As you've seen previously, fertilisation of the sea urchin eggs triggers a dramatic
increase of protein synthesis activity. This increase is necessary and required
for the progression through the cell cycles of the early embryonic development.
It's important to be able to measure and visualise protein synthesis activity to be
able to understand the signalling pathways that act upon this translational control
and to do this, in the following experiment you will learn how to measure and visualise
protein synthesis activity by using a non-radioactive method called SUnSet.
SUnSet is based on the use of puromycin, an antibiotic that is an analogue of aminoacyl
tRNA synthetase and that will be incorporated into nascent polypeptides.
The incorporation of puromycin results in the termination of elongation and the
release of a labelled peptide from the ribosome. Then the labelled peptide
with puromycin is detected using a Western blot using an antibody against puromycin.
So it's important to understand that the low concentration of puromycin that is
used in this SUnSet experiment will not inhibit completely translation,
but will reflect the overall rate of protein synthesis.
In the following experiment, we will visualise the protein synthesis activity
triggered by fertilisation in absence or in presence of a specific inhibitor of the
mTOR pathway called P242. This inhibitor will inhibit the protein synthesis
that is dependant on the recruitment of the translation machinery on the cap of the mRNA.
We will monitor protein synthesis activity by the SUnSet method every 30 minutes
up to two hours post-fertilisation.
Patrick will walk you through this experiment. We will use dejellied sea urchin eggs that
have been prepared in lab video 1 and 2, we will need DMSO as a vehicule,
stock solution for the mTOR inhibitor PP242,
puromycin for the SUnSet labelling,
and dry sperm for fertilisation.
Eight Eppendorf tubes of 1.5 mL, corresponding to each time point
to collect, and two 100mL beakers.
The experiment is described in this diagram. We will fertilise eggs pre-incubated with
PP242 inhibitor or control DMSO, and label the newly synthesised proteins
with puromycin at four time points, unfertilised, 30 minutes, 60 minutes,
and 170 minutes post-fertilisation. Let's start.
Distribute an equal volume of 5% egg suspension into two beakers.
The PP242 inhibitor at a final concentration of 10 microM is added
to the egg culture suspension in one beaker.
The same amount of DMSO is added to the other one for the control experiment.
The egg suspensions are stirred to ensure proper oxygenation of the egg culture.
The first step is to proceed to the puromycin labelling of the eggs before fertilisation.
This will be our starting reference, the zero time point.
Collect 500 microliters from each beaker
in the corresponding T0- labelled Eppendorf tubes.
Then, add 5 microliters of puromycin at 100 microM final concentration.
Incubate on a rotating wheel for 10 minutes.
After this incubation time, spin the tubes for five seconds to pellet the eggs.
Remove the sea water,
and re-suspend the pellet with SDS-PAGE buffer.
Your sample is now ready for Western blot analysis.
The second step is fertilisation of the pre- incubated cultures and labelling of newly
synthesised proteins at the indicated times following fertilisation.
In parallel, cell cleavage can be followed under the microscope to assess the effect
of PP242 inhibitor on mitotic division.
We fertilise the eggs by adding the activated sperm to each culture
and start the timer.
Aliquots are collected at the indicated times and puromycin labelling is performed
as we did before for the zero time point.
Four samples for the DMSO control and four samples for the PP242-treated samples
are obtained, ready for analysis.
We next analyse the extracts by Western blot to reveal the incorporation of puromycin
into translating proteins. The first step is to separate the proteins in the extract
according to their molecular weight. After heat denaturation, the samples
are loaded on a 12% SDS-PAGE gel and constant voltage is applied to the gel.
At the end of the migration, you can see on the left side of the gel the molecular
weight markers that have been well-separated on the gel.
The second step is to electrotransfer the protein onto a nitrocellulose membrane.
At the end of the electrophoresis, the gel is removed from the running cassette
and is unmoulded from the plates.
The wealth and the stacking gel are removed carefully.
Prepare a large dish filled with transfer buffer.
And soak the gel in the transfer buffer so you can equilibrate it before the transfer.
You need to soak the elements that you will need for the transfer to make the sandwich.
You need four filter papers,
two fiber pads,
and the nitrocellulose membrane that is cut at the size of the gel.
On the transfer cassette, place the pad, two filter papers, the gel, the membrane,
two other filter papers, and the last pad. Make sure that no bubbles are trapped
into the sandwich, as it will prevent correct transfer of the proteins from
the gel to the membrane. Close the cassette and place it into the transfer module,
making sure that the black side of the cassette is facing the black side of the module.
Add an ice pack in the tank,
fill the tank with the transfer buffer,
and add a magnetic stirrer.
Put the lid back on,
put it on the stirrer plate,
and transfer for one hour at 100 volts.
Once the transfer is done,
carefully unclip the cassette and remove each layer
until you reach the nitrocellulose membrane.
The membrane is rinsed in water,
and the transfer of your samples is revealed by Ponceau red staining.
The last step of the Western blot is the incubation with the antibodies.
In the next slides you will see the results of the experiment.
The transferred proteins are revealed after Ponceau red staining.
Here you can see that equal amounts of proteins from each extract have been loaded.
The last step of the Western blot is the incubation of the membrane
with an antibody that is directed against puromycin, and the signal obtained after
chemiluminescence reaction is shown here.
As you can see in the unfertilised eggs corresponding to the zero time point,
a very weak signal is observed with the antibody labelling, reflecting a very low
level of protein synthesis activity.
In the control embryos, the signal increases after fertilisation, reflecting
the increase of protein synthesis activity triggered by fertilisation.
In contrast, in the PP242-treated embryos, the signal shows a modest increase after
fertilisation. The mTOR inhibitor PP242 has inhibited the increase of protein
synthesis activity triggered by fertilisation.
These results demonstrate that the mTOR activity is necessary for translation
activation induced by fertilisation.
In this video, you've learned how to visualise and measure protein synthesis following
fertilisation by a method called SUnSet. This allows you to investigate protein
translation activity and how its regulated. For the first three cell cycles, the protein
synthesis increase is required and relies on the use of maternal messenger RNAs
which are stored in the egg cytoplasm.
This protein synthesis increase is then necessary for the progression through
these cell cycles. Afterwards, the protein translation relies on the newly transcribed
mRNA, which are produced by the reactivation of the zygotic genome,
and this translation is necessary for the correct progression through the cell cycles
of the embryonic development. To go further on specific translation analysis,
you can identify the translated mRNA
among all the mRNAs that are present in the egg or embryo, by sequencing them
by sequencing the mRNA that are associated to actively translating ribosomes
that are called polysomes. Such genome- wide analysis will be informative for two things:
1 - how many and which mRNA are regulated and translated, and 2 - which
molecular functions are activated or regulated during the course of
fertilisation and early development.