BIOLOGICAL OVERVIEW

The chromosomal passenger protein complex has emerged as a key player in mitosis, with important roles in chromatin modifications, kinetochore-microtubule interactions, chromosome bi-orientation and stability of the bipolar spindle, mitotic checkpoint function, assembly of the central spindle and cytokinesis (see Organization of the animal kinetochore). The inner centromere protein (Incenp; a subunit of this complex) is thought to regulate the Aurora B kinase and target it to its substrates. To explore the roles of the passenger complex in a developing multicellular organism, a genetic screen was performed looking for new alleles and interactors of Drosophila Incenp. A new null allele of Incenp has been isolated that has allowed a study of the functions of the chromosomal passengers during development. Homozygous incenp^EC3747 embryos show absence of phosphorylation of histone H3 in mitosis, failure of cytokinesis and polyploidy, and defects in peripheral nervous system development. These defects are consistent with depletion of Aurora B kinase activity. In addition, the segregation of the cell-fate determinant Prospero in asymmetric neuroblast division is abnormal, suggesting a role for the chromosomal passenger complex in the regulation of this process (Chang, 2006).

During mitosis, replicated chromosomes segregate equally to the two daughter cells on a complex molecular scaffold -- the mitotic spindle. Formation of the condensed mitotic chromosomes and the spindle involves reorganization of components of the cell nucleus and cytoskeleton, with changes occurring in different domains of the mitotic cell, in a coordinated sequence. These changes are triggered by the action of a number of protein kinases, including Cdk1:cyclin B, Plk1, and the Aurora kinases. Aurora B kinase is a chromosomal passenger protein that associates with chromosomes during the early stages of mitosis and transfers from chromosomes to the central spindle and cell cortex at the onset of anaphase (for a review, see Carmena, 2003). It is thus a strong candidate for an activity that triggers changes in different domains of the mitotic cell at different times during mitosis (Chang, 2006).

The chromosomal passenger proteins (Adams, 2001a; Vagnarelli, 2004) are present in a complex that includes Incenp (inner centromere protein; known as Sli15p in budding yeast) (Cooke, 1987; Kim, 1999), Aurora B kinase (Ipl1p in budding yeast), survivin (Bir1p in budding yeast) and borealin (see Drosophila Borealin-related). A fifth protein, telophase disc 60 (TD-60), appears to be functionally linked to the complex, but is not stably associated with it. Yeast homologs of borealin or TD-60 have yet to be identified. These five proteins are all mutually interdependent for their localization during mitosis, further supporting the notion that their functions are also interdependent. The chromosomal passengers are required for a number of key functions during mitosis, including chromatin modifications, regulation of kinetochore-microtubule interactions, chromosome bi-orientation and stability of the bipolar spindle, mitotic checkpoint function, assembly of the central spindle and cytokinesis (Chang, 2006 and references therein).

It is currently thought that the various components of the chromosomal passenger complex target and regulate Aurora B kinase activity (Carmena, 2003). Incenp binds to Aurora B kinase through its highly conserved C-terminal IN-BOX motif (Adams, 2001b; Honda, 2003). Incenp is phosphorylated by Aurora B kinase, and this activates the kinase through a positive-feedback loop (Bishop, 2002; Honda, 2003; Kang, 2001). In addition, because Incenp is a microtubule-binding protein (Wheatley, 2001a) that also has a well-defined centromere-targeting domain at its N-terminus (Ainsztein, 1998), it was suggested to have a role in targeting the kinase to particular locations within the mitotic cell (Chang, 2006 and references therein).

Genetic analysis of the chromosomal passenger complex in higher eukaryotes has thus far been limited to the mouse, where knockouts of survivin and Incenp have been described. Genes encoding survivin and Incenp are essential, and homozygous-null embryos die early, at the 32-64-cell stage. Survivin-null embryos have high levels of failed cytokinesis and polyploidy. Incenp-null embryos are characterized by the absence of discernible metaphase or anaphase stages, absence of midbodies, multinucleated cells, multipolar mitotic spindles, micronuclei, abnormal microtubule bundling and chromatin bridges (Cutts, 1999). However, in both cases, the early death of the embryos made it difficult to observe a sufficient number of mitotic cells to perform a thorough analysis of the phenotype (Chang, 2006).

The present study investigated the function of Incenp in normal cells and tissues during development in a multicellular organism. Genetic analysis in Drosophila was used to identify new interactors that will be used in future studies to further define the roles of the chromosomal passenger complex in mitosis and meiosis in Drosophila, and a new recessive allele of Incenp (incenp^EC3747) has been obtained. This mutation is embryonic lethal, with the maternal contribution allowing development to proceed essentially normally until embryonic stage 13. These studies reveal that Incenp is essential for phosphorylation of histone H3 on Ser10, and also for cytokinesis in developing neurons. In addition to these defects, abnormalities were observed in the segregation of cell-fate determinants during neuroblast division; this suggests a role for the passengers in the regulation of asymmetric cell division (Chang, 2006).

The chromosomal passenger protein Aurora B kinase, together with its auxiliary subunits Incenp, survivin and borealin/dasra is a key regulator of mitotic events. This complex has been widely studied in cultured cells, but the early lethality that is observed when its components are knocked-out by gene targeting or RNAi has thus far precluded the study of its role in development. Incenp is a key regulator responsible both for activation of the kinase and for its targeting (Chang, 2006).

This study used Drosophila genetics to isolate a new allele of Incenp (incenp^EC3747) that allowed investigation of the role of the chromosomal passenger complex in development. The mutagenesis screen was designed to identify new genetic interactors of Incenp. The screen used a previously isolated allele of Incenp (incenp^P(EP)2340) that shows a dominant phenotype in mitosis and meiosis. The dominant phenotype associated with the incenp^P(EP)2340 insertion can probably be explained by the production of an N-terminal truncated peptide, predicted to be 91 aa long. Studies in higher eukaryotes have shown that, the first 43 aa of the Incenp N-terminal contain an essential domain required for centromere targeting (Mackay, 1998). The incenp^P(EP)2340 truncated peptide contains this domain, but lacks the conserved in-box required for binding to Aurora B kinase. Therefore, this peptide potentially competes with wild-type Incenp for binding to the centromere. Alternatively, the truncated polypeptide could associate with wild-type Incenp and trigger its destruction. In accordance with these hypotheses, P(EP)2340 heterozygous flies show a 4-fold decrease in the amount of centromeric Incenp compared with wild-type controls (Chang, 2006).

The screen was based on the hypothesis that in flies heterozygous for incenp^P(EP)2340, chromosomal passenger function might be in a precarious equilibrium that can be thrown further out of balance by mutation of a gene coding for an interacting protein. EMS was chosen as a mutagen, with the aim of obtaining point mutations that specifically disrupt protein-protein interactions (Chang, 2006).

Although far from saturation, the EMS-screen yielded 16 mutants showing phenotypes characteristic of mitotic and/or meiotic defects when combined with incenp^P(EP)2340. Preliminary characterization of the trans-heterozygous combinations (mutant incenp^P(EP)2340) reveals a variety of distinctive phenotypes affecting different processes during cell division, including chromosome condensation, segregation and cytokinesis. Future characterization of these mutants, identification of the genes affected, their products and the nature of the interaction with Incenp should permit a functional dissection of the multiple roles of the passenger complex in a developmental context (Chang, 2006).

One of the new mutants, EC3747, contains a point mutation that generates a stop codon just before the coiled-coil region of Drosophila Incenp. The predicted molecular mass of the 456 aa protein product truncated at the C-terminal is approximately 61 kDa. Immunoblotting analysis of extracts from homozygous mutant embryos failed to detect any truncated Incenp polypeptide. This is probably due to instability of the polypeptide. Unlike incenp^P(EP)2340 heterozygous individuals, EC3747 heterozygous individuals do not show any defects in mitosis or meiosis, suggesting that EC3747 is a recessive null allele of Incenp. Incenp is an essential gene in Drosophila, and individuals homozygous for EC3747 died late during embryogenesis. One probable factor contributing to this embryonic lethality was abnormal development of the nervous system (Chang, 2006).

A maternal stockpile of Drosophila Incenp protein allowed incenp^EC3747 homozygotes to survive to late embryogenesis, therefore enabling a study of the role of this protein in development. During phenotypic analysis of incenp^EC3747, it was observed that maternal Incenp protein becomes generally undetectable at stage 13 of embryonic development. The only cells dividing at this stage are those of the nervous system, where abnormally large mitotic cells were observed, without any phosphorylated histone H3. This is the first demonstration in a developing organism that Incenp is essential for the activity of Aurora B kinase (Chang, 2006).

Mutant neuroblasts were polyploid, and had multiple centrosomes, a phenotype that can result as a consequence of failure in chromosome segregation and cytokinesis. However, a recent study has shown that the new chromosomal passenger borealin, which is found in a complex with Aurora B kinase, Incenp and survivin, is required for maintenance of bipolar spindle in mitosis (Gassmann, 2004). In addition, an elevated frequency of monopolar and multipolar spindles has been observed in S2-phase cells following dsRNAi knockdown of Drosophila Incenp. Therefore it is possible that the centrosomal abnormalities observed in stage 13 embryos reflect a requirement for Incenp in maintenance of bipolar spindle integrity (Chang, 2006).

The results reveal that Incenp is required for the asymmetric distribution of Prospero. This is likely to be a consequence of the involvement of Aurora B kinase in the regulation of asymmetric cell division rather than simply a consequence of defects in mitosis and cytokinesis. Importantly, mutations in the gene encoding pebble, a Rho GTP exchange factor that is essential for cytokinesis, do not disrupt the polarized localization of cell-fate determinants. Furthermore, the prometaphase delay resulting from the loss of Incenp-Aurora B kinase complex function, does probably not in itself alter Prospero localization because colchicine treatment does not have an effect on this process. It has been reported that cortical Prospero is highly phosphorylated and that this modification could be relevant for its localization. This raises the possibility that Prospero is an Aurora B kinase substrate. The observation of the partial colocalisation of Prospero with Incenp on chromatin in mitosis would agree with this (Chang, 2006).

An alternative role for the passenger complex in asymmetric cell division could be through regulation of myosin. At late prophase of neuroblast asymmetric mitosis, localization of Prospero and Numb in the basal crescent is coordinated with the orientation of the mitotic spindle and depends on two protein subcomplexes acting together with the actin cytoskeleton. Recently, it has been shown that myosin motors are involved in localization of fate determinants in different ways. The myosin VI Jaguar (Jar) is required for localization of Miranda. Myosin II [restricted to the apical cortex by lethal giant larva (Lgl)] is necessary for apical exclusion of fate determinants. Myosin II regulatory light-chain has been described as a substrate of Aurora B kinase. Thus a potential role of the chromosomal passenger complex is to regulate myosin-motor activity essential for localization of cell-fate determinants (Chang, 2006).

The Drosophila Incenp mutant incenp^EC3747 has allowed the role of passenger proteins in development to be studied for the first time. This has revealed a previously unsuspected requirement for Incenp in the regulation of the asymmetric distribution of cell-fate determinants during CNS development in Drosophila embryos. Thus, the chromosomal passenger complex appears to have an essential role not only in regulating basic functions of normal mitosis, but also in regulating the more elaborate process of asymmetric cell division, which is essential for metazoan development (Chang, 2006).

GENE STRUCTURE

cDNA clone length - 2604 bp

Bases in 5' UTR - 262

Exons - 6

Bases in 3' UTR - 74

PROTEIN STRUCTURE

Amino Acids - 755

Structural Domains

A candidate Drosophila INCENP cDNA (LD24414) was identified 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. The sequence and exon/intron analysis agrees exactly with that described by Celera Genomics for the hypothetical gene CG12165. (Adams, 2001b).

Drosophila INCENP 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 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 (Adams, 2001b).

date revised: 5 August 2021

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