Drosophila chromosome passengers

Table of Contents

1. Introduction
2. Sequence homology with vertebrate passenger proteins
3. Distribution during mitosis
4. Functional analysis by RNA interference
5. Unresolved questions

Introduction

To date, homologues of INCENP and aurora-B are known in Drosophila. A protein called deterin, that may be a survivin homologue, is known to have anti-apoptotic effects but its role in mitosis has not yet been studied.

DmAurora-B (originally called IAL) was identified by a group working on the JAK/STAT signal transduction pathway, as a neighbouring gene to the xxxgene. DmINCENP was identified as a potential transcript from the Celera genome sequence with homology at the C-terminus to vertebrate INCENPs.

Sequence homology with vertebrate passenger proteins

DmINCENP was identified on the basis of its homology in the C terminal 100 amino acids. This domain, now called the "IN-Box" is conserved from yeasts to humans. DmINCENP is also highly basic and contains a coiled coil in its central region. The N terminus however is poorly conserved with vertebrate INCENPS.
To see a protein sequence alignment of the IN-Box from flies and vertebrates, click here (45kB file).

Dm AuroraB has a similar domain structure to vertebrate Aurora-Bs but is much shorter, with only a 35aa N terminal domain. Nevertheless, this domain is probably sufficient to target it properly during mitosis.
To see a protein sequence alignment of the aurora-B kinases, click here (100kB file).

Deterin is a potential fly orthologue of survivin. It is a BIR domain containing protein and is of similar size and predicted structure to vertebrate survivin. To see an alignment of deterin with survivin, click here (90kB file).

All these sequence alignments need the Flash5 player to be viewed.

Distribution during mitosis

Both INCENP and aurora-B show classical patterns of chromosome passenger distribution. Click on the thumbnails below to see a gallery of images.

DmINCENP images		DmAurora-B images

Functional analysis by RNA interference

No mutants are yet available in either the DmINCENP or the DmAurora-B genes.
Recently the technique of RNA interference has become available in a number of embryonic cell lines, enabling a "null phenotype" to be generated by loss of the protein. We performed RNAi studies on both INCENP and auroraB, with the following main conclusions:
1. Loss of either gene causes a near identical phenotype.
2. Mitotic cells are aberrant in chromosome condensation, congression of chromosomes to the metaphase plate, sister chromatid separation and cytokinesis.
3. INCENP is required for correct localisation of aurora-B.
4. In the absence of aurora-B, INCENP localises to chromosomes during prophase but does not go the centromere and does not transfer to the spindle.
For some images from this study click on the thumbnail below:

Wild-type cell (left) still has INCENP (red); treated cell displays lagging anaphase chromatin.	These two images show CENP-A staining (red) at both poles, illustrating that centromere separation is not impeded by INCENP RNAi. Images by W.C. Earnshaw.		Aurora-B (red) is not at the midbody following INCENP RNAi.	INCENP remains diffuse on chromosomes and does not colocalize at the centromere in Aur-B RNAi cells.


This work has recently been published (Adams et al., 2001). Similar results were obtained by Giet and Glover.

Unresolved questions

1. What are the in vivosubstrates for aurora-B?
2. Why is aurora-B needed for alignment of chromosomes at the metaphase plate?
3. Is aurora-B phosphorylation of histone H3 needed for chromosome condensation or for chromosome segregation?
4. Is deterin a true survivin homologue in flies?
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   © Copyright Richard Adams 2001
   Images © Rockefeller press