Worm Breeder's Gazette 15(1): 61 (October 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
ABL-Basic Research Program, Frederick Cancer Research and Development Center,Frederick, MD 21702
A number of laboratories have recently described various
members of a highly conserved serine/threonine protein kinase family
that appears to play a role in chromosome segregation and mitotic
spindle dynamics in several different organisms (1,2,3,4). A search of
the C. elegans genome database revealed that there are at least two
nematode proteins that are highly related to this protein kinase
family. We refer to these proteins as AIR-1 and AIR-2 (Aurora/Ipl-1
related).
To date, the subcellular location of only one member of this
protein kinase family has been reported (3,4). The mammalian protein
Iak-1 has been shown to be associated with the centrosomes of the
mitotic spindle in tissue culture cells and with meiotic chromosomes in
mouse spermatocytes (J. Schumacher, unpublished). By performing
immunocytochemistry experiments on fixed C. elegans embryos with
antisera raised against specific peptides, we have found that the AIR-1
protein, like its mammalian counterpart, is also found on centrosomes
in mitotic cells. The protein is first detectable on centrosomes of
the first mitotic division following pronuclear fusion in the one-cell
embryo. It is clearly associated with duplicated centrosomes prior to
their migration to opposite sides of the nucleus and is found on
mitotic centrosomes up to the limits of resolution in two-fold stage
embryos. The location of the AIR-2 protein mimics that of Iak-1 in
meiotic cells. Like Iak-1 in spermatocytes, AIR-2 is also found on
chromosomes undergoing meiotic divisions both in oocytes and
spermatocytes. AIR-2 staining is diffuse throughout the cellularized
oocytes of the proximal gonad, but becomes localized to the chromosomes
in the oocyte that resides next to the spermatheca. The protein
persists on these chromosomes throughout meiosis and remains associated
with polar body chromatin following these divisions. AIR-2 is also
found on meiotic chromosomes during spermatogenesis in C. elegans
males. In addition, it is associated with mature sperm present in the
spermatheca, but at this stage it doesn!t appear to be localized to the
chromatin. Instead, it appears to surround the sperm, suggesting an
association with the cellular membrane. In embryos, diffuse AIR-2
staining is found in the cytoplasm, but is also clearly localized to
mitotic metaphase chromosomes. The protein may be present on
chromosomes at other stages of mitosis, but is difficult to detect on
less condensed chromatin. By telophase, AIR-2 is clearly localized to
midbody microtubules, and a small dot of staining persists on the cell
membrane once cytokinesis is complete.
To disrupt the function of each of these proteins during
embryogenesis, we injected antisense RNA corresponding to the entire
cDNA of each gene into the gonads of C. elegans hermaphrodites.
Injection of either RNA resulted in embryonic lethality and the
specific loss of each protein as detected by immunocytochemistry.
AIR-1 deficient embryos die with greater than 100 cells and are
severely aneuploid. Analysis of younger embryos revealed a variety of
chromosome segregation defects ranging from the loss of a single
chromosome to the missegregation of every chromosome to one daughter
cell in a particular division. Some cells also appeared to be severely
polyploid and contain multiple centrosomes, suggesting multiple cell
cycles that lack an intervening mitotic division. Disruption of AIR-2
resulted in the production of one-cell embryos that contained many
nuclei and centrosomes, as well as polar bodies that continue to
replicate and divide. One-cell embryos containing anywhere from one to
greater than 20 nuclei were found with equally abnormal numbers of
centrosomes. This dramatic phenotype again suggests the uncoupling of
DNA replication and centrosome duplication from the completion of
mitosis.
References:
1) Chan, C.S. and Botstein, D. (1993). Isolation and characterization
of chromosome-gain and increase-in-ploidy mutants in yeast. Genetics
135, 677-691
2) Glover, D. M., Leibowitz, M. H., McLean, D. A., and Parry, H.
(1995). Mutations in aurora prevent centrosome separation leading to
the formation of monopolar spindles. Cell 81, 95-105.
3) Gopalan, G., Chan C.S., and Donovan P.J. (1997). A novel mammalian,
mitotic spindle-associated kinase is related to yeast and fly
chromosome segregation regulators. J Cell Biol 138, 643-656
4) Kimura, M., Kotani, S., Hattori, T., Sumi, N., Yoshioka, T.,
Todokoro, K., and Okano, Y. Cell cycle-dependent expression and
spindle pole localization of a novel human protein kinase, Aik,
related to Aurora of Drosophila and yeast Ipl1. J Biol Chem 272,
13766-13771
Research sponsored by the National Cancer Institute, DHHS, under
contract with ABL.