Correspondence to: William C. Earnshaw, Center for Cell Biology, Institute for Cell and Molecular Biology, King's Building, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, Scotland, UK. Tel:44-0-131-650-7101 Fax:44-0-131-650-7100 E-mail:bill.earnshaw{at}ed.ac.uk.
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Abstract |
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We have performed a biochemical and double-stranded RNA-mediated interference (RNAi) analysis of the role of two chromosomal passenger proteins, inner centromere protein (INCENP) and aurora B kinase, in cultured cells of Drosophila melanogaster. INCENP and aurora B function is tightly interlinked. The two proteins bind to each other in vitro, and DmINCENP is required for DmAurora B to localize properly in mitosis and function as a histone H3 kinase. DmAurora B is required for DmINCENP accumulation at centromeres and transfer to the spindle at anaphase. RNAi for either protein dramatically inhibited the ability of cells to achieve a normal metaphase chromosome alignment. Cells were not blocked in mitosis, however, and entered an aberrant anaphase characterized by defects in sister kinetochore disjunction and the presence of large amounts of amorphous lagging chromatin. Anaphase A chromosome movement appeared to be normal, however cytokinesis often failed. DmINCENP and DmAurora B are not required for the correct localization of the kinesin-like protein Pavarotti (ZEN-4/CHO1/MKLP1) to the midbody at telophase. These experiments reveal that INCENP is required for aurora B kinase function and confirm that the chromosomal passengers have essential roles in mitosis.
Key Words: mitosis, cytokinesis, chromosomal passengers, chromosomes, RNAi
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Introduction |
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Successful mitosis depends on the coordination of chromosomal and cytoskeletal behavior. A group of proteins termed chromosomal passengers are prime candidates for a role in this coordination. Chromosomal passengers were originally defined by their dynamic distribution in mitosis (
To date, four chromosomal passenger proteins have been described in detail: inner centromere protein (INCENP)1 (
Two dominant-negative forms of vertebrate INCENP have been described. One of these, INCENP1405, interferes with prometaphase chromosome congression, sister chromatid disjunction at anaphase, and the completion of cytokinesis (
Aurora kinases were discovered in a screen of mitotic mutants in Drosophila (
Metazoans, including Drosophila, have two (three in vertebrates) aurora-related kinases (
Here, we report studies of chromosomal passenger function in Drosophila embryos and cultured cells. Drosophila INCENP and aurora B both behave as classical chromosomal passenger proteins, however they exhibit subtle differences in their targeting to chromosomes. Inactivation of INCENP and aurora B by RNAi in Drosophila cultured cells dramatically disrupted mitotic events with several significant differences from the results of recent studies in C. elegans (
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Materials and Methods |
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Molecular Biology Methods and DNA Constructs
Standard molecular biology methods were followed throughout this study. INCENP and aurora B/ial Drosophila cDNAs were purchased from Research Genetics. INCENP1755, INCENP1348, and INCENP654755 were amplified by PCR and cloned into pGEX 4T3 (Amersham Pharmacia Biotech). To produce pGEX-INCENP1755His6, an oligonucleotide encoding an His6 tag flanked by NotI adapters was inserted into the NotI site of pGEX-INCENP. DmAurora B was subcloned into pET 22b (Novagen) into the NdeI site at the 5' end and the XhoI site at the 3' end. All constructs were fully sequenced. After expression in E. coli BL21 (DE3) pLysS, DmAurora B was purified by Ni 2+-agarose chromatography.
Antibodies
Polyclonal anti-DmINCENP and antiDmAurora B antibodies were produced in rabbits. Rabbits were immunized with the following gel-purified proteins: glutathione S-transferase (GST)-INCENP 1348 (R801), GST-INCENP654755 (R803), or GSTDmAurora B158 (R963). Anti-INCENP sera were diluted 1:500 for use in immunoblotting and immunofluorescence. Antiaurora B antibodies were affinity purified by incubating serum with Affigel 10 beads (Bio-Rad Laboratories) to which aurora B158 had been bound. After washes in 0.5 M NaCl, 10 mM phosphate (pH 7.4) antibodies were eluted in 0.2 M glycine, pH 2.0, and the eluate was neutralized with one-tenth volume 1 M Tris, pH 8.0, and dialyzed overnight into PBS. Antibodies were concentrated by ultrafiltration and stored in 50% glycerol at 20°C. For immunofluorescence, antibodies were used at 2 µg/ml.
Other antibodies used were anti-tubulin (YL1/2 rat monoclonal, used at 1:50; Harlan Sera Labs; or mouse mAb B512, used at 1:2,000; Sigma-Aldrich), antiPAV-KLP (R3301, used at 1:500;
For the blocking experiment, the affinity-purified antiaurora B antibody was diluted 1:50 in PBS + 0.1% Triton X-100 (Bio-Rad Laboratories) with 10% FBS in the presence of GSTDmAurora B158 (0.3 mg/ml) and then incubated for 1 h at room temperature before being added directly to the cells and processed as usual.
Drosophila Embryos and Cells
Drosophila embryos derived from w1118 adults were fixed and processed for immunostaining exactly as described previously (30 min at room temperature in PBS + 10% FBS. Antibody incubation was done in PBS + 0.1% Triton X-100 for 1 h at 37°C, followed by 46-min washes in PBS five times. DNA was counterstained with 0.1 µg/ml DAPI for 5 min at room temperature and rinsed in PBS. Slides were mounted in Vectashield and sealed using nail varnish. Three-dimensional data sets of selected cells were collected using a DeltaVision microscope (Applied Precision), based on an Olympus IX-70 inverted microscope with a cooled couple-charged device camera (CH350L, Photometrics). Data sets were deconvolved if required, projected onto a single plane and, exported as TIF files to be processed using Adobe® PhotoshopTM.
Microtubule Binding Assay
30 µg pure bovine tubulin (Cytoskeleton) was polymerized at 37°C in 20 µl BRB80 (80 mM Pipes, pH 6.8, 1 mM MgCl2, 1 mM EGTA, 1 mM GTP) by stepwise addition of taxol to a final concentration of 20 µM. Either BRB80, 5 µg GST-DmINCENP, or 5 µg GST, was added to preformed microtubules to a final volume of 30 µl. Reactions were incubated at 20°C for 30 min and then centrifuged at 50,000 g for 20 min through a 30% (wt/wt) sucrose cushion containing 1 mM GTP and 5 µM taxol. The supernatant was aspirated and retained, and the cushion washed three times with BRB80. The pellets were resuspended in protein sample buffer before analysis on 10% SDSpolyacrylamide gels.
In Vitro Assay for DmINCENP/DmAurora B Binding
Full-length GST-DmINCENPHis6 was expressed in E. coli and purified by standard methods onto Ni2+-agarose beads (QIAGEN). After elution in 0.25 M imidazole, the protein was dialyzed into TEN buffer (10 mM Tris:HCl, pH 8.0, 100 mM NaCl, 2 mM EGTA) and bound to glutathionesepharose beads (Amersham Pharmacia Biotech). This procedure increased the yield of full-length INCENP over truncated INCENP. Beads were washed and incubated with TEN alone or 5 µg DmAurora B for 30 min at 4°C. To remove nonspecifically bound protein, beads were washed five times in 20 volumes of TEN200 (TEN + 100 mM NaCl) containing 0.05% Triton X-100. As a control, DmAurora B was incubated with GST-coated sepharose beads. After washing, proteins were eluted by boiling in protein sample buffer and electrophoresed in 10% SDS polyacrylamide gels.
Measurement of Relative Levels of Phospho-H3 and Mitotic Chromosome Condensation
Three-dimensional data sets were collected with the DeltaVision microscope and were in the linear range of the CH350L camera, deconvolved, and the image intensities of four image planes bisecting the midplane of the mitotic chromosomes were averaged using the Quick Projection algorithm. We then measured the integrated intensity of a box, 5 pixels on a side, at the appropriate wavelengths for detection of phospho-H3 and DAPI, respectively, using the Data Inspector tool. This assumes that the amount of DNA within the 25-pixel box is proportional to the overall level of condensation of the chromatin within that box. Three measurements were taken per metaphase, as were three measurements of the local background. These values were entered in a Microsoft® ExcelTM spreadsheet, and the intensities at 617 and 457 nm were corrected by subtracting the local background. For control cells at prometaphase, metaphase, anaphase, telophase, and interphase, this method yields the following relative degrees of condensation: 2.4 ± 0.55, 2.8 ± 1.26, 2.7 ± 0.49, 1.7 ± 0.49, and 1.0 ± 0.27, respectively. To produce the plot shown in Fig 7 G, we averaged the three corrected values for each cell, sorted the DAPI measurements based on increasing DAPI fluorescence, and performed a linear regression on each data set. For this experiment, we obtained deconvolved three-dimensional data sets from 44 prometaphase cells, of which 34 were normal and 10 were "dumpy." Average corrected phospho-H3 staining intensities were 2194 ± 1779 and 74 ± 123 for normal and dumpy chromosomes, respectively. These corresponded to DNA condensation values of 317 ± 109 and 474 ± 249, indicating that dumpy chromosomes were not less condensed than normal-looking chromosomes.
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RNA Interference
RNAi in Drosophila S2 and Dmel2 tissue culture cells was performed according to the published protocol (
Scoring of INCENP/Aurora-B Phenotype
Trypan blue (Sigma-Aldrich) was used to score nonviable cells. Growth curves were plotted considering only viable cells (levels of cell death were equivalent in all cultures). Microsoft® ExcelTM was used to plot all quantifications. Error bars represent the standard deviation, and all the values were plotted as numerical average. To determine the doubling time of the cells in the four experiments, we calculated the best fit for the growth curves (semilog scale), and the doubling time was calculated from the corresponding equations. Mitotic Index was determined as a percentage of mitotic cells in the total population. Mitotic stages were quantified in terms of percentage of mitotic cells. To determine the mitotic index in the presence of microtubule poisons, Dmel2 cells were incubated for 12 h in the presence of 1 µg/ml of colcemid.
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Results |
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Identification of DmINCENP
We identified a candidate DmINCENP cDNA (LD24414) by querying the Berkeley Drosophila Genome Project database with the conserved COOH-terminal 90 amino acids of vertebrate INCENP. LD24414 was completely sequenced, and the cDNA sequence was mapped onto genomic P1 clone
AC005425. The candidate DmINCENP gene maps to cytological region 43B1, contains six exons, and encodes a 2,441-bp cDNA with a continuous ORF of 2,265 bp (Fig 1). Our sequence and exon/intron analysis agrees exactly with that described by Celera Genomics for the hypothetical gene CG12165. (Sequence data are available from GenBank/EMBL/DDBJ under indicated accession nos.)
DmINCENP has a predicted molecular mass of 83.5 kD and a calculated isoelectric point of 9.63. Between residues 540 and 660, a coiled coilforming region is predicted. The major region of homology with vertebrate INCENPs is in the COOH-terminal IN-BOX (
DmINCENP and DmAurora B Kinase Are Chromosomal Passenger Proteins
To demonstrate whether the characteristic INCENP behavior was conserved in Drosophila mitosis, we raised antibodies to two nonoverlapping regions of the protein (Fig 1). Both sera recognized a single protein of 110 kD on immunoblots of embryo extract (Fig 2 A). This is larger than the predicted molecular mass of 83.5 kD but consistent with the behavior of INCENPs from other species, which also migrate anomalously on protein gels.
In syncytial embryos, DmINCENP associates with condensing chromatin during prophase (Fig 2, BE), before becoming focussed to the centromeric regions of metaphase chromosomes (Fig 2, FI, arrows). Upon entry into anaphase, the protein leaves the chromosomes to form a ring of spots between the segregating chromatids (Fig 2, JM). Each spot seems to be at the converging focus of bundles of microtubules (Fig 2 K, arrow). As telophase progresses, the DmINCENP ring decreases in diameter until it becomes a single midbody-like structure between the central spindle microtubule bundles. A similar distribution of INCENP was also observed in cellularized embryos (Fig 2 N) and Dmel2 cultured cells (Fig 2, OQ).
To localize DmAurora B in embryos and tissue culture cells, we raised antibodies against the NH2-terminal 58 amino acids of the protein fused to GST. Although neither of the two sera we raised detected a protein on immunoblots of embryo extract, both recognized the recombinant protein expressed in bacteria (data not shown). The affinity-purified antibodies worked well for indirect immunofluorescence in both embryos and cells. This immunostaining is likely to be specific for DmAurora B for three reasons. First, affinity-purified antibodies gave a staining pattern essentially identical to the highly characteristic pattern seen with antibodies to aurora B kinases in higher eukaryotes (Fig 3, AK) (
The distribution of DmAurora B resembled that of DmINCENP throughout mitosis. However, interphase nuclei showed no detectable staining for DmAurora B (they did for INCENP), and as nuclei entered prophase, DmAurora B appeared first at the centromere: no staining was observed along the chromosome arms. Fig 3 A shows a region of the embryo being traversed by a wave of mitosis. Nuclei just entering mitosis (i.e., adjacent to interphase nuclei) accumulated DmAurora B only at the centromere (Fig 3, compare enlarged nuclei in C and D). DmAurora B remained at the centromere until the metaphase to anaphase transition, when it transferred to the central spindle and subsequently to the midbody (Fig 3, EH). This distribution was also observed in cellularised embryos (Fig 3 B) and in cultured cells (Fig 3, IK). We conclude that DmINCENP and DmAurora B are chromosomal passenger proteins whose distribution in mitosis resembles their vertebrate counterparts.
DmINCENP Binds Directly to Microtubules and to DmAurora B
During anaphase, INCENP colocalizes with microtubules of the central spindle. Furthermore, INCENP overexpression in cultured vertebrate cells or disruption of murine INCENP leads to a dramatic remodeling of the microtubule network (
INCENP is stockpiled in Xenopus eggs in a complex with aurora B kinase (
The RNAi Method
We have used RNAi to eliminate DmINCENP and DmAurora B/ial from cultured cells. dsRNA was added to exponentially growing cultures of Dmel-2 tissue culture cells (see Materials and Methods), and at different time points samples were taken for analysis by immunoblotting and indirect immunofluorescence. Two negative controls were analyzed in parallel: cells to which no dsRNA had been added and cells to which we added dsRNA synthesized from a human intronic sequence, chosen at random to rule out unspecific effects of dsRNA on the cell cycle. The latter caused no defects and was indistinguishable from the untreated control.
Analysis of RNAi experiments is complicated by the fact that this technique causes a gradual depletion of the proteins under study, and that proteins are not necessarily lost from all cells in the population at the same rate. Furthermore, inhibition of INCENP or aurora B function causes cultures to become polyploid, and it is difficult to exclude that some of the aspects of aberrant mitosis seen in these experiments are caused by complications arising during polyploid mitosis. To minimize these complications, we examined the phenotypes described here at various times after the onset of RNAi treatment and could show that certain phenotypic aspects, such as defects in histone H3 phosphorylation and mitotic chromosome assembly, are observed in cells in some cases within the first cell cycle, before cultures become highly polyploid. In addition, where possible, we limited our phenotypic conclusions to cells that were demonstrably lacking INCENP or aurora B, detectable by indirect immunofluorescence.
Elimination of DmINCENP and DmAurora B/ial Function by RNAi
Immunoblotting analysis of cells treated with DmINCENP dsRNA showed that the levels of DmINCENP in the culture became greatly decreased 3648 h after the addition of dsRNA to the culture (Fig 5 A', top). In the best experiments, 95% of the protein was lost. The RNAi treatment for aurora B took effect more rapidly, the protein becoming undetectable by indirect immunofluorescence in most mitotic cells by 24 h (Fig 5 A'', and see Fig 7 H). In both cases, the loss of protein was transient, with levels beginning to recover at later times.
The phenotypes observed after INCENP and aurora B RNAi were complex, revealing defects at multiple stages of the mitotic cycle. To follow the appearance of the various phenotypes after the onset of RNAi, cultures were harvested at 24, 36, 48, and 72 h after exposure to dsRNA and assessed for the following parameters: cell number, frequency of dead (Trypan blue-positive) cells, frequency of overtly polyploid cells, mitotic index, percentage of mitotic cells negative for INCENP or DmAurora B by indirect immunofluorescence, and for the cells in mitosis, the distribution of the various mitotic phases.
RNAi treatment caused an increase in the cell doubling time from 21 h in Dmel2 cells (21.6 h in cells after exposure to control dsRNA) to 36.1 and 27.5 h in cultures after exposure to dsRNA to DmAurora B and DmINCENP, respectively (Fig 5 B). This was accompanied by an increase in polyploid cells starting at 24 h in the DmAurora B experiment (Fig 5 C, 36 h for DmINCENP). These correspond to the first times when significant numbers of mitotic cells were observed to be lacking DmAurora B and DmINCENP, respectively.
Strikingly, RNAi of INCENP abolished the ability of cells to achieve a metaphase chromosome alignment (Fig 5, compare D with E). A similar phenotype was observed in the aurora B RNAi (Fig 5 F). Instead, the population of mitotic cells came to be dominated by cells with a prometaphase-like chromosome arrangement. Importantly, this increase in the percentage of prometaphase cells did not reflect an arrest in mitosis, as the mitotic index of the culture remained constant at the control level of 5% throughout the entire experiment (Fig 5, DF). As discussed below, we believe that many cells in these cultures must exit mitosis directly from prometaphase without achieving a metaphase chromosome alignment.
DmINCENP and DmAurora B Are Mutually Dependent for Their Correct Localization in the Cell Cycle
INCENP is required for the correct localization of aurora B kinases in human cells and C. elegans early embryos (
Elimination of DmINCENP by RNAi completely abolished DmAurora B localization throughout mitosis: the protein was not detected on chromosomes, central spindle microtubules, or midbodies (Fig 6E and Fig F). In contrast, DmAurora B RNAi did not block DmINCENP association with the chromosome arms during prometaphase but impaired its ability to concentrate at centromeres (Fig 6 B) and eliminated the transfer to the midbody (Fig 6 C). Thus, aurora B function is not required for the initial stages of INCENP targeting to chromosomes during prophase, but it is necessary for INCENP behavior later in mitosis.
DmINCENP and DmAurora B Function Is Required for Histone H3 Phosphorylation, Mitotic Chromosome Structure, and Metaphase Chromosome Alignment
In untreated and control cultures, mitotic chromosomes invariably showed high levels of histone H3 phosphorylation on serine10, detected with a specific antibody (Fig 7, BD). Inhibition of DmAurora B or DmINCENP function led to both a decrease in the levels of detectable histone H3 phosphorylation (Fig 7A and Fig E) and an increase in the incidence of malformed chromosomes starting as early as 24 h after exposure to dsRNA (Fig 7 F). The phospho-H3 staining varied from cell to cell, but by 24 h after DmAurora B RNAi, the level of H3 phosphorylation was significantly reduced in 79% of the aurora-null prometaphase cells (74% of the INCENP-null cells at 36 h after DmINCENP RNAi; Fig 7A and Fig E). This result suggests that, as in C. elegans (
Importantly, the level of histone H3-serine10 phosphorylation showed only a weak correlation with the overall degree of chromatin condensation. Fig 7 G shows a plot in which quantitative measurements of phospho-H3 labeling and DAPI staining were compared for a series of prometaphase cells, showing a wide range of phospho-H3 labeling (see Materials and Methods). Although levels of histone H3 phosphorylation on serine10 do tend to increase with increasing chromatin condensation, it is evident that there is a huge scatter in the data from cell to cell. In other studies, we observed chromosomes completely lacking detectable aurora B kinase, which showed an apparently normal level of condensation (Fig 7, compare H with I).
An aberrant dumpy prometaphase chromosome morphology (Fig 7 F) was seen in 46% of INCENP-negative and 60% of aurora Bnegative cells after RNAi. These dumpy chromosomes had a 28-fold lower level of phospho-H3 staining, as detected with specific antibody, than did the chromosomes with a normal morphology (see Materials and Methods). Dumpy chromosomes had an amorphous shape, and defined sister chromatids were not seen. In many cases, the dumpy chromosomes appeared to correspond to an abnormal prometaphase arrangement, characterized by a disassembled nuclear lamina (data not shown) and persistent high levels of cyclin B protein (Fig 7, compare J with K). Although they initially appeared less condensed than normal mitotic chromosomes (Fig 2 O, 3, I and L, and 7, BD), in fact, their level of condensation is normal (Fig 7 G, includes measurements from both normal and dumpy chromosomes; see also Materials and Methods). Instead, it appears that other aspects of chromosome higher order structure and behavior are aberrant. This may be due to defects in condensin binding (
Dumpy chromosomes have kinetochores, as defined by the presence of double dots of CENP-A/Cid staining. Cid is the Drosophila CENP-A orthologue (
Role of DmINCENP and DmAurora B in Anaphase and Telophase
As expected, given the lack of normal metaphase cells, we saw few if any normal anaphase cells that were negative for INCENP or aurora B. Instead, the anaphase/telophase cells had a range of abnormalities, including anaphase-like spindles with chromosomes distributed along their length (Fig 8A and Fig B), cells in various states of attempted cytokinesis with large amounts of amorphous lagging chromatin draped out behind the segregating chromatin (Fig 8C and Fig D), and bizarre cells in which banana-shaped nuclei were surrounded by a mitotic-like bipolar microtubule array (Fig 8, EG). Fig 8 E shows two adjacent mitotic cells in the INCENP RNAi, one of which is still expressing INCENP and is at normal metaphase, and the other of which is INCENP negative and is forming an elongate banana-shaped nucleus.
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In cells with chromosomes distributed along the spindle or with banana-shaped nuclei, centromeres were seen to cluster either near opposite poles (Fig 8, AD) or at the opposing pointed ends of the elongate nuclei (Fig 8F and Fig G). This strongly suggests that kinetochores had attached to microtubules and that anaphase A movement of chromosomes had occurred.
In cells that appeared to be in telophase, we often noticed one or more pairs of centromeres that appeared to be stalled midway between the spindle poles (Fig 8C and Fig D; and data not shown). This organization is what would be predicted if these centromeres had successfully become bioriented but were then unable to disjoin at the onset of anaphase chromosome movement. This was never seen in normal anaphases where the centromeres were typically grouped in a tight cluster at the leading edge of the segregating chromatids (Fig 7C and Fig D). Consistent with difficulties in disjunction of sister kinetochores, we also saw numerous paired kinetochore spots near the spindle poles, as though nondisjoined chromatid pairs had moved together to a single pole (Fig 8A'', B'', and F'', double arrows).
With increasing time after RNAi treatment, we observed a dramatic increase in the number of polyploid cells in both the INCENP and aurora B RNAi so that by 72 h most of the cell population had become highly polyploid (Fig 5 C). The simplest explanation for the origin of the many binucleate cells that we observed (Fig 9C and Fig F) is that chromosome segregation and nuclear reassembly occurred, but that cytokinesis was then defective. We also observed cells with one giant nucleus (Fig 9 G). These are likely to have arisen as a consequence of repeated failures in chromatid segregation. In addition to the chromosomal defects, we also observed spindle abnormalities in INCENP and aurora B RNAi.
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Together, these observations suggest that DmINCENP and DmAurora B might be essential for a variety of anaphase/telophase events, including sister chromatid and kinetochore disjunction, chromosome structure during anaphase, and mitotic spindle architecture.
DmINCENP Is Not Essential for the Initiation of Cytokinesis
It was possible to observe cells lacking detectable DmINCENP in which constriction of the cleavage furrow had advanced considerably and a midbody had formed (Fig 9 B). These cells showed an accumulation of actin at the cleavage furrow similar to that in untreated cells (Fig 9D and Fig E), although more actin was dispersed throughout the remainder of the cell than normal. In binucleate cells, there was no longer a focus of actin staining between the nuclei, indicating that the contractile ring had disassembled (Fig 9 F). In contrast, binucleate cells consistently showed an abnormally high density of tubulin between the two nuclei (Fig 9 C). This is likely to be a remnant of the central spindle.
Pavarotti, an Essential Spindle Midzone KLP Localizes Normally to the Midbody Despite Disruption of the Chromosomal Passenger Complex
In aurora B/AIR-2 ts mutants of C. elegans, the kinesin-related protein ZEN-4 fails to localize properly, and a spindle midzone fails to form (
In untreated cells, PAV-KLP was invariably associated with the central spindle throughout cytokinesis (Fig 10 A). In DmINCENP depleted cells, PAV-KLP staining was present at the midbody of 94% of cells undergoing cytokinesis (n = 95), however the staining was occasionally weaker than in untreated cells. To monitor the effect of the DmAurora B RNAi on PAV-KLP localization, dsRNA-treated cells from the same well were split and stained for DmAurora B and PAV-KLP on the same slide. In 90% of telophases, PAV-KLP was detected at the midbody, whereas DmAurora B staining was absent from 80% of telophases (Fig 10C and Fig E). PAV-KLP was occasionally present in binucleate cells, where the cleavage furrow had regressed (Fig 10 D).
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We conclude that PAV-KLP localization is relatively unchanged after the loss of DmINCENP or DmAurora B, at least in cells that form recognizable midbody structures.
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Discussion |
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We have characterized the mitotic distribution and function of Drosophila homologues of the chromosomal passengers INCENP and aurora B in early embryos and cultured cells. These studies reveal that INCENP and aurora B have essential roles in mitotic chromosome assembly, chromosome alignment at the metaphase plate, chromatid segregation at anaphase, and the completion of cytokinesis.
DmINCENP and DmAurora B Are Chromosomal Passenger Proteins That Are Interdependent for Their Targeting during Mitosis
INCENP targeting in mitosis depends on motifs at the NH2 terminus of the protein (
Here, we have shown for the first time that DmAurora B is required for some, but not all, aspects of INCENP localization in mitosis. In the absence of DmAurora B, INCENP localizes normally to chromosomes during pro-phase; however, it is subsequently unable to concentrate at centromeres and transfer to the central spindle or midbody. As predicted from previous studies, INCENP is essential for aurora B targeting: after INCENP RNAi, DmAurora B does not localize to chromosomes, midzone microtubules, or midbodies. Thus, the chromosomal passenger proteins are interdependent on one another for proper targeting during mitosis.
This interdependence, plus the fact that the two proteins are stockpiled in an 11S complex in Xenopus eggs, suggests that they could function in vivo in a protein complex. DmINCENP binds microtubules in vitro and may be responsible for targeting aurora B to the central spindle, as the kinase appears to lack microtubule binding activity of its own (data not shown). However, the differences in centromere targeting in Drosophila early embryos suggest that the two proteins may not function in an obligate complex, at least during prophase.
DmINCENP Is Required for DmAurora B to Function Efficiently as a Histone H3 Kinase and for Mitotic Chromosome Assembly
S. cerevisiae aurora/Ipl1p and C. elegans aurora B/AIR-2 are required for H3-serine10 phosphorylation in mitosis (
The availability of mitotic cells containing chromosomes with a range of levels of H3 phosphorylated on serine10 enabled us to assess the widely held hypothesis that H3 phosphorylation is correlated with the degree of chromatin condensation. When phospho-H3 levels and the degree of chromatin compaction were compared by quantitative fluorescence microscopy, only a weak correlation between the two values was observed. Instead, interference with INCENP and aurora B function appears to correlate much more strongly with difficulties in assembling mitotic chromosomes of normal morphology. Mitotic chromosomes deficient in phospho-H3 had a characteristic dumpy morphology, with no evidence of resolved sister chromatids. This resembles the defects seen in Drosophila mutants in the SMC4 subunit of condensin (
INCENP and Aurora B Are Required for Mitotic Chromosomes to Achieve a Stable Metaphase Alignment
At later times, after addition of dsRNA, we observed a dramatic increase in the percentage of mitotic cells in prometaphase coupled with a corresponding decrease in the number of metaphase cells. This was particularly dramatic in the INCENP RNAi, where we failed to observe any INCENP-negative cells in metaphase. Surprisingly, this did not lead to an increase in the mitotic index of the cultures. Therefore, in the absence of INCENP and/or aurora B function, Drosophila Dmel2 cells must exit mitosis from prometaphase. Elimination of INCENP and aurora B function does not trigger a mitotic checkpoint in Dmel2 cells. However, as these cells do not arrest in mitosis in colcemid (Fig 5 D), they appear to lack a robust metaphase checkpoint anyway.
What is the ultimate fate of these prometaphase cells? We have excluded the possibility that they are removed by cell death. Although the number of dying cells in the culture showed a transient increase at 1224 h in all cultures, including controls (presumably due to some aspect of the serum-shock protocol used to induce dsRNA uptake), it then declined and remained constant in all cultures at 5% throughout the remainder of the experiment (data not shown).
An alternative explanation for the lack of an increase in mitotic index would be if the cells transit directly from prometaphase into anaphase or telophase, as is the case for budding yeast cells mutant in the essential kinetochore protein Ndc10p (
Why does abrogation of INCENP and/or aurora B function prevent cells from attaining a stable metaphase chromosome alignment? One obvious possibility is that kinetochore function is impaired, much as when anticentromere antibodies from patients with scleroderma spectrum disease are microinjected into human cultured cells (
Arguing against this model is our observation that kinetochores assemble correctly, at least as far as CENP-A/Cid binding is concerned, and move to the spindle poles at anaphase/telophase. This implies that the ability of kinetochores to bind microtubules and to undergo anaphase A movement are preserved after abrogation of INCENP and aurora B function. However, other aspects of kinetochore function, namely the ability to form bipolar spindle attachments and disjoin at anaphase (see below), appear to be defective. How RNAi of INCENP or aurora B leads to defects in chromosome biorientation is unknown, but this is unlikely to be a result of interference with binding of the condensin subunit barren, as barren mutants successfully complete metaphase chromosome alignment (
Normal INCENP and/or Aurora B Function May Be Required for Sister Kinetochore Disjunction and Chromosome Stability in Anaphase
Anaphase/telophase cells after RNAi for INCENP or aurora B exhibited three highly unusual chromosomal phenotypes. First, they often had one or more pairs of sister kinetochores located in the central spindle or flanking the midbody. Second, the foci of CENP-A/Cid staining at or near the spindle poles were often present as pairs, suggesting that sister kinetochores remained paired despite having undergone anaphase Alike poleward movement. Third, separated masses of chromatin were often connected by a mass of lagging chromatin. We refer to this as chromatin and not chromosomes because the material was amorphous, and little or no evidence of a condensed mitotic chromosome morphology could be observed.
The first two phenotypes can be explained if centromeres fail to disjoin at anaphase onset. Under these circumstances, centromeres of bioriented chromosomes would tend to accumulate near the spindle equatorlater, near the midbodyand be stretched apart by the spindle forces. Monooriented kinetochores would move as pairs to one or the other spindle pole. If this occurred in cells that had attained metaphase, then the bulk of kinetochores would remain as pairs in the spindle midzone. However, as described above, abrogation of INCENP and/or aurora B function prevents cells from reaching metaphase and would therefore be expected to lead to the observed phenotype, with most centromeres at poles and only a few remaining in the midzone. Defects in sister kinetochore disjunction could arise if INCENP and/or aurora B is involved in regulation of the cohesin complex at centromeres; experiments are under way to determine whether cohesin components are substrates for aurora B.
The presence of massive amounts of lagging chromatin is highly characteristic of anaphase/telophase after loss of INCENP and/or aurora B function. This lagging chromatin is distinct from that seen when massive problems with sister chromatid disjunction are caused by loss of topoisomerase II function. Loss of topo II function in S. pombe resulted in anaphase cells with a small subset of the chromatin, including centromeres at the poles and the bulk of the tangled chromatin trapped at the spindle midzone (
This lagging chromatin might arise from difficulties in sister chromatid disjunction, but we believe it more likely that it represents a failure of the chromosomes to move as integral units under the physical stress of anaphase movement. If the dumpy chromosomes observed in prometaphase cells lack an organized infrastructure then when centromeres begin to move polewards, the chromatin of the arms might simply unravel and be left behind as a smear of amorphous chromatin. This would be consistent with the observation that interference with aurora B function in Drosophila cells interferes with the binding of the condensin subunit barren to mitotic chromosomes (
Cells with Reduced Levels of DmINCENP Can Assemble a Contractile Ring with Targeted pavarotti KLP but Are Defective in the Completion of Cytokinesis
After RNAi for DmINCENP or DmAurora B, the dramatic increase of binucleate and polyploid cells in the cultures suggests strongly that a large percentage of attempts at cytokinesis ultimately fail. However, we did observe cells lacking detectable INCENP and aurora B in the final stages of cytokinesis with a contractile ring and well-developed midbody structure (Fig 9B and Fig E). This suggests that normal levels of INCENP cannot be essential for formation of a central spindle and contractile ring. Because of the nature of the RNAi method, however, we cannot exclude that at least a portion of these cells initiated mitosis with low levels of INCENP, and that this enabled them to assemble the central spindle and contractile ring.
These observations appear to exclude an essential role for the chromosomal passenger proteins in the earliest stages of cytokinesis. This is consistent with the phenotypes induced by two dominant-negative INCENP mutants in cultured cells, both of which caused a failure in the completion of cytokinesis (
The situation in Drosophila cells is apparently different. The Drosophila ZEN-4 orthologue, Pavarotti KLP (PAV-KLP) is essential for cytokinesis but functions earlier in the process. PAV-KLP mutants do not form central spindles, do not assemble a stable contractile ring, and do not initiate furrowing (
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1 Abbreviations used in this paper: CB, cytoskeleton buffer; dsRNA, double-stranded RNA; GST, glutathione S-trasferase; INCENP, inner centromere protein; KLP, kinesin-like protein; RNAi, dsRNA-mediated interference.
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Acknowledgements |
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We thank P. Vagnarelli for communicating her results before publication; W. Whitfield for cyclin A and B antibodies; S. Henikoff for anti-Cid antibodies; P. Fisher for antilamin antibodies; and M. Heck, A. Merdes, C. Morrison, H. Ohkura, K. Sawin, and S. Vass for scientific discussions and for criticism of the manuscript.
R.R. Adams is a postdoctoral fellow of the Medical Research Council. H. Maiato was supported by a studentship from the Programa Gulbenian de Doutoramento em Biologia e Medicina and the Portuguese government. Work in the W.C. Earnshaw laboratory was supported by the Wellcome Trust, of which W.C. Earnshaw is a Principal Research Fellow.
Submitted: 6 December 2000
Revised: 29 March 2001
Accepted: 29 March 2001
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