Download Investigating elongated centrioles in human cells

March 2014
Investigating elongated centrioles in human cells
Gönczy Laboratory, School of Life Sciences, Swiss Institute for Experimental Cancer Research, École
Polytechnique Fédérale de Lausanne – EPFL, CH-1015, Lausanne, Switzerland
Iman Ibragic (Kantonsschule Olten), Isabelle Kehrli (Gymnasium Oberaargau)
Mentor:
Nicola Jane Brown
1. Abstract
We observed the effects of CPAP and the importance of PN-N region of CPAP on centriole
duplication and centriole growth. By using primary and secondary antibodies, we were able
to localise different proteins at centrioles. In our first experiment we observed the localisation of different centriole proteins at elongated centrioles caused by increased levels of
CPAP. We decided that POC5 would be a good protein to localize it because it localises along
the length of the elongated centrioles. The size of the localised proteins should tell us something about the size of the centrioles. By analyzing the cells with microscopy, we’ve seen
some anomalies: increasing the dose of CPAP leads to elongated centrioles and the CPAP
mutant in which the PN-N region is deleted leads to three different kinds of structures within
the cells, some of which are elongated centrioles.
2. Introduction
All cells which are capable of doing cell division have a centrosome. It organises microtubules, which are necessary to move the chromosomes apart during mitosis (figure 2). In the
centrosome, there are normally two centrioles; a mother and a daughter centriole. During
the cell cycle, the centrosomes and so the centrioles, duplicate.
figure 1. The centrosome consists of two centrioles, and
organises microtubules.
figure 2. The cell cycle in U2OS cells. Microtubules are labelled
in magenta by immunofluorescence with antibodies that
recognise alpha-tubulin. DNA is labelled in blue using Hoechst
dye that binds to DNA.
March 2014
The centrioles itself are made of different proteins. One of them, which is very important for
the mitosis, is the CPAP protein. CPAP is required for centriole duplication.1 We also know
that CPAP is important in brain development. A defect or a mutation of CPAP besides other
proteins causes (autosomal recessive primary) microcephaly;2 and overexpression of CPAP
leads to elongated centrioles.3
PN-N is the part of the CPAP which binds tubulin. We’ve deleted the PN-N and observed what happened.
The aim of the project:
Is the PN-N region required for CPAP function in centriole elongation?
3. Methods
RNAi protocol
1. 60 nMsiRNA (small interferingRNA) plus 4 μlLipofectamine RNAi Max transfection reagent in
2 ml final volume in each well (6-well plate). siRNAs used: control (non-specific sequence) or
CPAP (sequence specific to CPAP).
2. Remove cells (U2OS cells) from the flask using trypsin (enzyme that cuts the proteins that
the cells use to attach to the flask).
3. Collect the cells using fresh growth medium and count the cells.
4. Plate 120,000 cells in each well (with the Lipofectamine-siRNA mixture). Each well contained
2 glass coverslips in order to perform immunofluorescence microscopy.
Experiments:
1. U2OS cells + control siRNA
2. U2OS cells + CPAP siRNA (to deplete CPAP protein)
3. U2OS cells expressing GFP-CPAP + CPAP siRNA (to deplete endogenous CPAP protein,
but not the GFP-CPAP)
4. U2OS cells expressing GFP-CPAPΔPN-N + CPAP siRNA (to deplete endogenous CPAP
protein, but not the GFP-CPAPΔPN-N)
Immunofluorescence Protocol
5.
6.
7.
8.
Rinse cells with PBS
Fix in -20°C MeOH for 10 min
Rehydrate in PBS 0.01% Triton X-100
Permeabilise with PBS 0.2% TX for 20 min.
1
Kohlmaieret al. 2009
Bond et al. (2005); Gul et al. (2006)
3
Kohlmaier et al. (2009); Tang et al. (2009); Thorsten et al. (2009)
2
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9. Incubate with blocking solution (PBS, 2% FBS, 1% BSA), 1 hour incubation at room temperature.
10. Primary antibodies diluted in blocking solution and incubated for 1 hour at room temperature, or overnight at 4°C.
11. Wash with PBS 0.01% TX (x3).
12. Secondary antibodies diluted in blocking solution and incubated for 1 hour at room temperature.
13. Wash with PBS 0.01% TX (x3).
14. Incubate with PBS 0.01% TX, 1 μg/ml Hoechst. 2 minutes at room temperature.
15. Rinse coverslip with PBS then dH2O.
16. Mount coverslip onto a glass slide with mounting medium (glycerol) and seal the edge of the
coverslip with nail polish.
Fixation and permeabilisation are needed to allow antibodies to go inside the cell and bind
to the proteins that we want to look at. Antibodies cannot go through the cell membrane, so
we can’t use them to do immunofluorescence in living cells. Fixation using methanol works
by dehydrating the cells (removing the water), but the proteins stay in the same place as
when the cells were living (just that the proteins don’t function anymore and the cells are
dead).
Triton X-100 is a detergent.
PBS is Phosphate Buffered Saline
FBS is Foetal Bovine Serum
BSA is Bovine Serum Albumin
(BSA and FBS are used in the blocking solution as a source of non-specific protein in order to
reduce non-specific antibody interactions with proteins in the cells.)
dH2O is distilled water
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4. Results and Conclusions
10 μm
By immunofluorescence microscopy, we’ve decided to use POC5 antibodies to localise the
centrosome. On the images, we also can see, that elongated centrioles are caused by too
much CPAP. 4
GFP-CPAP
GFP-CPAP∆PN-N
10µm10 µm
Figure 3. control
GFP, POC 5 and merged
Figure 4. deleted PN-N
GFP, POC 5 and merged
POC5 localises all the way along the GFP structures. These structures are likely to be centrioles.
GFP-CPAP∆PN-N
POC 5 localises at the bottom of the strutures. GFP-CPAP∆PN-N fibres which do not
have POC5 staining are not likely to be centrioles.
We’ve also counted the POC5 foci in U2OS cells treated with control RNAi
or CPAP RNAi. CPAP is required for centriole duplication, so in order to
check that the RNAi was working we counted POC5 foci (figure 5). We
observed fewer POC5 foci in cells treated with CPAP RNAi compared to
the control (graphs on the next page).
Figure 5. POC5 foci mark
centrioles
4
Kohlmaier et al. (2009); Tang et al. (2009); Thorsten et al. (2009)
March 2014
We came to the conclusions
that
Number of elongated centrioles per cell
100%
80%
GFP-CPAP POC5
positive 59
60%
GFP-CPAPΔPN-N
positive 81
40%
20%
GFP-CPAP ΔPN-N
POC5 focus 92
0%
0
1
2
3
4
1. There must be fewer
centrioles, or
2. POC5 cannot go to the centrioles.
Number of POC5 foci per mitotic cell
50%
40%
30%
Control RNAi
n=63
20%
10%
0%
0
1
2
3
4
>4
5. Discussion
GFP-CPAP and GFP-CPAPΔPN-N Our exciting preliminary data shows that here is a difference
between GFP-CPAP and GFP-CPAPΔPN-N structures. We need to do further experiments to
see if the GFP-CPAPΔPN-N structures 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 furtherexperimentstodeterminewhethertherearefewercentriolesorwhetherthereis a decrease in POC5 protein, or a problem with its localisation.