BIOLOGICAL OVERVIEW

The chromosomal passenger complex (CPC) is a key regulator of mitosis in many organisms, including yeast and mammals. Its components co-localise at the equator of the mitotic spindle and function interdependently to control multiple mitotic events such as assembly and stability of bipolar spindles, and faithful chromosome segregation into daughter cells. This study reports the first detailed characterisation of a CPC mutation in Drosophila, using a loss-of-function allele of borealin (borr). Like its mammalian counterpart, Borr colocalises with the CPC components Aurora B kinase and Incenp in mitotic Drosophila cells, and is required for their localisation to the mitotic spindle. borr mutant cells show multiple mitotic defects that are consistent with loss of CPC function. These include a drastic reduction of histone H3 phosphorylation at serine 10 (a target of Aurora B kinase), and a pronounced attenuation at prometaphase and multipolar spindles. The evidence suggests that borr mutant cells undergo multiple consecutive abnormal mitoses, producing large cells with giant nuclei and high ploidy that eventually apoptose. The delayed apoptosis of borr mutant cells in the developing wing disc appears to cause non-autonomous repair responses in the neighbouring wild-type epithelium. These responses involve Wingless signalling, which ultimately perturbs the tissue architecture of adult flies. Unexpectedly, during late larval development, cells survive loss of borr and develop giant bristles that may reflect their high degree of ploidy (Hanson, 2005).

The chromosomal passenger complex is conserved from yeast to humans, and consists of at least three components that regulate multiple mitotic events. Its name stems from the observation that CPC proteins colocalise on condensing chromosomes during prophase, and are carried along to centromeres and to the equator of the mitotic spindle during metaphase. After metaphase, the CPC components re-localise to the midzone and midbody of the spindle, where they remain until the completion of cytokinesis. The CPC components include Aurora B kinase, inner centromere protein (Incenp) and Deterin/Survivin, an inhibitor of apoptosis-like protein (Bolton, 2002), as well as the recently discovered (Gassmann, 2004; Sampath, 2004) Borealin/Dasra protein (Hanson, 2005).

Borealin/Dasra was identified in human cell lines and in Xenopus extracts, respectively, and found to colocalise with other CPC proteins throughout mitosis (Gassmann, 2004; Sampath, 2004). The correct localisation of human Borealin in mitotic cells depends on the function of the other CPC components; conversely, RNAi-mediated depletion of Borealin in HeLa cells causes mislocalisation of Aurora B, Incenp and Survivin (Gassmann, 2004). Human Borealin binds directly to Incenp and Survivin in vitro, and forms a complex with the other CPC components in vivo. Its loss of function, like that of other CPC components, causes multiple mitotic defects, including failures in chromosome attachment to the spindle, multifocal spindles and uneven chromosome segregation. This typically results in multinucleate cells, aneuploidy and polyploidy, as well as, ultimately, apoptosis (Gassmann, 2004; Sampath, 2004). However, cells that lack CPC function can also occasionally escape apoptosis since they appear to be defective (Lens, 2003; Yang, 2004) for their spindle attachment checkpoint (Hanson, 2005).

Little is known about the role of the CPC during development, except for its function in the early C. elegans embryo (Kaitna, 2000; Kaitna, 2002; Romano, 2003). This study present the first detailed characterisation of a CPC mutation in Drosophila, using a loss-of-function allele of borealin. This gene was identified independently in a recent RNAi screen for cytokinesis defects in cultured Drosophila cells, and was named borr (borealin-related) (Eggert, 2004). Evidence is provided, based on its subcellular localisation and function during the cell cycle, that Borr is the functional counterpart of vertebrate Borealin/Dasra. borr is an essential gene, and loss of borr function causes mitotic defects, including multipolar spindles that result in large polyploid cells and often in delayed apoptosis. The developmental consequences of these defects include striking cell-autonomous and non-autonomous defects in cell-type specification and tissue architecture (Hanson, 2005).

Four independent lines of evidence argue that Borr is the functional ortholog of vertebrate Borealin/Dasra. (1) Based on stringent database searches, it was found that borr is the only gene in the Drosophila genome with significant sequence similarity to Borealin/Dasra, and vice versa. (2) Like vertebrate Borealin/Dasra and other CPC components (Andrews, 2003; Carmena, 2003; Gassmann, 2004; Sampath, 2004), Borr colocalises with endogenous Incenp and Aurora B in transfected mitotic Drosophila DmD8 cells. (3) Like its vertebrate counterpart (Gassmann, 2004), borr is required for the correct subcellular localisation of Incenp and Aurora B in dividing cells. (4) Borr loss causes similar mutant phenotypes in mitotic Kc cells and in developing embryonic and larval cells as does depletion of other Drosophila CPC components in tissue culture, or depletion of Borealin/Dasra and other CPC components in mammalian cell lines. These phenotypes include abnormal spindles and uneven chromosome segregation, leading to giant multi-nucleate and/or polyploid cells and, usually, to apoptosis. A noticeable molecular consequence of Borr loss is also the reduction in the P-H3 levels – given that this phosphorylation event is mediated by Aurora B, this links Borr function specifically to the activity of this CPC component. It is noted that the C. elegans protein CSC-1 appears to be another functional ortholog of Borealin/Dasra (Romano, 2003), despite showing very limited sequence similarity to these proteins, based on its mutant phenotypes in the embryo and on its functional interactions with other CPC components (Hanson, 2005).

One striking mutant phenotype of mitotic borr mutant VNC cells is a significant reduction of their P-H3 levels. Normally, this phosphorylation appears during prophase and spreads throughout the chromosomes, with peak levels during metaphase, followed by dephosphorylation during anaphase and telophase. Although the function of P-H3 is not known, correlations have been noted in many species between the P-H3 levels and the degree of DNA condensation, and a T. thermophila strain with a non-phosphorylatable version of H3 shows perturbed chromosome condensation and abnormal chromosome segregation. This has led to the hypothesis that S10 phosphorylation of H3 may be necessary for chromosome condensation (Hanson, 2005).

However, in borr mutant embryos, the condensation of the chromosomes in mitotic VNC cells is barely affected, yet their P-H3 staining is often strongly reduced. This argues that H3 phosphorylation occurs in parallel or subsequent to chromosome condensation, rather than driving it. Consistent with this, others have also reported a lack of correlation between chromosome condensation and P-H3 levels, including Yu (2004). These reports have observed normal levels of P-H3 on undercondensed chromosomes in greatwall mutants of Drosophila. Indeed, it has been suggested that P-H3 may be a sort of licensing factor, namely a mark placed on mitotic chromosomes to indicate their readiness to undergo separation during the subsequent stages of the cell cycle (Hanson, 2005).

The striking reduction of the P-H3 levels in borr mutant embryonic cells, and in Borr-depleted cultured Drosophila cells (Eggert, 2004), is in contrast to the situation in HeLa cells in which RNAi-mediated depletion of Borealin does not affect their P-H3 levels (Gassmann, 2004). This author suggested that, in these cells, H3 phosphorylation may be mediated by a Borealin-independent subcomplex of Aurora B and Incenp (Gassmann, 2004). More work is required to determine whether this apparent discrepancy between human and Drosophila Borealin function in mediating phosphorylation of H3 is genuine and cell type- or species-specific, or whether it is simply due to methodological differences in the analyses (Hanson, 2005).

borr is an essential gene in Drosophila, and borr loss results in multiple successive defects during mitosis, including a reduction of P-H3, a severe attenuation prior to metaphase, multipolar spindles and uneven chromosome segregation. These defects may all reflect a function of Borr in the attachment of kinetochores to the mitotic spindle, given that this process often fails in Borealin-depleted HeLa cells (Gassmann, 2004). However, it is also possible that they reflect additional underlying activities of the CPC during the progression of mitosis. However, all of these mitotic defects are probably due, ultimately, to the observed failure of other CPC components such as Aurora B to localise correctly to the mitotic spindle (Hanson, 2005).

Multifocal spindles as observed in borr mutant cells are expected to cause aneuploidy, and may trigger checkpoint function. They should thus be cleared from the developing tissue by apoptosis. The observation of apoptotic borr mutant cells in larval imaginal discs provides direct support that cell death is often the ultimate consequence of borr loss at the cellular level. However, borr mutant cells can also clearly evade apoptosis, and can undergo several consecutive abnormal divisions, given that the surviving (and dying) borr mutant cells in imaginal disc epithelia are typically large, with giant nuclei and greatly increased ploidy. Consistent with this, mammalian cells lacking CPC function appear to be defective for their spindle attachment checkpoint and can thus escape apoptosis (Lens, 2003; Yang, 2004). A similar defect in the checkpoint function of borr mutant epithelial cells would explain why these cells can survive multiple abnormal mitoses, instead of entering apoptosis in response to the uneven chromosome segregation of a single abnormal mitosis. However, the survival capacity of the mutant cells is clearly limited, and most of them die ultimately – except in late larval and pupal discs in which they survive, possibly because of the slowing down of mitotic activity and/or growth at these stages, which perhaps provides a more permissive environment for the abnormally dividing borr mutant cells (Hanson, 2005).

borr mutant epithelial cells can cause major non-autonomous disruptions of the patterning of adjacent wild-type cells. This is unusual as imaginal discs can tolerate considerable cell death without compromising the development of normal adult tissues. The reason for this appears to be that apoptotic imaginal disc cells activate transient bursts of extracellular signalling by Wg and Dpp, to induce compensatory cell divisions in their wild-type neighbours. However, if apoptosis is suppressed through inhibition of caspase activity (which creates 'undead' cells), this produces more sustained signalling, which in turn causes gross pattern abnormalities in the resulting adult tissue. It thus appears that interfering with, or suspending, the apoptotic pathway leads to over-compensatory responses (Hanson, 2005).

A similar situation is proposed to arise in the case of borr mutant imaginal disc cells: given that these can survive multiple abnormal divisions, they may be doomed -- i.e., on a suspended apoptosis path -- for an extended period of time and thus mimic some characteristics of 'undead' cells. Like the latter, doomed borr mutant cells may induce a burst of compensatory responses in their neighbours by stimulating the expression of extracellular signals such as Wg. This is suggested by analysis of larval discs bearing early-induced mutant clones that revealed examples of overexpressed Wg in giant borr mutant cells, and also lateral expansion of Wg in twin spot areas whose associated borr mutant cells have died. Doomed borr mutant cells may also affect signalling by other pathways, e.g., the Notch pathway, given some of the borr mutant phenotypes, but this has not been observed directly (Hanson, 2005).

This analysis further suggests that cell rearrangements can take place as a result of dying, or dead, borr mutant cells. These could be a consequence of compensatory signalling, and they may be aimed at repairing the substantial gaps in epithelial integrity expected to arise after the death of a giant borr mutant cell (Hanson, 2005).

borr loss also affects the lineage divisions of the external sensory organs: evidence from late-induced borr mutant clones indicates that surviving giant borr mutant cells develop large bristles without sockets. This phenotype suggests a defect or block in the division of the pIIa precursor cell that normally gives rise to the trichogen and tormogen. It is less likely that the division of pI (the initial sensory organ precursor cell) is blocked by borr loss in these instances, because evidence from the analysis of embryonic sensory organs suggests that blockage of the first lineage division should result in the precursor cell adopting a neural fate (Hanson, 2005).

Why a borr mutant cell should adopt the bristle fate at the expense of the socket fate is not immediately obvious. One possibility is that the determining factor is its increased DNA content and large size. Notably, the trichogen cells that produce the stout bristles of the wing margin undergo at least one round of endoreplication during their differentiation (though in other external sensory organs the tormogen does as well). Thus, borr loss could mimic an aspect of normal trichogen development, and could actively promote the acquisition of the bristle fate. It is thus conceivable that endoreplication is instructive during the process of sensory organ development (Hanson, 2005).

Borealin directs recruitment of the CPC to oocyte chromosomes and movement to the microtubules

The chromosomes in the oocytes of many animals appear to promote bipolar spindle assembly. In Drosophila oocytes, spindle assembly requires the chromosome passenger complex (CPC), which consists of INCENP, Borealin, Survivin, and Aurora B. To determine what recruits the CPC to the chromosomes and its role in spindle assembly, a strategy was developed to manipulate the function and localization of INCENP, which is critical for recruiting the Aurora B kinase. An interaction between Borealin and the chromatin was found to be crucial for the recruitment of the CPC to the chromosomes and is sufficient to build kinetochores and recruit spindle microtubules. HP1 colocalizes with the CPC on the chromosomes and together they move to the spindle microtubules. It is proposed that the Borealin interaction with HP1 promotes the movement of the CPC from the chromosomes to the microtubules. In addition, within the central spindle, rather than at the centromeres, the CPC and HP1 are required for homologous chromosome bi-orientation (Wang, 2021).

GENE STRUCTURE

cDNA clone length - 1309 bp

Bases in 5' UTR - 128

Exons - 3

Bases in 3' UTR - 222

PROTEIN STRUCTURE

Amino Acids - 319

Structural Domains

The CG4454 locus consists of three exons that encode a protein of 319 amino acids, without recognisable domains or known sequence motifs. Stringent Psi-Blast searches revealed a significant similarity between CG4454 and Borealin/Dasra, the only protein with any detectable sequence relationship to CG4454 (see also Gassmann, 2004). This suggests that CG4454 may be the Drosophila ortholog of Borealin/Dasra (Hanson, 2005).

date revised: 12 September 2022

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