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 (). 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.)
Figure 1 Map of the DmINCENP locus and constructs used in this study. DmINCENP (CG12165) is located on P1 clone, AA005425 and contains six exons. A gene encoding a bromodomain protein (CG1845) is transcribed in the opposite direction to the DmINCENP gene; (more ...)
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 coil–forming region is predicted. The major region of homology with vertebrate INCENPs is in the COOH-terminal IN-BOX (Adams et al. 2000
), a 40–50-amino acid domain that defines the INCENP family from yeasts to humans. The NH2
-terminal 540 amino acids are poorly conserved relative to vertebrate INCENPs, however it is this region that contains all the previously known functional domains for INCENP, such as the heterochromatin protein 1 (HP-1) and β-tubulin binding domains, the centromere targeting region and the spindle targeting domain (Mackay et al. 1993
; Ainsztein et al. 1998
; Wheatley et al. 2001
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 (). Both sera recognized a single protein of 110 kD on immunoblots of embryo extract ( 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.
Figure 2 (A) Immunoblot of total fly embryo protein extract probed with antibody R801, raised against GST-INCENP1–348. A single band of 110 kD is visible. (B–Q) Localization of DmINCENP in embryos and cultured cells. All panels are stained for (more ...)
In syncytial embryos, DmINCENP associates with condensing chromatin during prophase (, B–E), before becoming focussed to the centromeric regions of metaphase chromosomes (, F–I, arrows). Upon entry into anaphase, the protein leaves the chromosomes to form a ring of spots between the segregating chromatids (, J–M). Each spot seems to be at the converging focus of bundles of microtubules ( 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 ( N) and Dmel2 cultured cells (, O–Q).
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 (, A–K) (Schumacher et al. 1998
; Terada et al. 1998
). Second, preincubation of the antibody with purified recombinant GST-aurora B1–58
abolished all specific staining in cultured cells ( L). Third, treatment of Dmel2 cells with DmAurora B dsRNA obliterated all staining in many mitotic cells after 24 h, confirming that the epitope visualized in fixed cells is the product of the DmAurora B
gene (see I).
Figure 3 (A–L) Immunolocalization of DmAurora B in embryos and cultured cells. DNA (blue), DmAurora B (red), and tubulin (green) are shown. (A) Syncytial nuclei entering mitosis. Black arrow indicates a nucleus (enlarged in C) just ahead of the mitotic (more ...)
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. 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 (, 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 (, E–H). This distribution was also observed in cellularised embryos ( B) and in cultured cells (, I–K). 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 (Mackay et al. 1998
; Cutts et al. 1999
). To test whether DmINCENP binds microtubules directly, we expressed soluble full-length GST-DmINCENP in bacteria, purified it, and incubated it with taxol-stabilized microtubules prepared from purified tubulin. As a control, GST alone was incubated with polymerized tubulin. The reaction mixture was then sedimented through a sucrose cushion. GST-DmINCENP, but not GST, cosedimented with the microtubules ( A). This suggests that the binding of DmINCENP to microtubules in mitosis is likely to be direct.
Figure 4 (A) DmINCENP binds directly to microtubules. (lane M) Molecular weight markers. (lanes 1–5) Coomassie blue–stained gel of microtubule pellet after sedimentation through a sucrose cushion. 64% of the added INCENP (lane 4) but no GST (lane (more ...)
INCENP is stockpiled in Xenopus
eggs in a complex with aurora B kinase (Adams et al. 2000
contains two recognizable aurora-like protein kinases: the founder member of the family, Aurora (DmAurora A), which is required for centrosome separation, and the recently described DmAurora B/ial (Reich et al. 1999
), whose function and localization are unknown. To determine whether DmAurora B can associate with INCENP, we incubated DmAurora B expressed in bacteria with beads laden with GST-DmINCENP. As a control, the kinase was incubated with beads carrying GST alone. Under these conditions, DmAurora B bound specifically to GST-DmINCENP but not to GST alone ( B).
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 36–48 h after the addition of dsRNA to the culture ( 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 ( A″, and see H). In both cases, the loss of protein was transient, with levels beginning to recover at later times.
Figure 5 Summary of effects of RNAi on cell growth and mitosis. (A′ and A″) Efficacy of RNAi was assessed by immunostaining (INCENP and aurora B RNAi) and by immunoblotting (INCENP RNAi only). Graph shows percentage of cells lacking detectable (more ...)
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 ( B). This was accompanied by an increase in polyploid cells starting at 24 h in the DmAurora B experiment ( 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 (, compare D with E). A similar phenotype was observed in the aurora B RNAi ( 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 (, D–F). 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
early embryos (Adams et al. 2000
; Kaitna et al. 2000
). However, it was not known whether aurora B kinases have a role in INCENP localization.
Elimination of DmINCENP by RNAi completely abolished DmAurora B localization throughout mitosis: the protein was not detected on chromosomes, central spindle microtubules, or midbodies ( and ). In contrast, DmAurora B RNAi did not block DmINCENP association with the chromosome arms during prometaphase but impaired its ability to concentrate at centromeres ( B) and eliminated the transfer to the midbody ( 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.
Figure 6 INCENP and DmAurora B are mutually dependent for their correct localization. (A) Control metaphase showing INCENP (red) in discrete centromeric spots. (B) Rare metaphase from DmAurora B dsRNA-treated cells. INCENP is more diffusely distributed on the (more ...)
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 (, B–D). Inhibition of DmAurora B or DmINCENP function led to both a decrease in the levels of detectable histone H3 phosphorylation ( and ) and an increase in the incidence of malformed chromosomes starting as early as 24 h after exposure to dsRNA ( 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; and ). This result suggests that, as in C
(Hsu et al. 2000
), DmAurora B is at least partially responsible for the histone H3 kinase activity in Drosophila
Importantly, the level of histone H3-serine10 phosphorylation showed only a weak correlation with the overall degree of chromatin condensation. 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 (, compare H with I).
An aberrant dumpy prometaphase chromosome morphology ( F) was seen in 46% of INCENP-negative and 60% of aurora B–negative 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 (, compare J with K). Although they initially appeared less condensed than normal mitotic chromosomes ( O, 3, I and L, and 7, B–D), in fact, their level of condensation is normal ( 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 (Giet and Glover 2001
Dumpy chromosomes have kinetochores, as defined by the presence of double dots of CENP-A/Cid staining. Cid is the Drosophila
CENP-A orthologue (Henikoff et al. 2000
), and provides a marker for the kinetochore inner plate (Warburton et al. 1997
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 ( and ), cells in various states of attempted cytokinesis with large amounts of amorphous lagging chromatin draped out behind the segregating chromatin ( and ), and bizarre cells in which banana-shaped nuclei were surrounded by a mitotic-like bipolar microtubule array (, E–G). 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.
Figure 8 Aberrant centromere disjunction and lagging chromatin in the INCENP and aurora B RNAi. (A and B) Aberrant anaphases with paired CENP-A/Cid spots (red) at poles and lagging chromatin (INCENP RNAi, 36 h). (C and D) Aberrant telophases with Cid spots at (more ...)
In cells with chromosomes distributed along the spindle or with banana-shaped nuclei, centromeres were seen to cluster either near opposite poles (, A–D) or at the opposing pointed ends of the elongate nuclei ( and ). 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 ( and ; 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 ( and ). 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 (″, 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 ( C). The simplest explanation for the origin of the many binucleate cells that we observed ( and ) is that chromosome segregation and nuclear reassembly occurred, but that cytokinesis was then defective. We also observed cells with one giant nucleus ( 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.
Figure 9 Removal of DmINCENP causes cytokinesis defects. (A, D, and D′) Telophase figures from untreated cells. All other panels show cells after INCENP RNAi treatment. (A–C) Merged images with INCENP (red), tubulin (green), and DNA (blue). After (more ...)
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 ( B). These cells showed an accumulation of actin at the cleavage furrow similar to that in untreated cells ( and ), 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 ( F). In contrast, binucleate cells consistently showed an abnormally high density of tubulin between the two nuclei ( 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
, the kinesin-related protein ZEN-4 fails to localize properly, and a spindle midzone fails to form (Severson et al. 2000
). As a result, cytokinesis begins, but the furrow regresses, and binucleate cells are produced. A similar phenotype is seen with the ZEN-4 ts mutant. In Drosophila
, however, the ZEN-4 homologue PAV-KLP appears to act at an earlier stage, as pavarotti
mutants do not form a stable contractile ring and fail to initiate cleavage (Adams et al. 1998
In untreated cells, PAV-KLP was invariably associated with the central spindle throughout cytokinesis ( 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 ( and ). PAV-KLP was occasionally present in binucleate cells, where the cleavage furrow had regressed ( D).
Figure 10 Pavarotti-KLP distribution is normal after DmINCENP or DmAurora B RNAi. (A) Wild-type cell showing PAV-KLP (red) at the midbody, tubulin (green), and DNA (blue). (B) PAV-KLP (arrow) is present at the midbody after INCENP RNAi. (C and D) Aberrant telophases (more ...)
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.