Article |
Address correspondence to Ron Vale, Department of Cellular and Molecular Pharmacology, 513 Parnassus Ave., University of California, San Francisco, San Francisco, CA 94143. Tel.: (415) 476-6380. Fax: (415) 476-5233. E-mail: vale{at}phy.ucsf.edu
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Abstract |
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Key Words: EB1; microtubule; mitosis; spindle; dynamics
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Introduction |
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Microtubules are intrinsically dynamic, which allows the microtubule cytoskeleton to rapidly rearrange in response to internal or external cues. Within a population of microtubules at steady-state, individual microtubules undergo transitions between phases of prolonged polymerization and depolymerization. This behavior, known as "dynamic instability," is enabled by the hydrolysis of GTP after monomeric tubulin becomes incorporated into the microtubule (Desai and Mitchison, 1997). Dynamic instability is modulated by various microtubule-associated proteins (MAPs)* and motor proteins, some of which act to promote microtubule assembly and stability, whereas others induce their depolymerization (Desai et al., 1999). Although many MAPs bind along the length of microtubules, two classes of MAPs localize selectively to the plus ends of growing microtubules: the Cap-Gly proteins (e.g., CLIP-170, p150glued subunit of dynactin) and the EB1 protein family (Schuyler and Pellman, 2001). The mechanism by which these proteins interact selectively with microtubule plus ends and their biological roles are poorly understood. Current work, however, suggests that microtubule plus endbinding proteins mediate interactions between microtubule ends and the cell cortex, kinetochores, endosomes, and dynein motor complexes (Tirnauer and Bierer, 2000; Schuyler and Pellman, 2001).
EB1 was first discovered in a yeast two-hybrid screen for proteins that interact with the human adenomatous polyposis coli (APC) tumor suppressor protein (Su et al., 1995). Homologous proteins have been identified subsequently in many organisms including budding and fission yeast, Drosophila, and Caenorhabditis elegans (Tirnauer and Bierer, 2000). The budding yeast EB1 homologue, BIM1, has received the most attention to date. In yeast, Bim1p is a nonessential gene product that performs at least three related functions: (1) it localizes to the plus ends of cytoplasmic microtubules, where it increases dynamic instability (Tirnauer et al., 1999); (2) Bim1p links microtubule ends to the cell cortex to facilitate orientation of the spindle toward the bud site by binding to a multiprotein complex containing Kar9 and myosin (Myo2p) (Korinek et al., 2000; Lee et al., 2000; Miller et al., 2000; Yin et al., 2000); and (3) through its participation in spindle orientation, Bim1p indirectly participates in a checkpoint that delays cytokinesis pending mitotic exit (Muhua et al., 1998). A mitotic function also has been assigned to the EB1 homologue Mal3 in Schizosaccharomyces pombe (Beinhauer et al., 1997).
In higher eukaryotes, the functions of EB1 proteins remain poorly understood. In epithelial cells of the early Drosophila embryo, EB1 is required to direct the axis of cell division (Lu et al., 2001), although the mechanism by which it performs this function was not resolved. In vertebrate cells, the only activity attributed to EB1 is its ability to bind the COOH terminus of the APC tumor suppressor protein and target it to the tips of growing microtubules (Mimori-Kiyosue et al., 2000a,b). The functional significance of these interactions has not been ascertained, although truncations of the COOH-terminal EB1 binding domain of APC are frequently associated with sporadic and familial colorectal cancers (Polakis, 1997).
Given the high degree of evolutionary conservation, EB1 proteins very likely perform important functions in higher eukaryotes. However, given that budding yeast and higher eukaryotes exhibit considerable differences both in their interphase microtubule organization and in their mechanisms of mitosis (Segal and Bloom, 2001), extrapolating results from yeast BIM1 to metazoan cells becomes precarious. In this study, we investigated the role of EB1 in Drosophila cells in culture by decreasing EB1 protein levels using RNA-mediated inhibition (RNAi) technology and in Drosophila embryos by injecting antibodies against EB1. These complementary techniques and preparations have allowed us to demonstrate that EB1 influences microtubule dynamics and plays a particularly critical role in the assembly, dynamics, and positioning of the mitotic spindle. Interference of EB1 function in these metazoan cells shows similar yet distinct phenotypes from those described in lower eukaryotes.
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Results |
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As tools for immunolocalization and RNAi studies, we generated polyclonal antibodies against a Dm EB1GST fusion protein. The affinity-purified antibodies recognized a protein with a molecular weight of 31 kD on immunoblots of extracts from Drosophila embryos and Schneider (S2) tissue culture cells (Fig. 1 a). To ensure that the 30-kD immunoreactive band was Drosophila EB1, S2 cells were treated with dsRNA corresponding to a 600-bp sequence of Dm EB1. Quantitative immunoblots showed that the band recognized by our antiDm EB1 antibodies decreased over time to 1% of controls after 6 d of dsRNA treatment. In contrast, this band was unaltered in cells treated for 6 d with dsRNA corresponding to either GFP (Fig. 1 b) or the most homologous member of the other three EB1 proteins, CG18190 (unpublished data). From these results, we conclude that our antibodies specifically recognize Drosophila Dm EB1 and that the RNAi treatment was effective in eliminating virtually all Dm EB1 protein from S2 cells.
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To improve the cytology of the S2 cells, we tested various substrates for their ability to promote cell adhesion and spreading. One of the substrates tested, concanavalin A, promoted S2 cell attachment to coverslips and caused them to adopt a flattened, discoid morphology (20 µm in diameter) within 12 h. In these preparations, S2 cells elaborated a well-developed, radial interphase microtubule network with readily discernible tips extending toward the cell periphery (Fig. 1 d). Because of the considerable improvement in cytology, we employed this cell preparation for subsequent examination of Dm EB1 and microtubules.
In concanavalin Atreated cells, Dm EB1 staining clearly coincided with individual microtubules and exhibited a comet-like gradient of staining, with the greatest intensity at the most distal tip of the microtubule (Fig. 1 d). During all stages of mitosis, Dm EB1 also was localized at microtubule plus ends (Fig. 1, eh). Additionally, puncta of Dm EB1 staining were found at the duplicated centrosomes of prophase cells as they began to separate from one another (Fig. 1 e). During metaphase, Dm EB1 localization to the tips of astral microtubules was particularly prominent (Fig. 1 f). In addition, as cells progressed to telophase, EB1 staining was enriched on the interpolar microtubule bundles that separated each chromosomal mass (Fig. 1 h). The distribution of Dm EB1 in S2 cells is, therefore, very similar to the localization that has been described in vertebrate cell lines (Morrison et al., 1998; Tirnauer et al., 1999; Mimori-Kiyosue et al., 2000b).
We also examined the distribution of Dm EB1 in synctitial blastoderm embryos. Consistent with observations in S2 cells, antibodies against Dm EB1 decorated the mitotic spindle and showed prominent staining of the spindle poles and astral microtubules (Fig. 1 i). Embryos in late anaphase and telophase also showed a dramatic accumulation of Dm EB1 staining on interpolar microtubule bundles and midbodies (Fig. 1 j).
Depletion of Dm EB1 affects microtubule dynamics but causes minimal perturbation of microtubule organization in interphase cells
To gain insight into the cellular functions of EB1, we investigated whether RNAi depletion of Dm EB1 affected microtubule organization by fluorescence microscopy. As discussed above, 6 d of dsRNA treatment was sufficient to reduce Dm EB1 protein to very low levels (Fig. 1 b). When plated on concanavalin Acoated coverslips, Dm EB1 dsRNAtreated cells attached and spread as well as control cells and displayed no obvious morphological abnormalities. Tubulin staining revealed that the interphase microtubule organization in these cells was indistinguishable from controls (see Fig. 3 g).
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Loss of Dm EB1 function causes defects in mitotic spindle structure
Given the role of EB1 family members in mitosis in yeast, we also examined how RNAi inhibition of Dm EB1 expression affected mitosis in Drosophila cells. Mitosis in untreated or GFP dsRNAtreated cells progressed in a very reproducible manner. At prophase, the two spindle poles were in close proximity to condensing chromosomes and always nucleated asters of long, radial microtubules (Fig. 3 a). As the cells proceeded to prometaphase (Fig. 3 b), the spindles assumed a typical bipolar organization and chromosomes were positioned between each pole. At this stage, and for all successive stages, bipolar spindles nucleated highly developed radial arrays of astral microtubules, many of which extended to the cell cortex. The chromosomes congressed to the metaphase plate (Fig. 3 c), and subsequently migrated to the spindle poles during anaphase (Fig. 3 d) and telophase (Fig. 3 e). At cytokinesis (Fig. 3 f), the two incipient cells assumed a more rounded shape.
In cells lacking Dm EB1, defects in microtubule organization were readily apparent. During preprophase, Dm EB1deficient cells duplicated centrosomes normally and the two centrosomes migrated to opposite sides of the nucleus as in control cells (Fig. 3 g). At this stage, each centrosome nucleated a normal radial array of long microtubules that extended toward the cell periphery. However, when dsRNA-treated cells progressed to prophase, the long cytoplasmic microtubules disappeared (Fig. 3, compare h with a), and instead, only very short (<1 µm) astral microtubules were observed clustered around the two poles. Short microtubule fragments unattached to the poles were often present in the cytoplasm of Dm EB1deficient cells. These phenotypes were observed in 74% of the Dm EB1deficient prophase cells examined (n = 100), but were never observed in untreated (n = 100) or GFP RNAi control cells (n = 100). From these observations, we conclude that Dm EB1 is required for stabilizing microtubules and creating astral arrays in mitosis.
The loss of Dm EB1 also produced aberrant spindle phenotypes in metaphase cells that could be classified into four general categories. The most common defect was a complete loss of astral microtubules (Fig. 3 i) (35% of cells, n = 264). These spindles maintained their bipolar symmetry, but commonly exhibited detachment of centrosomes from the spindle (Fig. 3 i, arrow). The second class of defects (observed in 33% of the cells) lacked astral microtubules and exhibited an overall compaction of the spindle into a basket-like meshwork of microtubules surrounding the chromosomes (Fig. 3 j). In these structures, the poles could not be clearly distinguished, but mitotic chromosomes maintained their position at the center of the spindle. The third type of defect (30% of the cells) was a detachment of a spindle pole from the bundles of microtubules that were connected to the kinetochores (Fig. 3 l). These spindles exhibited a "splayed" morphology. The fourth category of defect (2% of cells) was "barrel-shaped" spindles that maintained their symmetry, but failed to focus the microtubules at the poles and also lacked astral microtubules. These phenotypes did not appear to be due to gross centrosome defects, as immunofluorescent staining with antibodies against centrosomin protein revealed spindle poles to be present and intact (unpublished data). In all four classes of defective spindles, the distance from pole to pole was significantly smaller (5.4 ± 1.1 µm) than in GFP dsRNAtreated cells (7.7 ± 0.9 µm, P < 0.0001, t test). These results indicate that Dm EB1 plays a critical role during spindle assembly.
The mitotic defects we observed in the Dm EB1 RNAitreated cells were severe enough that we suspected they might affect cell cycle progression by activating the spindle checkpoint. To test this possibility, fixed cells were stained for DNA and the number of cells with mitotic figures was scored as a percentage of the entire cell population. In Dm EB1 dsRNAtreated cultures, the mitotic index was 5.9% (n = 1,500 cells), approximately double that of control cultures at 2.7% (2,700 cells) (Table II). Although significant (P < 0.0001), this difference was not as dramatic as might be expected if mitotic progression were completely blocked. If the mitotic checkpoint were activated for prolonged periods of time, an increase in apoptotic cells might be expected. However, S2 cells exhibit macrophage-like properties (Ramet et al., 2002), and we observed that they consume their apoptotic neighbors, as judged by nuclear morphology (unpublished data). This property of S2 cells could give rise to artificially low mitotic index measurements. To determine at which stage of the cell cycle mitotic progression was interrupted, we next categorized all of the mitotic cells in these samples according to their stage of mitosis. In control-treated cultures, cells appeared to spend approximately the same amount of time in each stage of mitosis (Table II). In Dm EB1depleted cells, however, there was an accumulation at metaphase (40% compared with 22% in controls) and in telophase (
43% compared with 38% in controls). These data suggest that inhibition of Dm EB1 activated the spindle checkpoint. Further work will be required to understand potential checkpoint activation in response to loss of EB1 function, perhaps by live cell imaging.
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In control embryos, dynamics of the mitotic spindles followed a well-characterized, documented progression (Karr and Alberts, 1986; Kellogg et al., 1988; Sullivan and Theurkauf, 1995). During interphase of cycle 12, duplicated centrosomes moved to opposite sides of the nucleus to positions separated by 120°. Upon entry into prometaphase, the nuclear envelope broke down and the nuclear space was invaded by microtubules emanating from opposite poles (Fig. 5 c). These microtubules formed attachments either with chromosomes to form kinetochore fibers or intercalate with microtubules of opposite polarity to form interpolar bundles. A few minutes after chromosomes congressed to the metaphase plate, the spindles transited to anaphase and sister chromosomes segregated to opposite poles to complete mitosis. The pole-to-pole distances are highly reproducible in spindles throughout cycle 12 (Fig. 5). After nuclear envelope breakdown of cycle 12, the length of the spindle is
8 µm. As the cells progressed to metaphase, spindles elongated at a rate of
0.03 µm/s until reaching a separation of
12 µm. Upon anaphase onset (Fig. 5 c, asterisk), spindles further elongated at a rate
0.07 µm/s until reaching a maximal length of
16.5 µm. These measurements are in close agreement with a previous description of Drosophila embryo spindle dynamics (Sharp et al., 2000a).
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Spindles further from the injection site exhibited a pole-to-pole distance that more closely resembled controls, but also frequently displayed structural defects such as frayed (Fig. 5 a, II) and monopolar half spindles that had both centrosomes present at a single pole (Fig. 5 a, II). Observation of these defects over time revealed that spindle structure was dynamic and these frayed and monopolar spindles could sometimes correct themselves and complete mitosis (unpublished data). Regions of these embryos distal to the injection site supported formation of morphologically normal spindles that progressed through mitosis similar to controls.
We quantitated the effects of Dm EB1 inhibition on spindle elongation by measuring the pole-to-pole distances of spindles proximal to the injection site over time (Fig. 5 b). During the prophase-to-metaphase transition, spindles elongated twofold slower (0.015 µm/s) and achieved a shorter length (8.1 ± 0.5 µm) at metaphase. At anaphase, spindles elongated threefold slower (
0.01 µm/s) and elongated to a maximal length 40% less than controls (9.2 ± 0.6 µm) (Fig. 5 b). In addition to reduced rates of elongation, spindles proximal to the injection site exhibited a striking overall reduction in associated microtubules and failed to form normal interpolar microtubule bundles or a midbody at the end of anaphase (Fig. 5 d).
If interference with Dm EB1 activity disrupted normal spindle elongation at anaphase, we speculated that proper chromosome segregation could be affected as well. To test this hypothesis, we coinjected Dm EB1 antibodies and rhodamine-labeled histones into embryos expressing GFPtubulin to simultaneously observe the behaviors of chromosomes and microtubules. In control embryos, fluorescent histones incorporated into chromatin and allowed observation of chromosome condensation at prometaphase, chromosome congression to the metaphase plate, and sister chromatid separation and segregation to each pole during anaphase (Fig. 6 a; Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200202032/DC1). Injection of Dm EB1 antibodies, however, disrupted chromosome segregation and produced a range of phenotypes (Fig. 6 b; Table III; Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200202032/DC1). The mildest defect caused by Dm EB1 antibody injection was the generation of lagging chromosomes during anaphase (30%). More deleterious effects were produced when the chromosomes began to segregate but failed during anaphase, producing bilobed (8.6%) or stretched (31%) chromosomal masses that failed to segregate and decondensed midway between the poles at the end of mitosis. The most extreme defect observed was complete inhibition of chromosomal segregation, leading to the formation of a tetraploid nucleus in between two spindle poles. Taken together, out results from microinjecting antiDm EB1 antibodies into preblastoderm embryos indicate that Dm EB1 plays a crucial role in mitotic spindle formation and elongation and is needed for the proper segregation of mitotic chromosomes during anaphase.
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Discussion |
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EB1 influences microtubule dynamics in distinct ways in interphase and mitosis
During interphase, loss of Dm EB1 does not alter microtubule length or distribution and produces no obvious effect on cell morphology. However, by imaging GFPtubulin, we find that loss of Dm EB1 causes the majority (60%) of microtubules to enter a "paused" state in which they are neither growing nor shrinking. Microtubules assembled from purified tubulin rarely exhibit such static behavior (Walker et al., 1988). Therefore, pausing most likely reflects the action of a cellular factor that suppresses microtubule dynamics, possibly by capping the microtubule end. Dm EB1 appears to promote dynamic behavior, at least in part, by antagonizing the actions of this yet unknown factor, either by directly competing for tubulin sites or by inducing a conformation at the microtubule end that prevents capping. Interestingly, our findings are similar to those obtained for Bim1p in S. cerevisiae, which show that microtubules in bim1-null cells are less dynamic in G1 of the cell cycle, spending >60% of their lifetimes in a paused state (Tirnauer et al., 1999). Although these effects of EB1 on interphase microtubule dynamics are not crucial to the formation of the microtubule network in S2 cells, we speculate that they may be important for dynamic rearrangements of the microtubule cytoskeletal network that occur during cell migration and other polarized cell shape changes.
Although Dm EB1 loss does not dramatically change the number of microtubules during interphase, it does decrease microtubule lengths and numbers in mitosis. In a study of microtubule dynamics at the G2/M transition in vertebrate cells, Zhai et al. (1996) observed that microtubule polymer levels dramatically decrease upon entry into prophase, but polymer levels increase as mitosis progresses and chromosomes become attached to microtubules. In EB1-depleted S2 cells, the extent of microtubule disassembly in prophase is more severe than in control cells, and may reflect an inability of the cell to reestablish microtubule polymer levels later in mitosis. This decrease in microtubule polymer was not observed with RNAi of another plus endbinding protein, CLIP-190 (Lantz and Miller, 1998; unpublished data). The basis for the mitotic-specific effect of Dm EB1 on microtubule stability may be due either to a change in how Dm EB1 interacts with microtubules or in the activities of other microtubule-associated proteins. We favor the latter possibility because it has been shown that assembly-promoting factors, such as XMAP215/TOG, are downregulated in mitosis, which allows depolymerization factors, such as the KIN-I kinesins or stathmin/OP18, to predominate (Andersen, 2000; Tournebize et al., 2000). Thus, EB1 may play a particularly important role in mitosis in counteracting microtubule depolymerization factors. The most direct way to test these ideas would be to observe microtubule behavior in Dm EB1depleted mitotic cells in real time. However, due to the loss of astral microtubules and the bundling of microtubules in the interpolar regions, we were unable to resolve the behavior of individual microtubules in GFPtubulin-transfected, Dm EB1depleted cells.
This finding for DM EB1 differs from that obtained for Bim1p in S. cerevisiae, as bim1 cells do not exhibit significant defects in microtubule behavior in preanaphase or anaphase, even though there are subtle changes in microtubule dynamics and spindle positioning (Tirnauer et al., 1999). The role of Dm EB1 also differs from its orthologue Mal3 in fission yeast, as null mutants exhibit abnormally short cytoplasmic microtubules but no defects in their spindle morphology. These differences may not be due to different molecular mechanisms of EB1, but rather due to the distinct processes for creating the spindle and executing chromosome movements.
EB1 is needed for proper formation and positioning of mitotic spindles
The most frequent phenotype observed in Dm EB1depleted mitotic spindles is the failure to form astral microtubules, which may underlie many of the aberrant spindle phenotypes produced by Dm EB1 RNAi and antibody-injected embryos, although other unknown roles of EB1 (e.g., interactions with other proteins) may play a role as well. In the absence of Dm EB1, the central spindle still contains kinetochore fibers, often they are partially or fully detached from the centrosomes, which gives rise to defocused or "splayed apart" microtubules at the poles. In wild-type spindles, we speculate that astral microtubules nucleated from the spindle poles intercalate with microtubule bundles in the central spindle to focus them to the poles via microtubule cross-linking proteins or through motor proteins such as Ncd or cytoplasmic dynein (Sharp et al., 2000b).
Loss of astral microtubules is also likely to underlie the spindle positioning defects that we observe in Dm EB1depleted cells. Spindle positioning has been speculated to involve a balancing of forces generated either by growing astral microtubules pushing against the cell cortex or by cortically bound motor complexes containing dynein and Lis1 pulling on astral microtubules (Faulkner et al., 2000; Segal and Bloom, 2001; Dujardin and Vallee, 2002). Similarly, yeast Bim1p has been shown to be important in orienting the mitotic spindle into the bud neck by linking microtubules to the cortically bound Kar9p complex and the actin cytoskeleton (for review see Bloom, 2000). Mammalian EB1 also has been shown to interact with dynein intermediate chain and with subunits of the dynactin complex, and so it may mediate motor microtubule linkages at the plasma membrane of higher eukaryotes as well (Berrueta et al., 1999).
Our results also shed light upon the recent observations of Lu et al. (2001) who demonstrated that Dm EB1 is required for spindle orientation in epidermoblasts of the Drosophila embryo. In this cell type, cell divisions are normally oriented within the plane of the tissue in response to lateral polarity cues established by adherens junctions formed between neighboring cells. When Dm EB1 was reduced by RNAi, epidermal cells instead divide randomly with respect to the plane of the tissue. It was generally assumed that this effect was due to impaired interactions of microtubules with adherens junction components that served as polarity cues. Although this may be true, our results also reveal a drastic reduction in the number and length of astral microtubules that also may underlie the defect observed in these asymmetric cell divisions.
Anaphase chromosome motion is impaired after inhibition of EB1 function
Inhibition of Dm EB1 in synctitial Drosophila embryos by injection of anti-EB1 antibodies also revealed important roles for this protein during the later stages of mitosis. In these cells, the most severe mitotic defects were observed closest to the injection site, and these included dramatically reduced rates of spindle elongation throughout mitosis and defective chromosome segregation. Spindles distal to the injection site exhibited less severe structural defects, but also exhibited lagging chromosomes during anaphase. These phenotypes were not directly observed in S2 cells depleted of Dm EB1, and we postulate that this is due to activation of the spindle checkpoint as the result of damage to the spindle.
Why do mitotic spindles fail to elongate during anaphase? The forces that drive spindle elongation during anaphase B are derived, at least in part, from the activities of cortical cytoplasmic dynein pulling on astral microtubules and from bipolar kinesins that push spindle poles apart by sliding antiparallel interpolar microtubule bundles. We demonstrated that inhibition of Dm EB1 suppresses the formation of both astral microtubules and interpolar microtubules and eliminates the formation of midbodies during late telophase. A role for Dm EB1 in the formation or stabilization of these subpopulations of spindle microtubules is supported by our immunolocalization data showing the protein enriched on astral microtubules and in interpolar bundles and midbodies in S2 cells and embryos (Fig. 1 h; unpublished data). The inhibition of anaphase after EB1 depletion may be a consequence of the failure to produce spindles that form the specialized microtubule structures required for elongation in anaphase. Another possible mechanism is suggested by the observation that anaphase B is accompanied by microtubule polymerization in the central spindle that may contribute to the forces that drive spindle poles apart (Shelden and Wadsworth, 1990). As Dm EB1 appears necessary to promote microtubule growth during mitosis, it may be that in the absence of this protein, anaphase microtubule polymerization is inhibited and spindle elongation fails. These two potential mechanisms are not mutually exclusive.
The question of why chromosome segregation fails when Dm EB1 is inhibited is also an important one. The simplest explanation is that, in the absence of EB1, spindle elongation during anaphase is crippled to such an extent that chromosome-to-pole movement is insufficient to drive their segregation, leading to an increased number of 4N nuclei. Alternatively, it is possible that Dm EB1 mediates interactions between kinetochores and microtubules and in the absence of this interaction, anaphase A is affected. This is an interesting possibility in light of recent work identifying APC as a kinetochore component (Fodde et al., 2001; Kaplan et al., 2001), although no evidence exists for a direct interaction between Drosophila APC/APC2 and Dm EB1 (Lu et al., 2001; unpublished data).
In conclusion, our studies reveal that Dm EB1 is not essential for creating the microtubule network in interphase but is essential for microtubule organization in mitosis. Such cell cycle specificity, which is not common among MAPs, raises the possibility that EB1 might constitute an attractive target for small molecule inhibition of cell division in cancer chemotherapy. At least three different genes for EB1 family proteins exist in the human genome; one of which is ubiquitously expressed, and the other two are tissue specific. Selective inhibition of these mammalian genes will be required to evaluate the utility of EB1 inhibition as means of interfering with cancer growth.
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Materials and methods |
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Antibodies
We obtained an EST, clone LD08743, from the Berkeley Drosophila Genome Project that contained the full reading frame for Drosophila EB1 (Research Genetics). Primers containing a 5' BamHI site and a 3' EcoRI site were used to amplify the EB1 coding sequence. This PCR product was inserted in frame into the expression vector pGEX-6-2P (Amersham Biosciences) to make a fusion protein with GST. The recombinant GSTDm EB1 was expressed in Escherichia coli and purified by glutathione-Sepharose affinity chromatography per the manufacturer's instructions. Anti-Dm EB1GST antisera were produced in rabbits by Covance, Inc. Polyclonal antibodies against GST were first removed by applying the serum to a GSTSepharose column; the flowthrough was applied to a GSTDm EB1Sepharose column and the Dm EB1 polyclonal antibodies were eluted with low pH.
Immunofluorescence microscopy
For microtubule staining, S2 cells were rinsed in BRB80 (80 mM Pipes, pH 6.9, 1 mM MgCl2, 1 mM EGTA) and fixed in the same buffer containing 0.5% glutaraldehyde (EM Sciences), 3% formaldehyde (EM Sciences), and 1 mg/ml saponin for 10 min. The cells were then permeabilized in PBS containing 0.5% SDS, treated with sodium borohydride, and blocked with 5% normal goat serum in PBS/0.1% Triton X-100. In experiments examining the localization of Dm EB1, cells were fixed for 10 min by immersion in a solution of 90% methanol, 3% formaldehyde, 5 mM sodium carbonate (pH 9) chilled to -80°C. Samples were then rehydrated into PBS/0.1% Triton X-100 and blocked as above. All antibodies were diluted into 5% normal goat serum in PBS/Triton (DM1, 1:500; rabbit anti-EB1, 1:1,000) and applied to the fixed cells for 1 h followed by extensive washing with PBS/Triton X-100. Fluorescent secondary antibodies (Cy2-conjugated antirabbit and rhodamine-Xconjugated antimouse; Jackson ImmunoResearch Laboratories) were used at a final dilution of 1:300. After antibody staining, cells were treated with DAPI (0.5 µg/ml in PBS) for 10 min, briefly rinsed with distilled water, and mounted in 90% glycerol, 10% 0.1 M borate, pH 9.0, plus 5% n-propyl gallate. Specimens were imaged by confocal microscopy (TCS; Leica) and presented as maximum intensity projections.
Double-stranded RNAi
RNAi was performed according to the methods of Clemens et al. (2000) using target sequences that exhibited minimal homology with other genes as determined by BLAST comparison. Templates for in vitro transcription were generated by using the primers 5'-GAATTAATACGACTCACTAT-AGGGAGAATGGCTGTAAACGTCTACTCCACAAATGTG-3' and 5'-GAA-TTAATACGACTCACTATAGGGAGATGCCCGTGCTGTTGGCACAGGCG-TTTA-3' to amplify the first 600 bp from the coding sequence of Dm EB1 from Drosophila EST clone LD08743 (Research Genetics), and the primers 5'-TAATACGACTCACTATAGGGAGAGATGTTAATGGGCACAAATTTTCT-3' and 5'-TAATACGACTCACTATAGGGAGATTTGTATAGTTATCCATGCCATG-3' to amplify a 650-bp segment of EGFP from the vector EGFP-C1 (CLONTECH Laboratories, Inc.). PCR products were used as templates for in vitro transcription using the Megascript T7 kit (Ambion) according to the manufacturer's instructions.
Microtubule dynamics in live S2 cells
In the experiments in which microtubule dynamics were observed, cells were treated with 15 µM dsRNA every 3 d for 6 d and transfected with a plasmid encoding GFP-tubulin (gift from Nicole Grieder, University of Basel, Basel, Switzerland) in the pAc5.1/His-V5B vector (Invitrogen) using the Cellfectin transfection reagent (Invitrogen) according to manufacturer's instructions. The cells were cultured for 2 d more in the presence of dsRNA, and then plated onto concanavalin Acoated coverslips 2 h before observation. Coverslips were fastened to microscope slides using warm VALAP (equal parts vaseline, lanolin, and paraffin) and fragments of broken coverslips as spacers. Microtubule dynamics were observed on a Nikon TE300 inverted microscope with a 100X/1.4 N.A. objective lens using an Orca II cooled CCD camera (Hamamatsu). Images were acquired for a period of 5 min at a frame capture rate of every 5 s using Simple PCI software (Compix, Inc.). Image sequences were converted to movies and the ends of microtubules were tracked over time using ImageJ (http://rsb.info.nih.gov/ij/). Microtubule dynamics were calculated as described in Tirnauer et al. (1999).
Embryo microinjection and live cell microscopy
Embryo microinjection was performed essentially as previously described (Sharp et al., 1999). Rabbit antiDm EB1 polyclonal antibodies were affinity purified on the day of injection and concentrated to 25 mg/ml using Ultrafree centrifugal concentrators (Millipore). Control embryos were injected with PBS alone. In some experiments, antibodies were coinjected with rhodamine-labeled histones prepared as previously described (Valdes-Perez and Minden, 1995). Time-lapse fluorescence microscopy was performed using an Ultraview spinning disk confocal microscope (PerkinElmer). Image series were manipulated and quantitated as previously described (Sharp et al., 1999).
Online supplemental material
Supplemental videos 14 are available online at http://www.jcb.org/cgi/content/full/jcb.200202032/DC1. Videos 1 and 2 show examples of microtubule behavior in cells expressing GFPtubulin. Video 1 is a sequence acquired from a control cell, whereas Video 2 was acquired from a cell treated with RNAi to inhibit DmEB1. Videos 3 and 4 show mitotic spindle dynamics in embryos expressing GFPtubulin and injected with rhodaminehistones to visualize chromosomes. Video 3 is a sequence of a control-injected embryo, whereas Video 4 is of an embryo injected with antibodies raised against DmEB1.
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Footnotes |
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* Abbreviations used in this paper: APC, adenomatous polyposis coli; MAP, microtubule-associated protein; RNAi, RNA-mediated inhibition.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (38499) and the Howard Hughes Medical Institute.
Submitted: 7 February 2002
Revised: 19 July 2002
Accepted: 19 July 2002
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References |
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