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© The Rockefeller University Press,
0021-9525/1997//615 $5.00
The Journal of Cell Biology, Volume 138, Number 3,
, 1997 615-628
Article |
Spindle Assembly in Xenopus Egg Extracts: Respective Roles of Centrosomes and Microtubule Self-Organization
In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end–directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.
DURING cell division, the correct organization of microtubules in bipolar spindles is necessary to distribute chromosomes to the daughter cells. The slow growing or minus ends of the microtubules are focused at each pole, while the plus ends interact with the chromosomes in the center of the spindle (Telzer and Haimo, 1981; McIntosh and Euteneuer, 1984). Current concepts of spindle assembly are based primarily on mitotic spindles of animal cells, which contain centrosomes. Centrosomes are thought to be instrumental for organization of the spindle poles and for determining both microtubule polarity and the spindle axis. In the prevailing model, termed "Search and Capture," dynamic microtubules growing from two focal points, the centrosomes, are captured and stabilized by chromosomes, generating a bipolar array (Kirschner and Mitchison, 1986). However, while centrosomes are required for spindle assembly in some systems (Sluder and Rieder, 1985; Rieder and Alexander, 1990; Zhang and Nicklas, 1995a,b), in other systems they appear to be dispensable (Steffen et al., 1986; Heald et al., 1996). Furthermore, centrosomes are not present in higher plant cells and in female meiosis of most animal species (Bajer and Mole, 1982; Gard, 1992; Theurkauf and Hawley, 1992; Albertson and Thomson, 1993; Lambert and Lloyd, 1994). In the absence of centrosomes, bipolar spindle assembly seems to occur through the self-organization of microtubules around mitotic chromatin (McKim and Hawley, 1995; Heald et al., 1996; Waters and Salmon, 1997). The observation of apparently different spindle assembly pathways raises several questions: Do different types of spindles share common mechanisms of organization? How do centrosomes influence spindle assembly? In the absence of centrosomes, what aspects of microtubule self-organization promote spindle bipolarity?
To begin to address these questions, we have used Xenopus egg extracts, which can be used to reconstitute different types of spindle assembly. Spindle assembly around Xenopus sperm nuclei is directed by centrosomes (Sawin and Mitchison, 1991). Like other meiotic systems (Bastmeyer et al., 1986; Steffen et al., 1986), Xenopus extracts also support spindle assembly around chromatin in the absence of centrosomes through the movement and sorting of randomly nucleated microtubules into a bipolar structure (Heald et al., 1996). In this process, the microtubule-based motor cytoplasmic dynein forms spindle poles by cross-linking and sliding microtubule minus ends together. Increasing evidence suggests that the function of dynein in spindle assembly depends on its interaction with other proteins, including dynactin, a dynein-binding complex, and NuMA1 (nuclear protein that associates with the mitotic apparatus) (Merdes et al., 1996; Echeverri et al., 1996; Gaglio et al., 1996). In this paper, we demonstrate that both in the presence and absence of centrosomes, spindle pole assembly occurs by a common dynein-dependent mechanism. We show that when centrosomes are present, they are tethered to spindle poles by dynein. In the absence of dynein function, microtubules are still sorted into an antiparallel array, indicating that other aspects of microtubule self-assembly independent of pole formation promote spindle bipolarity around mitotic chromatin. Since centrosomes are dispensable for pole formation in this system, what is their function? We show here that if only one centrosome is present, it acts as a dominant site for microtubule nucleation and focal organization, resulting in a monopolar spindle. Therefore, although centrosomes are not required in this system, they can influence spindle pole formation and bipolarity.
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Materials and Methods |
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Immunofluorescence and Video Experiments
For immunofluorescence of sperm DNA and chromatin bead spindles, 20-µl reactions were diluted with 1 ml 30% glycerol, 1% Triton X-100 in BRB80 (80 mM Pipes, 2 mM MgCl2, 1 mM EGTA) and spun onto coverslips in modified corex tubes as described (Mitchison and Kirschner, 1984). For DMSO aster reactions and for visualizing dissociating centrosomes, 15% glycerol was used instead of 30%. For samples to be processed for dynein heavy chain immunofluorescence, 4% formaldehyde was included. After spinning, coverslips were fixed in methanol at –20°C for 5 min and then blocked in 3% BSA for 10 min at room temperature. Primary antibodies used were raised against Xenopus proteins and affinity purified. Polyclonal anti-NuMA antibodies were provided by A. Merdes (University of California, San Diego, CA). Polyclonal anti–
tubulin antibodies were raised to a COOH-terminal peptide by T. Ashford (European Molecular Biology Laboratory, Heidelberg, Germany), and anti– dynein heavy chain antibodies raised to a conserved sequence in the motor domain (Vaisberg et al., 1993) were provided by S. Reinsch (European Molecular Biology Laboratory). Rhodamine-conjugated secondary antibodies were used, and in some cases DNA was stained with propidium iodide. Rhodamine-labeled seeds were prepared and video microscopy was performed as described (Heald et al., 1996). Seed movement data was acquired using NIH Image. Seed distance from the center of DMSO asters was measured at 5-s intervals.
Dynein Inhibition
The monoclonal IgM anti–dynein intermediate chain antibody (mAb 70.1) and control IgM anti–mouse IgG ascites were obtained from Sigma Chemical Co. (St. Louis, MO) and dialyzed against 50 mM potassium glutamate, 0.5 mM MgCl2, then concentrated to 20 mg/ml, flash frozen, and stored in small aliquots at –80°C. For spindle pole disruption experiments, antibodies were diluted 1:10 in assembly reactions, which were then diluted and spun after 5 or 10 min as described above. To block pole assembly, mAb 70.1 was added 1:10 to extracts before spindle assembly.
Preparation of EM Samples
Chromatin bead spindles in 200 µl of extract were magnetically retrieved at 20°C. The extract was removed, and the spindles were gently resuspended in 100 µl of hooking solution containing 1.5 mg/ml calf brain tubulin, 0.5 M Pipes, pH 6.9, 1.5 mM MgCl2, 1 mM EGTA, 1 mM GTP, and 2.5% DMSO. After a 5–7-min incubation at 20°C, spindles were again magnetically retrieved, and the hooking solution was removed. 1 ml of 25% glycerol, 0.2% glutaraldehyde in BRB80 was added, and then more glutaraldehyde was added immediately afterward, bringing the final concentration to 2%. After 30 min at 20°C, the spindles were pelleted by centrifugation and washed several times with PBS. Samples were then dehydrated, imbedded in epon and sectioned, and observed with an electron microscope (Carl Zeiss, Inc., Thornwood, NY). For data acquisition, hook handedness was assessed in 50–100 microtubules in each section, and three sections were analyzed for each condition.
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tubulin (Lajoie et al., 1994; Stearns and Kirschner, 1994; Debec et al., 1995; Li and Joshi, 1995) and NuMA (Maekawa et al., 1991; Gaglio et al., 1995; Merdes et al., 1996). These proteins were localized similarly in all three cases (Fig. 1). Tubulin was enriched at poles but was also present throughout the microtubules. NuMA was highly focused in the center of the poles. Thus, by morphological criteria, centrosomal and noncentrosomal poles appear to be quite similar.
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tubulin (Fig. 5 b). Centriolar-like structures were often visible, tenuously linked to spindle microtubules. These experiments indicate that centrosomes are discrete entities independent of spindle poles, tethered to the spindle by cytoplasmic dynein activity (Fig. 5 c).
Sorting of Microtubules into an Antiparallel Array Does Not Require Pole Formation
Our results thus far indicate that cytoplasmic dynein plays a central role in spindle organization. We wondered to what extent dynein function and pole assembly contributed to the polarized organization of microtubules in spindles, with microtubule minus ends at the poles and antiparallel microtubule interactions in the center of the spindle. We have shown previously that in the presence of mAb 70.1, a parallel array of microtubules formed around chromatin beads with frayed, unfocused ends, and the beads located in the center of the array (Heald et al., 1996; Fig. 6 a). We wanted to know whether microtubules in such bundles were randomly oriented or sorted into an antiparallel array around chromatin, with plus ends at the chromatin and minus ends at spindle termini. To address this question, we assessed microtubule polarity in spindles assembled in the presence or absence of mAb 70.1 using two independent approaches. First, we examined the localization of NuMA, a protein normally associated with the minus ends of microtubules in polarized arrays (Fig. 1) (Maekawa et al., 1991). We found that NuMA was still localized to the two frayed unfocused ends of the spindle formed in the presence of mAb 70.1, albeit more diffusely (Fig. 6 a). This result indicated that microtubule minus ends were still sorted away from chromatin in the absence of pole formation. Second, we determined directly whether microtubule polarity was uniform or not by the hooking technique. In this technique, microtubules are incubated with pure tubulin under conditions that promote addition of hooked protofilament appendages to the microtubule walls (Heidemann and McIntosh, 1980). The polarity of individual microtubules can then be determined by examination of hook handedness in serial sections by electron microscopy (Euteneuer and McIntosh, 1981; Euteneuer et al., 1982). Here we analyzed single sections to determine hook handedness in chromatin bead spindles assembled in the presence of control antibodies or mAb 70.1 (Fig. 6). Under both conditions, sections that were cut through beads, likely to correspond to the spindle centers, contained microtubules with approximately equal proportions of right- and left-handed hooks, indicating that microtubules were of mixed polarity (Fig. 6, b–d). In control spindles, sections through focused microtubule bundles, corresponding to poles, contained hooks of which 90% were the same handedness, indicating that microtubules were of almost uniform polarity (Fig. 6, b–d). Although spindles assembled in the presence of mAb 70.1 did not contain poles, microtubule bundles, likely to be close to spindle termini, contained microtubules of >95% uniform hook handedness. Therefore, both NuMA staining and hook analysis show that microtubules are sorted into antiparallel arrays around chromatin beads. This sorting is independent of dynein activity and pole formation, indicating that other microtubule-based motors are contributing to the antiparallel organization of microtubules in spindles.
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15 min later, when microtubules began to grow close to the chromatin beads. This nucleation phenotype corresponds to the first step in spindle assembly in the absence of centrosomes (Heald et al., 1996). Thus, poles assembled around chromatin beads do not become permanent microtubule organizing centers, whereas centrosomes retain this property.
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90% of the microtubule structures formed in the presence of mAb 70.1 were sorted into an antiparallel array around chromatin with NuMA localized at each end. NuMA staining was no longer focused because spindle poles did not form in the absence of dynein activity. However, these structures were bipolar in the sense that microtubule minus ends were at the two spindle termini. 10% of the structures had a monopolar distribution of NuMA with sperm chromatin asymmetrically located. These results indicate that bipolarity is the favored self-assembly state of microtubules around chromatin. However, the presence of a single centrosome influences microtubule organization, leading to monopolarity. Centrosomes therefore create dominant sites for pole formation. |
Discussion |
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Another protein known to be required for microtubule organization into poles is NuMA (Kallajoki et al., 1991, 1992, 1993; Yang and Snyder, 1992; Compton and Cleveland, 1993; Compton and Luo, 1995; Gaglio et al., 1995). Recently, experiments in Xenopus egg extracts have shown that dynein and NuMA interact and that depletion of NuMA results in a spindle pole defect very similar to that of dynein inhibition (Merdes et al., 1996). A model consistent with these results is that NuMA is transported to microtubule minus ends in a complex with dynein, where NuMA could help cross-link microtubules and give cohesion to the spindle pole (Merdes et al., 1996). However, our results indicate that at least some NuMA was still enriched at spindle ends in the absence of dynein activity (Fig. 6 a). Furthermore, we have found that inhibition of NuMA blocks movement of microtubule seeds on spindle arrays (Heald, R., and A. Merdes, unpublished data). These data support the idea that, at least in Xenopus, a complex of dynein and NuMA is essential for spindle pole formation. Our results indicate that NuMA is targeted to microtubule minus ends by another mechanism in addition to its association with dynein, perhaps in association with another minus end–directed motor, as proposed (Gaglio et al., 1996), or NuMA could attain a polarized distribution independently of motor activity (Maekawa et al., 1991). Taken together, the data emerging from several laboratories indicate that dynein functions in a multimeric complex with NuMA and dynactin. However, the molecular mechanism by which these proteins interact to form spindle poles is not yet understood.
Microtubule Sorting
In the process of spindle assembly around chromatin beads, randomly nucleated microtubules are organized into a bipolar array with microtubule minus ends at poles and plus ends at chromatin (Heald et al., 1996). We have referred to this process as microtubule sorting and proposed that microtubule-based motors could function as sorting devices by recognizing microtubule polarity and moving microtubules relative to one another and to chromatin. One possibility is that sorting occurs concomitantly with spindle pole assembly, as dynein collects microtubule minus ends into arrays of uniform polarity. Here we have shown by two independent means that microtubule sorting occurs in the absence of dynein activity and pole formation, resulting in an antiparallel array of microtubules around chromatin. While NuMA staining indicated that microtubule minus ends were enriched at the frayed spindle ends, hooking analysis revealed that microtubule bundles were of uniform polarity. Therefore, other motors besides dynein must contribute to microtubule sorting during spindle assembly. One possibility is that plus end–directed motors associated with chromatin sort microtubules by moving towards plus ends, thereby pushing microtubule minus ends away from chromatin (Vernos et al., 1995). Alternatively, or concurrently, spindle tetrameric plus end–directed motors of the BimC family (Walczak and Mitchison, 1996) or motors shown to promote antiparallel microtubule sliding such as MKLP1 (Nislow et al., 1992) could also fulfill this role. Since polarity-marked seed motility appears to be driven predominantly by dynein, other assays need to be developed to visualize the activities of other motors in spindles.
The Nature of a Spindle Pole
The precise nature of spindle poles and centrosomes and the relationship between them has long been a source of confusion and controversy in cell biology (see Mazia, 1984). One reason for the confusion is that centrosomes and mitotic poles are structurally similar, each containing a focus of microtubule minus ends. However, several observations suggest that centrosomes and mitotic poles are functionally different. First, several proteins, including NuMA (Merdes et al., 1996) and the dynein/dynactin complex (Gaglio et al., 1996; this work), are required for pole formation but not for microtubule nucleation by centrosomes. Second, we have shown that after microtubule depolymerization, centrosomes persist as microtubule organizing centers, while self-assembled poles do not (Fig. 7). Thus, focal nucleation and motor-dependent organization are two functionally different mechanisms for generating a focus of microtubule minus ends.
A concept emerging from these studies is that the organization of microtubules into a spindle pole is a process that is superimposed on the centrosomal aster (Karsenti, 1991; Gaglio et al., 1996). Several observations support a dynamic association of spindle microtubules with centrosomal microtubules at poles. First, poleward microtubule flux requires that microtubule minus ends are free to depolymerize (Mitchison and Sawin, 1990). Second, the centrosome and spindle pole appear to organize separate populations of microtubules. These two populations are easily recognized in spindles assembled around sperm nuclei in Xenopus egg extracts because there are two sources of microtubules, chromatin and centrosomes, that can be separated by dynein inhibition (Fig. 5 b). In other meiotic or embryonic systems, such as Lepidoptera and Drosophila, the centrosomes are physically located a significant distance away from the spindle poles (Wolf and Bastmeyer, 1991). These two different microtubule populations seem to exist in somatic systems as well. Serial sectioning of somatic spindles revealed that most spindle microtubules ended a significant distance away from the centrosomes (Mastronarde et al., 1993). In some centrosome-dependent systems, loss or removal of centrosomes does not damage spindle integrity once the spindle is in metaphase or in anaphase (Mitchison and Salmon, 1992; Murray et al., 1996; Nicklas et al., 1989). How are two populations of microtubules created in such systems, in which chromatin-induced nucleation of microtubules does not occur? Spindle microtubules could be generated by release of microtubules from centrosomes. This has been shown to occur in Xenopus egg extracts (Belmont et al., 1990) and somatic cells in interphase (McBeath and Fujiwara, 1990). Thus, centrosomes can provide a source of microtubules and not themselves be poles.
The Role of Centrosomes
Since centrosomes are not always required to form spindle poles, what is their role? We and others have shown that a single centrosome can create a dominant site for pole assembly, enforcing monopolarity (Mazia et al., 1981; Bajer, 1982; Sawin and Mitchison, 1991; Fig. 8). In Xenopus egg extracts, an explanation for this observation is that by functioning as an efficient microtubule nucleator, a centrosome generates microtubules before they appear to grow around chromatin (Fig. 7). Once microtubules begin to grow around chromatin, they may be preferentially shuttled by dynein towards the centrosome, which constitutes a preformed focal point of microtubule minus ends, before they can be sorted into an antiparallel array. Therefore, centrosomes could be dominant for kinetic reasons, suppressing aspects of microtubule self-organization and thereby directing the sites of spindle pole formation. We propose that the critical function of centrosomes is to determine pole assembly sites. Centrosome positioning then provides a mechanism for determining the orientation of the cleavage plane, which is important in many cell types and during development (White and Strome, 1996).
Why are centrosomes required in some systems and not in others? One distinguishing feature of early embryonic systems such as Xenopus results from the presence of stored components in eggs that may be limiting in somatic cells. Therefore, centrosomes may be required in most systems because of the lack of stored microtubule nucleating material in the cytoplasm, which is present only on centrosomes, preventing microtubule growth around chromosomes (discussed in Karsenti et al., 1996). Therefore, the difference between meiotic and mitotic spindle assembly may reflect differences in microtubule nucleation rather than different principles of spindle organization.
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Acknowledgments |
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Submitted: 10 February 1997
Revised: 28 May 1997
1. Abbreviation used in this paper: NuMA, nuclear protein that associates with mitotic apparatus.
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