (Pickett-Heaps et al., 1997; Scholey et al., 2001; Bloom, 2002; Kapoor and Compton, 2002). One of the most remarkable features of the spindle midzone in fungi and animal cells is its highly ordered geometrical arrays of antiparallel MTs (Ding et al., 1993; Mastronarde et al., 1993; Winey et al., 1995). We found that Ase1p binds and bundles MTs, is required for spindle elongation, and is immobile within the spindle midzone. Thus, Ase1p appears to have many of the characteristics for a proposed spindle matrix.
One view of the spindle matrix envisions a static and MT-independent structure: essentially another cytoskeleton to organize the MT-based spindle (Pickett-Heaps et al., 1997; Walker et al., 2000; Scholey et al., 2001; Bloom, 2002; Kapoor and Compton, 2002). At present, there is little functional evidence to support the presence of such a structure in budding yeast. One spindle-associated protein, Fin1p, was recently found to assemble into MT-independent filaments in vitro. However, deletion of FIN1 had no discernible mitotic defect (Bloom, 2002; van Hemert et al., 2002). More recently, the spindle matrix model has been expanded to encompass the idea that the spindle matrix might in fact be a dynamic assemblage of mitotic motor proteins, such as the bipolar tetramers related to BimC (Scholey et al., 2001). However, the very high dynamicity and low processivity of BimC motors are at variance with the notion that these proteins are the spindle matrix (Crevel et al., 1997; Gheber et al., 1999; Kapoor and Mitchison, 2001; Kapoor and Compton, 2002). Further, the unexpectedly low lateral mobility of Eg5 on spindle MTs suggested the presence of another immobile spindle component, hypothesized to be the genuine spindle matrix.
Whatever the molecular composition of this proposed spindle matrix, our work demonstrates that in the absence of Ase1p, the hypothetical matrix cannot maintain the stability of the anaphase spindle. An essential role for Ase1p-related proteins in anaphase spindle stability has also been observed in C. elegans and vertebrate tissue culture cells (O'Connell et al., 1998; Verbrugghe, K., and J. White, personal communication; Mollinari et al., 2002). Thus, in widely varied cell types, a MAP-independent matrix, if present, cannot maintain spindle bipolarity in the absence of an Ase1p-related protein.
Based upon our experiments, we propose a model for spindle midzone organization. We suggest that networks of immobile MAPs, particularly the Ase1p-related proteins, form the molecular basis of an MT-dependent spindle matrix in the spindle midzone in most cell types. Immobile cross-linking MAPs are ideally suited to couple MT organization with MT polymerization. This network of static cross-bridges within a highly organized MT lattice might work in concert with highly dynamic motor proteins to maintain spindle bipolarity and promote spindle elongation in anaphase B.
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Materials and methods |
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Protein sequence analysis and multiple sequence alignments
Coiled-coil domains were identified using "MacStripe 2.0" (Lupas et al., 1991). Protein homology was determined using the standard settings of BLASTp (Altschul and Gish, 1996). Multiple sequence alignments were performed using T-COFFEE (Notredame et al., 2000). Values for pair-wise identities were determined using BLAST2 (Tatusova and Madden, 1999). The tree diagram was generated using the BLOCKS Multiple Sequence Alignment Processor (Henikoff et al., 2000).
Protein biochemistry
Native yeast extracts prepared by liquid nitrogen lysis, coimmunoprecipitations, and hydrodynamic measurements and calculations were performed as previously described (Schuyler and Pellman, 2002). The lysis buffer was 50 mM Hepes-NaOH (pH 7.4), 150 mM NaCl, 1 mM PMSF, and Complete Mini-EDTA Free protease inhibitor mix (Roche). Large-scale production of recombinant protein in insect cells was performed as previously described (Zaloudik et al., 1997). A frozen pellet of 1.23 x 109 Ase1p-expressing Sf9 cells from 1 liter of culture was resuspended in 10 ml of lysis buffer (as above, with 1.0% Triton X-100). The cells were extracted in detergent by incubation on ice for 10 min. The suspension was homogenized for 2 min using a tight-fitting Pyrex pestle (Fisher Scientific). Cell lysates were spun at 13,000 g at 4°C. The supernatants were collected and filtered using a 0.8-µm sterile syringe filter (Millipore). A dot-blot assay was used to follow the recombinant protein during purification.
The supernatant was loaded onto a 6-ml MONO S column (Bio-Rad Laboratories) preequilibrated in lysis buffer lacking detergent. Protein was eluted from the column with a linear NaCl gradient. Ase1pMYC6xHis elutes at 500 mM NaCl. Ase1p-containing fractions were pooled and run through a 2-ml Ni-NTA column (QIAGEN) preequilibrated with 25 mM Hepes-NaOH (pH 7.4), 75 mM NaCl, and 25 mM imidazole. Protein was eluted with an imidazole gradient. Fractions were pooled and loaded onto a 1-ml MONO Q (Bio-Rad Laboratories) preequilibrated in 25 mM Hepes-NaOH (pH 7.4) and 75 mM NaCl. Ase1pMYC6xHis was eluted with a NaCl gradient, at 200 mM NaCl. Fractions were pooled and diluted with an equal volume of ddH2O and then loaded onto a 250-µl MONO S polishing column (Bio-Rad Laboratories) to concentrate the protein. The purified Ase1pMYC6xHis was eluted with a step gradient of NaCl.
MT binding and bundling was performed as previously described (Butner and Kirschner, 1991; Goode and Feinstein, 1994; Desai et al., 1999). Negative staining of MTs was performed as previously described (Desai et al., 1999).
Time-lapse video microscopy and FRAP
Fluorescence time-lapse imaging was performed at room temperature as previously described (Tirnauer et al., 1999). For photobleaching, a 377-nm Nitrogen pulse laser was used (Photonics Instruments, Inc.). Incoherent light was synchronized and amplified using a coumerin blue 440 chemical chamber, which emits at a wavelengh of 440 nm. The emitted beam was focused and projected onto the back of a dichroic mirror in the filter house and reflected through the objective lens onto the sample. For photobleaching GFPAse1p, cells were typically exposed to 10 pulses at 26 ns per pulse during an 800-ms window. Laser targeting and calibration were performed on a mirror. The laser was attenuated with a graded neutral density filter. After setting the filter to maximum attenuation, the attenuation was decreased until a hole was spotted in the mirror after a single laser pulse. The average target size had a diameter of 667 nm at the 1/2-max of a Gaussian profile, as determined by using the "profiler" option in Openlabs (Improvision). This method of targeting gives an average target area of 0.35 ± 0.2 µm2. The intensities of the prebleached target site and a region adjacent to it were measured. The intensity values of these sites were set as 100% fluorescence. The adjacent region was measured at each time point to measure the photobleaching caused by imaging. The intensity of the post-bleach target area was measured, and this value was set as 0% fluorescence.
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Footnotes |
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* Abbreviations used in this paper: APC, anaphase-promoting complex; CM, conserved motif; HU, hydroxyurea; MAP, microtubule-associated protein; MT, microtubule.
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Acknowledgments |
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D. Pellman is a scholar of the Leukemia and Lymphoma Society, and this work was supported by a grant from the National Institutes of Health, GM55772.
Submitted: 4 October 2002
Revised: 6 January 2003
Accepted: 14 January 2003
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References |
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