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