As we said, sea urchins represent a powerful tool for the analysis
of the cell cycle for at least three reasons:
First, that unfertilised eggs are physiologically blocked in G1.
Second, fertilization triggers the entry into the cell cycle with DNA duplications
and the first mitotic divisions,
and third, mitotic divisions of the sea urchin emrbyos are rapid and synchronous.
CDK1, the Cyclin-Dependent Kinase 1, when associated with cyclin B,
plays a crucial role for the control of the entry and the progress through mitosis.
The objective of the following experiment is to see the in vivo activity of
cyclin B/CDK1 complex by a non-radioactive method.
For this purpose, we will use the catalytic submit of protein phosphatase 1 that is
now known to be a natural substrate of the active CDK1/cyclin B complex.
The idea is to monitor the activity of the cyclin B/CDK1 complex by observing the
endogenous phosphorylation status of PP1Cα (on threonine 318 in sea urchins).
Measurements will be performed by Western Bloting using antibodies which
specifically distinguish PP1Cα phosphorylated at this threonine.
Therefore, during this video you will see how you have to prepare the eggs
when you want to perform some biochemical approaches.
The first step would be to prepare the eggs by removing the egg jelly
that is present around each egg.
We will see how to organise a classical experiment where you will have to pick up
samples every 10 minutes from one hour until four hours post-fertilization.
Finally, you will see example of data that we usually get by these approaches.
For this experiment, we need some natural filtered sea water,
the eggs that we obtained in the previous sequence and that have been kept at ~16°C,
dry sperm that has been kept in ice or the fridge,
we need citric acid at 1M concentration, diluted in sea water to remove the egg jelly,
several plastic tubes,
a beaker,
20 Eppendorf tubes that we previously annotated with a permanent marker,
concentrated loading buffer that contains sodium dodecyl suphate and DTT,
of course we need a set of micropipettes,
and the corresponding tips.
The eggs are surrounded by a transparent gelatinous matrix, the egg jelly.
This can be shown by adding Indian ink directly to seawater.
The ink thus makes it possible to visualise the contours of the matrix under a microscope.
The egg jelly must be removed in part because it may hinder us in the later
stages of the experiment. Therefore, we will remove the egg jelly by a
rapid acidic treatment and washing. 35 microliters of citric acid (1M initial
concentration) is added in 50mL of egg suspension.
Eggs are dejellied by swirling for 30 seconds and rapidly centrifuged at 1000 rpm
for less than one minute. Sea water is rapidly removed
and the pellet of eggs is rinsed three times in filtered seawater before fertilisation.
At the end of his step, eggs are suspended in filtered sea water at a concentration of
5% cells per volume, and glycine is added at the final concentration of 0.1%.
This diagram is to remind you that during this experiment, total extracts will be
prepared from embryos taken from 60 minutes after fertilisation and every 10
minutes until four hours post-fertilisation.
In the end, proteins will be resolved by SDS -PAGE, and phosphorylated PP1C will be
revealed by Western blot using a specific antibody.
Before fertilisation, egg suspension is transferred into a beaker, and eggs are
gently agitated.
100 microliters of diluted sperm are then added to 50 mL of egg suspension.
The chronometer is then started and the fertilisation time is noted.
Precisely 60 minutes later,
500 microliters of the egg suspension are transferred into the corresponding tube.
Eggs are rapidly pelleted by low speed centrifugation using a capsule, for instance.
The supernatant is then carefully removed by aspiration.
Finally, 150 microliters of SDS-fix buffer is added directly to the pellet.
Cells are lysed by a few up and down in the micropipette tip.
At the different times that we showed previously in the diagram, the samples
are treated in the same manner.
Therefore, at the end of the experiment we have 20 tubes containing embryo lysates
in SDS-fix buffer.
Before use, these tubes can be kept for several days or weeks at -20°C in the freezer.
Proteins from the different extracts are resolved by SDS-PAGE and transferred onto a membrane.
Western blot analysis is then performed using a primary antibody directed against
the phosphorylated form of the catalytic subunit alpha of the protein phosphatase 1.
Times are indicated in minutes after fertilisation.
On the upper panel, white arrows indicate the different times when synchronous
cleavages are observable under a microscope.
First cleavage arises at two hours after fertilisation, and the second and third
cleavages, respectively, at three and four hours post-fertilisation.
The signal obtained after the chemiluminescence reaction﻿
corresponds to the phosphorylation of PP1C.
While it is absent 60 minutes after sperm addition, the signal increases 10 minutes
later until the first cleavage time.
The signal is hardly detectable during the first cleavage,
then the signal oscillates with each cell cycle.
The phosphorylation level of PP1C provides a read-out of the CDK1 global activity.
Therefore, we can conclude that CDK1 activity oscillates with the cell cycle.
The lower panel corresponds to a Western blot using a primary antibody
that recognises the protein CDK1 specifically.
We can observe here that the protein level of CDK1 is constant during the experiment.
Therefore, taken together, these data show that in sea urchin embryos cell cleavage is
preceded by a wave of CDK1 activity, independently of any variation of the
global level of CDK1 protein.
You now know how to monitor CKD1 activity that occurs in vivo following fertilisation.
As you have seen, you can use the phosphorylation of PP1C as a readout
of the CDK1 activity.
This non-radioactive method is very interesting because it can be performed
quite easily, for instance with students during a practical session.
Therefore you should remember that it is possible to monitor CDK1 activity that
occurs in vivo following fertilization.
This is possible because egg divisions occur rapidly and are synchronous
for an egg population.
And finally, that the large number of eggs that can that we can obtain from a single
sea urchin female allows this kind of biochemical approach.
One can now imagine different approaches that would make it possible to analyse
signalling pathways that act upstream of the CDK1/cyclin B activity.
You can also imagine how to test different drugs that specifically target CDK1 activity
and monitor their effect on cell cycle.
This is important for understanding the cell's life in physiological conditions and
physio-pathological situations such as cancer.