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© The Rockefeller University Press,
0021-9525/1997//975 $5.00
The Journal of Cell Biology, Volume 139, Number 4,
, 1997 975-983
Article |
XMAP310: A Xenopus Rescue-promoting Factor Localized to the Mitotic Spindle
To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Domínguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289–1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5–10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985–993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation.
THE function of the mitotic spindle is to ensure even segregation of the duplicated chromosomes to the two daughter cells (Kirschner and Mitchison, 1986; McIntosh, 1994; Inoué and Salmon, 1995; Hyman and Karsenti, 1996; Waters and Salmon, 1997). Assembly of the mitotic spindle involves a dramatic reorganization of the radial interphase microtubule (MT)1 network into an elliptical, bipolar shape. This reorganization results from a significant increase in MT turnover at the onset of mitosis (Olmsted et al., 1989; Belmont et al., 1990; Verde et al., 1990, 1992; Vale, 1991; Shiina et al., 1992a; Sawin and Mitchison, 1994; Hyman and Karsenti, 1996; McNally et al., 1996; Zhai et al., 1996; Tournebize et al., 1997). MT turnover is largely due to the dynamic instability of MTs (Kirschner and Mitchison, 1986), meaning that MTs are not stable rods but instead alternate between phases of growth and shrinkage. Therefore a given turnover state for MTs will be determined by the respective values of four parameters: the growth rate (vg), the shrinkage rate (vs), and the transition between growth and shrinkage, a catastrophe which occurs with a certain frequency (fcat). The opposite event, when a MT transit from shrinkage to growth, a rescue also occurs with a certain frequency (fres) (Walker et al., 1988). Spindle MTs are stabilized and have reduced dynamicity compared with their cytoplasmic counterparts (Karsenti et al., 1984; Saxton et al., 1984; Toso et al., 1993; Vasquez et al., 1994; Dogterom et al., 1996; Heald et al., 1996; Zhai et al., 1996). Microtubule- associated proteins (MAPs) may have an important function in the stabilization of spindle MTs, since they in general dampen MT dynamics and since their activity can be regulated by phosphorylation. However, little is known about the properties of non-neuronal MAPs in general (for reviews see Olmsted, 1986; Matus, 1990; Wiche et al., 1991; Bulinski, 1994; Hirokawa, 1994; Mandelkow and Mandelkow, 1995; Hyman and Karsenti, 1996). Most probably, several different MAPs are involved in regulation of MT turnover during the cell cycle and in the spindle. However, only XMAP230, MAP4, and recently XMAP215 have been localized to the spindle and functionally characterized in vitro (Gard and Kirschner, 1987; Andersen et al., 1994; Vasquez et al., 1994; Ookata et al., 1995; Charrasse, S., M. Schroeder, C. Gauthier-Rouviere, L. Cassimeris, D.L. Gard and C. Larroque. 1996. Mol. Biol. Cell. 7:222a).
To proceed with the identification and characterization of non-neuronal MAPs, we have raised mAbs against MT proteins bound to taxol stabilized MTs isolated from interphase Xenopus laevis egg extracts (Andersen et al., 1994). Several studies indicate that the MT binding activity of MAPs is finely tuned during the cell cycle, rather than modulated by a strict on- off- switch (Zieve and Solomon, 1982; Olmsted et al., 1989; Shiina et al., 1992b; Andersen et al., 1994; Masson and Kreis, 1995). Therefore, from the interphase MAP fraction, it should be possible to identify MAPs associated with the interphasic MT network, the mitotic spindle MTs, or both. Here we report on the structural and functional in vitro characterization of a new 310-kD Xenopus MAP (XMAP310) that localizes to the mitotic spindle. Interestingly, this MAP turns out to be a strong rescue factor.
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Materials and Methods |
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Purification of XMAP310 from Xenopus Egg Extracts
Interphase egg extracts were prepared by activation of dejellied eggs with calcium ionophore A23187 (Sigma Chem. Co., St. Louis, MO) at a 1/10,000 dilution (stock 2 mg/ml in DMSO) in MMR plus 200 µg/ml cycloheximide for 5 min (see also Murray, 1991). Concentrated extracts were frozen in liquid nitrogen in 200-µl aliquots and stored at –80°C. Concentrated extracts (5–7 ml/purification) were rapidly thawed at 37°C, transferred to ice, and diluted with 1 vol 50 mM sucrose, 10 mM K-Hepes (pH 7.7), 1 mM EGTA, 1 mM MgCl2, 0.1% β-mercaptoethanol, protease inhibitors. This mix was centrifuged at 40,000 rpm (192,000 g) for 40 min at 4°C in a SW50 rotor. 20 µM (final) taxol (10 mM stock in DMSO; Molecular Probes) was added to this clarified extract on ice, together with 1/10 vol prepolymerized MTs: 400 µl cycled tubulin at 9 mg/ml was mixed with 400 µl 66% glycerol, BRB80 (80 mM K-Pipes, 1 mM EGTA, 1 mM MgCl2, pH 6.8), 10 mM MgCl2, and 1 mM GTP final and incubated for 30 min at 37°C; Taxol was then added to 20 µM (final) and incubation continued for 5 min before addition to the extract. The extract with the prepolymerized MTs was incubated on ice for 10 min, and then loaded on 40% sucrose cushions, BRB80, 20 µM taxol, and protease inhibitors at a 1:1 volume ratio. The MTs were spun through the cushion at 40,000 rpm (70,000 g) for 20 min at 4°C in a TLA100.2 rotor. The supernatant was discarded and the MT pellets resuspended in 1/4 the volume of the clarified extract (2 ml) using buffer A (20 mM K-Pipes (pH 7), 50 mM NaCl) plus 5 mM CaCl2, protease inhibitors and 0.1% β-mercaptoethanol. This mix was incubated for 10 min on ice to allow depolymerization of the taxol stabilized MTs and then centrifuged twice for 10 min, 50,000 rpm (110,000 g), 4°C. The supernatant (see Fig. 3, lane 3), enriched in XMAP310 and tubulin, was applied onto a PC 1.6/5 MonoS column using the SMART system (Pharmacia LKB Biotechnology, Uppsala, Sweden) at 4°C and 100 µl/min; After loading the column was washed with 1 ml 5% B buffer (20 mM K-Pipes (pH 7), 800 mM NaCl), a step to 20% B resulted in elution of XMAP310 along with other proteins; XMAP310 was enriched in the early fractions after shift to 20% B; pooled fractions (100–150 µl) from the MonoS column (see Fig. 3, lane 5) were loaded on a PC 3.2/30 Superose 6 column equilibrated in 100 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10% glycerol, 10 mM K-Hepes (pH 7.7) using the SMART system at 4°C and 40 µl/min. Fractions containing pure XMAP310 (see Fig. 3, lane 6) were stored separately or pooled with later fractions (see Fig. 3, lanes 7–8), aliquoted by 20 µl and stored in liquid nitrogen. The concentration of XMAP310 in the extract is 
200 nM.
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4 µl were used. Two strips of double-sided tape were fixed on a microscope slide (76 x 22mm, 0.8 mm thick, Select Micro Slide model BS3836-1975; Chance Propper Ltd., UK) 3 mm apart and perpendicular to the long axis of the slide. A 22 x 22mm coverslip (gold seal cover glass model 3306; Clay Adams, UK) was then placed on top of the two tape strips and firmly pressed to attach it well. Slide and coverslip were ethanol and water washed before use. The channel formed by the tape strips, the slide and the coverslip made up the chamber. Before use, centrosomes (Bornens et al., 1987) were diluted to 3 x 103/µl with working concentration of tubulin, and injected into the chamber and left for 5 min on ice. 10 µl 5 mg/ml Casein in BRB80 was then by capillary forces flowed through the chamber and left for 5 min on ice. The chamber was then washed with 15 µl BRB80 and the sample was injected. The volume of the sample was always 30 µl and mixed immediately before use. For most of the preparations the sample consisted of: 10.0 µl Superose 6 column buffer with XMAP310 or without XMAP310 (control), 15.1 µl BRB80, 4.83 µl cycled tubulin at 87 µM (final tubulin concentration 14 µM [using BSA as Standard]), 0.3 µl 0.1 M GTP. After application of the sample the chamber was sealed at both ends with grease and placed on the stage of a Zeiss Axiovert 10 photomicroscope (Zeiss GmbH, Oberkochen, Germany) equipped with a Zeiss 100x achrostigmat 100x/1.25 oil objective and a Hamamatsu Phototonics (Japan) C3077 CCD camera. Objective, condenser, and stage were heated to 37°C. The temperature in the chamber was 35°C. Measurements of MT dynamics were performed as described (Andersen et al., 1994) using 4-s time interval between individual video frames and NIH 1.55 and Microsoft Excel 5 programs. No samples were observed for more than 40 min. Tubulin at 40 µM plus 2 µM rhodamine-labeled tubulin (Hyman et al., 1991) in BRB80 and 1 mM GTP was mixed on ice with 1/2 vol MAP or control buffer (same dilution of MAP as used in the video experiments) and incubated for 15 min at 37°C. The sample was then processed for negative stain electron microscopy. Alternatively, the sample was diluted 200-fold into BRB80 plus 20 µM taxol at room temperature (without fixation), loaded on top of a 3 ml 15% glycerol cushion, BRB80, 20 µM taxol in modified Corex tubes (Evans et al., 1985) and then centrifuged at 12,000 rpm for 20 min at 20°C using a HB4 rotor. Coverslips were fixed for 5 min in –20°C methanol and mounted in 90% glycerol, 50 mM Tris-HCl (pH = 8), and analyzed. For negative stain EM, 3 µl MT-MAP mix was absorbed onto a glow-discharged 300 mesh carbon grid for 1 min at room temperature, followed by three 5-µl washes and staining with 1% uranyl acetate for 1 min and analysis with a Zeiss 10C electron microscope.
Miscellaneous
For microsequencing XMAP230 and XMAP310 were prepared in preparative amounts by immunoprecipitation. Dynabeads (model 110.12; Dynal GmbH, Hamburg, Germany) with the Q4 or L7 mAb precoupled were incubated in extracts for 30 min at 4°C. Beads were washed 10 times for 1 min, each time with 1 ml PBS, 150 mM NaCl, 0.5% TX-100, 1 mM PMSF at 4°C before analysis on 4% SDS-PAGE. The bands corresponding to XMAP230 and XMAP310 were excised and digested with trypsin. Peptides were separated as described by Tetaz et al. (1993). Edman degradation was performed using an Applied Biosystems Machine (model 477A; Foster City, San Francisco, CA). These microsequences as well as the identity of the Q4 immunoprecipitations, the purified XMAP310 protein (see Fig. 3, lane 6), and EF1-
(see Fig. 3, lane 5) were confirmed by Matrix-assisted laser desorption ionization mass spectroscopy (MALDI MS). MALDI MS consists of tryptic cleavage of the protein followed by mass spectroscopy of the peptides (Jensen et al., 1996). Therefore, using MALDI MS one gets a fingerprint spectrum of the tryptic cleavage sites of the protein. The spectrum can then be compared with hypothetical tryptic cleavage spectra of proteins in the data bases. Very conserved proteins will show homology by this method because trypsin sites will be conserved in a conserved sequence. Calf brain tubulin was prepared as described by Ashford et al. (1998). The relative molecular mass for XMAP310 of 310 kD was estimated on the basis of the migration on 4% SDS-PAGE of bovine brain MAP1B (Mr 325 kD). The hydrodynamic data for XMAP230 and XMAP310 were determined as described by Wilhelm et al. (1997). Gel electrophoresis and Western blotting were performed as described by Andersen et al. (1994). Western blots were developed using alkaline phosphatase conjugated secondary antibodies or ECL (Amersham Corp.). Protein concentrations were measured with Bradford (Bio-Rad Labs., Hercules, CA) or BCA (Pierce, BA oud-Beijerland, Holland) reagents. Tissue extracts were prepared as described in Andersen et al., (1994). Unless otherwise indicated, chemicals were from Sigma or Boehringer Mannheim GmbH.
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Results |
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), XMAP230 (o), XMAP310 (*). We raised mAbs against XMAP310, and obtained one clone (Q4) that recognized XMAP310 in extracts of the Xenopus tissue culture cell line XL177 (Miller and Daniel, 1977), in total Xenopus egg extracts, and in the MAP fraction (data not shown). When Xenopus tissue extracts were probed with the Q4 mAb, XMAP310 was very abundant in brain and testis tissues but could not be detected in heart, lung, stomach, or muscle tissues (data not shown).
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50% of the taxol MTs (Fig. 3 B, lanes 2 and 3; the large band at 50 kD is tubulin), and most of XMAP310 was released from the MT pellet by this treatment, although a heterogeneous fraction remained bound to MTs (Fig. 3 A, *, lanes 3 and 2, note the doublet in lane 2). XMAP230 was also eluted after Ca2+ depolymerization (Fig. 3 A o, lane 3). However, XMAP215 was highly enriched in the Ca2+-stable MT fraction (Fig. 3 A, , lane 2). The Ca2+-eluted XMAP310 was then loaded on a MonoS column, and eluted with a salt gradient (Fig. 3 A, lanes 4 and 5). The MonoS fractions enriched in XMAP310 (Fig. 3 A, lane 5) contained one major contaminant of 49 kD (Fig. 3 B, lane 5), that was identified as EF1- by MALDI MS (data not shown, Cormier et al., 1991; Hershey, 1991; Jensen et al., 1996). EF1-, was eliminated from the preparation by gel filtration through a Superose 6 column (Fig. 3 A and B, lane 6 and later fractions, lanes 7 and 8). The purification was monitored by western blotting with the Q4 mAb. A fraction appeared to remain bound to MTs after Ca2+-depolymerization (Fig. 3 C, lane IV). However, analysis of the same fractions by Coomassie staining (Fig. 3 A, lane 3) revealed that most of the protein was eluted from the MTs. This could be due to the presence of different proteins of the same molecular weight, the presence of isoforms (see Fig. 3 A, lane 2; occasionally three bands were resolved) or the loss of the epitope during the purification. To resolve this issue we checked by MALDI MS whether the protein recognized by the Q4 mAb and the purified protein were the same. The MALDI MS analysis showed that the protein immunoprecipitated by the Q4 mAb and the protein purified on the Superose 6 column were the same (data not shown). We conclude that the poor recognition of pure XMAP310 by the Q4 mAb reflects the presence of isoforms (as previously reported for XMAP230) and perhaps reduced recognition of the epitope on the pure XMAP310.
We checked that XMAP310 is a bona fide MAP by assaying the capacity of the pure protein to bind back to taxol stabilized MTs. The experiment showed that XMAP310 bound back quantitatively to MTs (Fig. 3 D, arrow). These experiments also showed that EF1- does not rebind to MTs (data not shown), and that the apparent dissociation constant of XMAP310 for MTs was 200 nM (data not shown).
XMAP310 Causes Microtubule Cross-linking
As a first step in the characterization of the effect of pure XMAP310 on MTs, we polymerized rhodamine labeled tubulin, either in the presence or absence of XMAP310. The morphology of the MTs obtained suggested that they formed bundles in the presence of XMAP310 (data not shown). This was confirmed by electron microscopy, which showed long bundles of 2–5 MTs, in the presence of XMAP310 (Fig. 4). The short distance between the individual MTs resembles the cross-links obtained with 280 kD Syncolin (Feick et al., 1991), but is different from the larger spacing reported for brain MAPs (Hirokawa et al., 1988; Sato-Yoshitake et al., 1989; Chen et al., 1992).
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was the same, indicating that EF1- does not influence MT polymerization (data not shown). These data show that XMAP310 stabilizes MTs by moderately decreasing the shrinkage rate and increasing the rescue frequency 5–10-fold.
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Interestingly, XMAP230 and E-MAP-115 (Masson and Kreis, 1995) are localized to spindle MTs although they do not have maximal binding affinity for MTs during mitosis. Preliminary results indicate that XMAP310 is a phosphoprotein and that its binding to MTs, like XMAP230 and E-MAP-115, is reduced by phosphorylation during mitosis (Andersen, S.S.L., unpublished observation). We previously hypothesized that MAPs may be locally less phosphorylated around chromosomes during mitosis, allowing their specific binding to spindle MTs (Andersen et al., 1994). It has been shown that mitotic chromatin can regulate the phosphorylation level of the MT polymerization inhibitor Stathmin/Op18, and it seems likely that a similar mechanism could locally regulate the binding activity/ phosphorylation of MAPs like XMAP310, XMAP230, and E-MAP-115 to spindle MTs (Sobel, 1991; Belmont and Mitchison, 1996; Andersen et al., 1997). However, more work will be required to determine how preferential binding of MAPs to spindle MTs is achieved during metaphase.
Effects of XMAP310 and other MAPs on MT Dynamics In Vitro
Brain MAPs and XMAP230 strongly reduce fcat and increase vg whereas XMAP310 primarily increases rescues (Drechsel et al., 1992; Pryer et al., 1992; Andersen et al., 1994; Itoh and Hotani, 1994; Trinczek et al., 1995). MAP4, a nonneuronal MAP resembling MAP2 and tau at the molecular level, strongly promotes rescues without having any effect on any of the other dynamic instability parameters (Ookata et al., 1995). However, the experimental conditions between our study and the former are so different that a direct comparison is difficult. Moreover, an important function of MAP4 seems to be the targeting of cdc2 kinase to MTs (Ookata et al., 1995). XMAP215 promotes MT turnover by strongly increasing vg at the MT plus end, increasing vs, and decreasing fres without affecting fcat (Gard and Kirschner, 1987; Vasquez et al., 1994). Comparatively, the brain MAPs together with XMAP230 are the most potent MAP MT stablizers, followed by XMAP310, MAP4, and then XMAP215.
The effect of XMAP310 on fres is unusual and raises interesting questions about the mechanism of MT polymerization. How could XMAP310 increase rescues and not growth? Protofilaments tend to change from a straight configuration during the assembly phase to a curved one during disassembly. This change in curvature is thought to drive MT depolymerization (Simon and Salmon, 1990; Mandelkow et al., 1991; Chrétien et al., 1995). We propose that XMAP310 does not increase vg because it does not bind along the length of tubulin dimers as brain MAPs are thought to do (Drechsel et al., 1992; Pryer et al., 1992). Based on our observations, XMAP310 rather cross-links MTs and protofilaments. As shown in the model (Fig. 6), XMAP310 could, by cross-linking protofilaments, reduce the tendency of protofilaments to change to a curved conformation during MT depolymerization. Together with a reduction in vs, this may increase the rescue frequency by allowing new tubulin-GTP to recap depolymerizing MTs (Fig. 6). This model fits also with the unusually high frequency of pauses observed in MTs assembled in the presence of XMAP310. The plus end of MTs with laterally cross-linked protofilaments could stay in a metastable state, some subunits being added to the end of some protofilaments while other subunits would be removed from other protofilaments.
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In mitotic Xenopus egg extracts lacking chromosomes, MTs display a high growth rate, a high catastrophe frequency and rescues and pauses are very infrequent (Belmont et al., 1990; Verde et al., 1992; Tournebize et al., 1997). One could wonder why, when a protein like XMAP310 is present in such extracts. The absence of rescues in mitotic extracts could be explained if during mitosis XMAP310 is inactivated globally by phosphorylation in the cytoplasm. In contrast, due to the presence of XMAP310 on spindle MTs, the rescue frequency of spindle MTs could be high. Therefore, it seems likely that XMAP310 and XMAP230 could be part of the machinery that ensures preferential growth of MTs towards chromosomes during spindle assembly (Andersen et al., 1997), XMAP310 by locally increasing fres and XMAP230 by locally reducing fcat (Andersen et al., 1994). In contrast, XMAP215 seems to be globally active during mitosis, since the MT growth rate remains high and XMAP215 probably is the main regulator of vg in the extract.
It is interesting to see that different MAPs affect different parameters of MT dynamic instability in a complex system like Xenopus. When dynamic instability was discovered, one of the immediate predictions was that different molecules could affect each parameter specifically, and now this is becoming reality. In the next phase, it will be very exciting to determine precisely the function of XMAP215, XMAP230, and XMAP310 in the global and local regulation of MT dynamics during mitosis and in spindle assembly. Before this becomes feasible, these molecules have to be cloned and efficient tools developed.
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Acknowledgments |
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Submitted: 26 June 1997
Revised: 18 August 1997
1. Abbreviations used in this paper: fcat, catastrophe frequency; fres, rescue frequency; mAb, monoclonal antibody; MALDI MS, Matrix Assisted Laser Desorption Ionization Mass Spectroscopy; MAP, microtubule-associated protein; MT, microtubule; pAb, polyclonal antibody; vg, growth rate; vs, shrinkage rate.
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