Organisms survive periods of environmental hypoxia or anoxia by entering into an ametabolic state of suspended animation, known as cryptobiosis. Cryptobiosis in developmental life history stages is seen in a variety of organisms, and embryonic diapause is seen in organisms ranging from nematode worms and insects up to fish. Fascinated by the molecular mechanisms that control episodes of cryptobiosis, Todd Nystul and colleagues have investigated the mechanisms by which cryptobiosis is achieved during environmental anoxia in nematode worms.
The researchers used RNA interference (RNAi) to screen the functional consequences of gene removal on anoxia tolerance by systematically removing the gene products from 2445 open reading frames (ORF) on chromosome I of C. elegans. RNAi uses short double stranded RNA molecules, homologous to the mRNA product of a gene, to bind those mRNAs and target them for removal from the cell, thereby nullifying the function of that gene.
By scoring for anoxia-specific lethality, they identified an ORF that, when nullified, caused a 70% decrease in survivorship after exposure to anoxia for 24 h. The function of this ORF is anoxia specific because nullification of the gene product did not produce any decreases in survivorship during exposure to hypoxic (0.5 kPa O2) or normoxic conditions. The research team named this ORF san-1, for `suspended animation 1'.
san-1 shares 27% sequence homology with the gene encoding yeast spindle checkpoint protein component Mad3P, and thus the team hypothesized that SAN-1 is a component of the spindle checkpoint in C. elegans. The spindle checkpoint prevents mitotic blastomeres from progressing from metaphase to anaphase with the consequence that cell division grinds to a halt. C. elegans embryos stained with SAN-1 antibodies, DNA stains,and antibodies to the kinetochore marker HCP-3 revealed that SAN-1 is localized to the nucleus during prophase, and to the poleward faces of the chromosomes during metaphase. This staining pattern is consistent with those of other proteins involved in spindle checkpoint activity. These observations suggest that SAN-1 induces cryptobiosis through arrest of the cell cycle.
To confirm the role of spindle checkpoint activity in cryptobiosis, RNAi was used to nullify additional spindle checkpoint components. In all cases,the spindle checkpoint was necessary for cryptobiotic survival of environmental anoxia.
The results of spindle checkpoint activity were observed in mitotic blastomeres of C. elegans embryos in normoxic and anoxic conditions. In wild-type worm embryos, metaphase blastomeres increased from 18.2% in normoxia to 42.9% in anoxia, but decreased from 20.3% to 0.7% in san-1 (RNAi) embryos in normoxic and anoxic conditions respectively. Spindle checkpoint activity during anoxia prevented abnormal anaphase and telophase nuclei from forming; abnormalities were observed in 30.7% of mitotic blastomeres in san-1 (RNAi) embryos compared to 0.2% abnormal nuclei in wild-type embryos. These mitotic abnormalities resulted in aneuploidy in san-1 (RNAi) embryos during anoxia. These observations indicate that during exposure to anoxic conditions, activation of the spindle checkpoint, thereby trapping mitotic cells in metaphase, protects the embryos from severe chromosomal abnormalities.
The factors involved in the initiation of cryptobiosis are complex. Nystul and colleagues have presented evidence for the role of the spindle checkpoint in anoxia-specific cryptobiosis that involves protecting dividing cells from incurring damaging chromosomal mis-segregation, but the regulation of spindle checkpoint genes remains to be investigated. Of significance is that the mechanism described here is anoxia tolerance specific; the spindle checkpoint is not involved in hypoxia tolerance, and other mechanisms (e.g. HIF-1 transcriptional activation) are important during environmental hypoxia.