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Project Progress

Desiccation tolerance in Rotifera One of the biggest challenges facing eukaryote extremophiles is the loss of water leading to desiccation. Resistance to desiccation as adults, juveniles, seeds, or spores is found in species of five animal phyla and four divisions of plants. Little is understood about the biochemistry of desiccation tolerance in eukaryotes, but ribosomes are likely to figure prominently in this phenomenon. The model for our investigations are animals from the phylum Rotifera which are capable from going from completely desiccated to active, swimming animals in minutes to hours (1, 2). We are examining how their ribosomes tolerate near complete dehydration, then can rehydrate and engage in translation within minutes. Our hypothesis is that specific proteins associate with ribosomes during desiccation, protecting them from damage and then dissociate upon rehydration. We want to enumerate these proteins and discover the underlying genes. In the future, this knowledge could be used to engineer desiccation tolerance into organisms that currently lack this ability.

Thermostable rotifer proteins. In monogonont rotifers, resting eggs have much greater thermotolerance than adults. We have isolated two thermostable proteins of 27 and 35 kD from resting eggs of the rotifer Brachionus manjavacas and used mass spectrometry to determine their identity. The 27 kD protein is similar to an LEA-1B (late embryogenesis abundant) protein known from the rotifer Adineta ricciae (3). It plays a key role in desiccation tolerance in rotifers (4, 5). This is the first observation that LEA proteins may play a role in thermostability. The 35 kD protein is similar to vitellogenin, a egg yolk protein widely present in the yolk of vertebrate and invertebrate animals, and the first report of a VTG-like protein in the phylum Rotifera. Both of these proteins exhibited increased expression in rotifer resting eggs when compared to their expression in amictic females. Our data suggests the possible existence of alternate pathways of desiccation and thermal resistance in brachionid rotifers.

Purification, isolation, and identification of rotifer ribosomal proteins. We have now successfully purified ribosomes from both B. calyciflorus adult rotifers and resting eggs (desiccated embryos) (6). Currently the rotifer genome is not published, so we have performed quantitative mass spectrometry analysis of the ribosomal proteins from both populations in collaboration with the Seyfried laboratory at Emory University. For the adult rotifers, we identified most of the ribosomal proteins known from both the small and large ribosomal subunits of eukaryotes. The samples were further analyzed by negative stain electron microscopy (EM) and showed intact 80S samples (in collaboration with the Schmidt-Krey laboratory at Georgia Tech).

Next we purified ribosomes from B. calyciflorus desiccated embryos and obtained a much lower yield of overall ribosomes. This is to be expected though as few, if any, macromolecules such as proteins and nucleic acids can exist intact in this dehydrated state. We found that ribosomes were indeed present in the desiccated embryos. From the EM studies, there seemed to be an equal amount of both small ribosomal subunits known as the 40S as well as the large ribosomal subunit, the 80S. Further time points are currently underway to determine if the 80S fraction is seen due to the lifetime of our experiments with the underlying hypothesis being that in the desiccated state, only 40S ribosomal subunit are present.
Interestingly when rotifer desiccated embryo ribosomes were subjected to MS analysis, far few ribosomal proteins were identified. This indicates that perhaps a specialized ribosome exists during this stress state. One possibility is that this ‘minimal’ ribosome is configured to withstand desiccation and then rapidly regain function upon rehydration, selectively translating ribosomal proteins to make a functional 80S ribosome that is normally present in adult rotifers. Experiments aimed to address this hypothesis include our EM studies at different times points in the process of rehydration as described above. These experiments are coupled with polysome profiling assays to determine whether 40S, 80S or polysomes are present and their relative abundance. Additional analyses include RT-PCR and western blots of the missing ribosomal proteins to further support the absence of such proteins in the desiccated state. This research is consistent with current thought in the eukaryotic ribosome field that specialized ribosomes are present at different points in the life cycle to promote translation of specific mRNA targets. Our research will be the first time that a ‘stress’ ribosome is characterized from a diapausing life stage of a bonafide extremophile. It may represent a model for how ribosomes of other extremophiles survive stress, whether it is desiccation, heat or nutrient deprivation.

References
1. Ricci C, Caprioli M, Fontaneto D, Melone G (2008) Volume and Morphology Changes of a Bdelloid Rotifer Species (Macrotrachela Quadricornifera) During Anhydrobiosis. J Morphol 269:233-239.
2. Marotta R, Leasi F, Uggetti A, Ricci C, Melone G (2010) Dry and Survive: Morphological Changes During Anhydrobiosis in a Bdelloid Rotifer. Journal of structural biology.
3. Denekamp NY, Thorne MA, Clark MS, Kube M, Reinhardt R, Lubzens E (2009) Discovering Genes Associated with Dormancy in the Monogonont Rotifer Brachionus Plicatilis. BMC Genomics 10:108.
4. Tunnacliffe A, Lapinski J, McGee B (2005) A Putative Lea Protein, but No Trehalose, Is Present in Anhydrobiotic Bdelloid Rotifers.315-321.
5. Denekamp NY, Reinhardt R, Kube M, Lubzens E (2010) Late Embryogenesis Abundant (Lea) Proteins in Nondesiccated, Encysted, and Diapausing Embryos of Rotifers. Biol Reprod 82:714-724.
6. Heggemann F, Schmelter R, Kleinow W (1997) Ribosomes of Brachionus Plicatilis (Rotifera) – Distribution in Homogenate Fractions and General Properties.11-24.