Isabelle Kehrli
Investigating centriole duplication in human cells:
Supervision: Nicola Jane Brown, EPFL
Introduction:
In the week at the EPFL in Lausanne we had the aim to investigate if the CPAP is required for
centriole duplication (Kohlmaier et. al 2009). The main questions were:
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What happens when we delete a sequence (PN-N) in the CPAP protein?
Is the PN-N region required for CPAP function?
The CPAP protein is built inside of the nucleus and is important for the cell cycle because it is
required for centriole and centrosome duplication. Two centrioles build the bricks for centrosomes
which are the poles of the spindle apparatus. During the cell cycle between the G1- and G2-phase the
centriole and the centrosomes are duplicated. CPAP apparents the first time between the G1- and
the S-Phase. I will not assume the cell cycle in this content.
Furthermore CPAP can cause human disease. But how and why it can come to this is still not
investigated enough. One disease which is known is microcephaly. Microcephaly is a rare
neurological condition in which the human head is smaller than the head of other people of the same
age and sex. These people are not in condition to live at their own. In order microcephaly can be
caused by other environmental factors.
Methods:
In the laboratory we worked with several methods which I am going to explain in the following
passage:
Cell Lines
We created three different cell lines which we wanted to investigate during the week. All of them
were built of human osteo sarcoma cells (U2OS) which are human cancer cells.
The first cell line was a normal U2OS cell line. We did not change anything.
In the second cell line we implanted a plasmid with the sequence from a normal CPAP protein in
order to this there was also a GFP sequence so that we could localise the protein under the
microscope.
Isabelle Kehrli
In the last cell line we implanted also a plasmid but this time we deleted the PN-N region.
But there was still the endogenous CPAP sequence which
normally builds the CPAP protein. We made it defective with RNA
interference (RNAi). With Lipofectamine we put a small interfering
RNA inside the cell so that it can interrupt the transcription of the
CPAP protein. I will not assume the whole process because it is
too difficult to explain.
To localise the CPAP
Immunofluorescence.
recognize the epitope
fluorescent secondary
primary antibodies.
protein under the microscope we used
We added primary antibodies which
of the different centrosome proteins and
antibodies which are able to localise the
Results and Conclusion:
Under the microscope it was obvious that too much CPAP makes extra-long centrioles (Kohlmaier et.
al 2009). This is a proof that CPAP is somehow appropriate for the length of centrioles.
In the CPAP protein with the deleted PN-N structure we observed three different samples
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The centriole protein (POC 5) localises all the way along the structures, which means that
there has to be centrioles.
The POC5 localises only at the bottom of the centriole which means we do not know how
long this centrioles are.
There are some fibres which are unknown to us. They are abnormal long so that they are not
likely to be centrioles.
We know that the CPAP∆PN-N protein can still build centrioles. There are different structures and
some of them are not investigated enough.
How I already mentioned CPAP is also detected during the mitosis. Because of this we investigated
how many POC5 is contained in mitotic cells. Our results showed that cells which are treated with
CPAP RNAi contain fewer POC5 foci.
As conclusion we know there are fewer centrioles or POC5 cannot localise the centrioles.
Discussion:
GFP-CPAP and GFP- CPAP∆PN: Our exciting preliminary data shows that there is a difference
between GFP-CPAP and GFP- CPAP∆PN structures. We need to do further experiments to see if the
GFP- CPAP∆PN with only a POC5 focus are really centrioles.
CPAP RNAi: Our data shows that CPAP RNAi reduces the number or POC5 foci in U2OS cells. We need
to do further experiments to determine whether there are fewer centrioles or whether there is a
decrease in POC5 protein, or a problem with its localisation.
Isabelle Kehrli
Finally I want to speak thanks to my supervisor Nicola Brown. She has been a great mentor during
the week at the EPFL. She explained everything very well and answered always very patiently our
questions. I’ve learned a lot of new and interesting things.
In order I want to say thanks to “Schweizer Jugend forscht”. Without this organisation this week
would not be formatted and I would not have had the possibility to learn new things and to collect so
many new experiences.