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<B01> Today I!m going to show how having sufficient
membrane in the cell is a new strategy that animals can use to
survive despite varying osmotic conditions. "
All animals must maintain the homeostasis of the body's water
content in a manner suitable to their environment. This is
achieved by regulating the osmotic pressure of bodily fluids, a
process called osmoregulation."
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<B02> One major mechanism of osmoregulation is to match
the body!s osmolality to that of the environment. This strategy,
called osmoconformation, is used by marine invertebrates, and
also some vertebrates such as sharks and rays. To match their
body!s osmolality to that of sea water, these animals have high
concentrations of special solutes in the body; for example urea,
in the case of sharks. However, due to their high body
osmolality these animals cannot survive in freshwater. In low
osmotic freshwater there is a huge imbalance of osmotic
pressure between their cells and the environment. Water will
flow into their cells, and the cells may even burst in such an
environment. "
<B03> We wondered whether it would be possible to engineer
the cell membrane to allow an exclusively seawater animal to
survive in a freshwater environment. "
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To test this possibility, we chose a “saltwater frog” found in
the Philippines, called Rana muscavora, because these
animals are easier to keep in the laboratory than sharks are.
Just like sharks, this frog is a osmoconformer, and has high
cellular osmolality approaching that of sea water. Muscavora
accomplishes this by having high concentrations of a solute
called N-Indolyl Guanidium, or “NIG” in its cells. This allows the
frog to flourish in seawater, but it cannot survive in a low
osmotic freshwater environment. "
<B04> In the next slide I will show you what happens this frog!s
eggs are placed in hypotonic fresh water. "
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As you can see the oocyte explodes when placed in
hypotonic condition. Because of the high osmolality of the cell,
water rushes into the egg and as you can see the volume of the
egg increases considerably. If you watch the temporal
sequence closely you may notice that the cell volume increases
about three fold, and stays that size for a few seconds, before
eventually bursting. "
<B05> So we wondered whether it would be possible to prevent
cell bursting and allow the eggs to survive by increasing the
elasticity of the cell membrane. !
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We focused on a particular property of the cell membrane called
“membrane reserve”. Membrane reserve is extra membrane
that is sequestered below the plasma membrane. Cells with
more membrane reserve can respond to the increase in the cell
volume by incorporating membrane reserve to the plasma
membrane and thus achieving a larger cell surface area. We
reasoned that if we artificially increased the membrane reserve
of this saltwater frog it would not have any deleterious effect on
the cells, and it would increase the elasticity of the cell.
Therefore, even when there is an influx of fresh water, the cell
might be able to sustain an enlarged volume. "
<B06> The question was how we could increase the membrane
reserve."
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We focused our attention on a recent study that identified a key
regulator of lipid biogenesis, called “lotsoflipid”, or LOL. LOL
controls production of cell membrane in cultured animal cells.
We thought that if we overexpressed LOL in muscavora oocytes
it might increase the membrane reserve so that the cell
membrane could support increased volume upon exposure to
freshwater. So we cloned the muscavora LOL gene, expressed
it in muscavora oocytes, and measured the amount of
membrane these oocytes have. "
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<B07> After LOL-overexpression, the diameter of the oocyte
did not change, and the amount of plasma membrane was
about the same as the control muscavora oocytes. We then
measured the amount of “total” surface membrane, which is the
sum of the plasma membrane and membrane reserve. In
control oocytes this value was not significantly different from the
amount of plasma membrane, indicating that these oocytes
have very small membrane reserve, if any. However, after LOL
overexpression, the total membrane content of these oocytes
was 3.5 times higher than the amount of the plasma membrane.
This meant that LOL expression had in fact created membrane
reserve in these oocytes. "
<B08> Having succeeded in increasing the membrane reserve
we next asked whether these membrane-enriched oocytes
could survive when placed in fresh water. "
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In hypotonic freshwater, the control muscavora oocytes swell
and explode within 3 minutes. LOL-overexpressing oocytes
also began swelling rapidly, and the volume reached about
three times the original volume. However, in contrast to the
control oocytes, LOL-overexpressing oocytes did not explode.
Rather, they maintained their expanded size upon continued
culture in fresh water. So, we concluded that increasing
membrane reserve allowed the oocytes to survive in low
osmotic pressure condition. Since the over-expression of LOL
is only transient in the oocyte expression system, the
unfertilized oocytes did not develop further. "
<B09> So we decided to create transgenic muscavora that
overproduce LOL permanently and test whether such animals
could grow and reproduce in a freshwater environment."
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To do that we constructed an LOL gene fused to a strong
constitutive promoter, and injected this transgene into fertilized
eggs. They were then allowed to grow to tadpoles in isotonic
salt water. We screened for animals whose genome had
integrated the LOL gene, identified three tadpoles with LOL
gene integration, and transferred these animals to fresh water an environment that is fatal for normal muscavora tadpoles."
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<B10> As you can see the transgenic tadpoles were swimming
happily in the fresh water. These animals, which we call “super
Rana muscorova”, didn!t have any morphological abnormalities,
except that their skin had a slightly swollen and wrinkled
appearance compared to normal animals and some unusual
red and yellow markings. Two of the three tadpoles
metamorphosed successfully to adults, "
<B11> so we tested whether they could reproduce in
freshwater. "
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We measured the survival rates of eggs grown in normal
saltwater and in fresh water. Whereas none of the normal, nontransgenic muscovora embryos survived in fresh water, super
muscavora embryos with overexpressed LOL survived not only
in the normal saltwater condition, but also in the freshwater
environment. Thus we have been able to convert a species that
can only survive in saltwater, into a species that can not only
survive but also breed in either saltwater or freshwater
conditions. This means that we have created a new “ecosystem”. "
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<B12> Today we have seen that a marine animal that has
osmolality equal to sea water can be engineered to survive in
fresh water simply by providing membrane reserve. Apparently
the animals can adjust to the new environment by expanding
their cellular membrane when water enters the cell in a
hypotonic condition. So, membrane synthesis is an extremely
important process in maintaining homeostasis in various
osmotic conditions. "
<B13> Although this can explain why super muscavora oocytes
did not explode when transferred to freshwater, our results
actually generates a new question. You might remember that
these frogs makes high concentration of a macromolecule
called N-IG to match their osmolality to that of sea water. In the
new fresh water environment, we don!t know the concentration
of N-IB yet, but it is likely that in freshwater the synthesis or
degradation of this molecule is also regulated so that the body
osmolality is maintained low. In future it will be interesting to
study the mechanism of N-IG regulation."
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14"
<B14> I!d like to thank Dr. Tatsumi Hirata for the LOL cDNA,
and Dr. Yasushi Hiromi for providing o-gui flies to feed our frogs.
All this work was done at the National Institute of Genetics,
which also provides graduate education as the Department of
Genetics, SOKENDAI. Thank you for your attention."