Download Sarah Scriven - Biochemical Society

Understanding how the cell cycle regulator, WEE1, is removed from
a DNA replication checkpoint in plants.
Sarah Scriven
Biochemical Society summer studentship report 2009
Supervisors: Dr H.J. Rogers and Dr D. Francis, Cardiff School of Biosciences
Introduction: The cell cycle, which makes a proliferative cell competent to divide,
comprises four successive phases: mitosis (M), G1 (post mitotic interphase), S phase (DNA
Synthetic phase) and G2, post-synthetic phase. For proliferative cells to remain viable,
checkpoint mechanisms exist that detect, repair and normalize DNA replication. In animals,
checkpoints are sensitive blocks at specific points during the cell cycle in response to DNA stress. In plants,
WEE1, a negative regulator of the cell cycle, is an important component of the G2/M checkpoint; this kinase
phosphorylates cyclin dependent kinases (CDKs) at their tyrosine15 residues and prevents cells from entering
mitosis. In plant cells, WEE1 expression is induced by agents that cause DNA damage. The mechanism of
WEE1 up-regulation following checkpoint induction is currently unidentified but its expression increases
following hydroxyurea treatment and is mediated by the Rad-3 related kinase (ATR). How plant cells recover
from a cell cycle checkpoint is unknown and a current aim of the Cardiff lab is to determine how WEE1 is
removed upon checkpoint recovery. In budding yeast, SWE1 is hyperphosphorylated by CDC28-CLB and then
ubiquitinated. This is regulated by the SCF complex (containing SKP1, CULLIN, RBX1 and an F-box protein)
which catalyses the covalent attachment of ubiquitin residues to the SWE1 substrate. The focus of the work
carried out during the bursary was to examine the extent to which WEE1 is ubiquitinated in tobacco BY-2 cells
with and without induction of checkpoint control by hydroxyurea treatment.
Aims:
1. Pull down WEE1 from extracts of tobacco BY-2 cells, during and following release from, the
checkpoint controls and challenge the WEE1 protein with a ubiquitin (UBQ) antibody.
2. Pull down ubiquitinated proteins and challenge with a WEE1 antibody.
Mitotic Index (%)
Departures from the original proposal: Some methods from the original proposal were slightly changed and
some were expanded upon; during the cell synchrony protein extracts were made at specific points during the
cell cycle, additionally the mitotic index was measured at hourly intervals along with the size of cells in
interphase and mitosis using Sigma scan Pro 5 software. Measuring cell size was not an aim of the original
proposal; however these additional results have proved to be interesting for the Cardiff group. Secondly the work
focussed on using WEE1 antibody to pull down WEE1 protein; initially pull downs were tested with the same
WEE1 antibody and then with the UBQ antibody. Pulling down ubiquitinated proteins and challenging with
WEE1 was not attempted due to lack of time.
50
45
40
WT BY-2
WT BY-2 + HU
35
30
25
20
15
10
5
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Time (hours) following removal of aphidicolin
Fig (1). The mitotic index in Tobacco BY-2
cells synchronized with aphidicolin +/hydroxyurea treatment added immediately after
aphidicolin release:
- No HU treatment: mitotic peak at 9 hours,
with 43.3% of cells
- HU treatment: mitotic peak at 14 hours, with
19.3% of cells
The arrows indicate at what time protein
extracts were taken; times corresponded to S,
G2/M and G1 phase of the cell cycle.
Work carried out: Cell synchronisations: BY-2 cells were
synchronised with aphidicolin at G1/S; two synchronies were
carried out with and without the addition of hydroxyurea. The
mitotic index (percentage of cells in mitosis) was measured at
hourly intervals (Fig. 1) along with the size of cells in both
interphase and mitosis (Fig. 2). There was a substantial delay in the
mitotic peak for hydroxyurea treated cells; the peak was also much
lower than in the untreated synchrony, i.e. less cells undergoing
mitosis. This result is comparable to previous work undertaken in
the Cardiff lab which suggests that hyroxyurea induces the DNA
replication checkpoint by upregulating the expression of ATR
kinase, which is involved in the mechanism that leads to the
phosphorylation of WEE1, preventing the cell from entering
mitosis. However, the delayed peak in the HU treatment suggests
that cells do recover from checkpoint induction.
A two sample t-test was performed on the sizes of BY-2 cells
with/without HU treatment; a P value of ≤0.001 was given
indicating that the mean size of hydroxyurea treated cells was
significantly higher than that of untreated cells. Therefore treatment
with hydroxyurea not only induces the DNA replication checkpoint,
as shown by the delay in the rise of the mitotic index but also induces an increase in cell size which has not been
formerly reported as an effect of hydroxyurea treatment.
Western Blots: Western blots were used to analyse
the amount of protein present at S, G2/M and G1
phase of the cell cycle. The protein was extracted at
times indicated in Fig. 1, throughout the two
synchronisations with and without hydroxyurea
treatment. WEE1 was then pulled down from the
protein extracts and subsequently probed with both
WEE1 and UBQ antibodies (Fig. 4A and 4B).
B
5000
Average cell size (µm 2)
A
4000
3000
2000
1000
0
WT Interphase
WT Mitosis
HU Interphase
HU Mitosis
The western blot in Fig. 4A illustrates the
efficiency of the pull down using WEE1 antibody.
The arrow shown corresponds to the approximate
size of WEE1 protein at 57KDa. Unfortunately the
Western was complicated by the likely presence on
the gel of the antibody used to pull down the
WEE1. This will in future have to be optimised
further. The next step was to pull down WEE1 and
probe it with UBQ antibody; Fig. 4B however,
lacks resolution compared with Fig. 4A as
problems were encountered whilst using the UBQ
antibody and blocking with BSA. This western
probed with a UBQ antibody indicates further
Fig. (4) Western blots of pulled down WEE1 from protein extracts
optimization is now required. Unfortunately there
corresponding to S, G2/M and G1 phase of the cell cycle with and
was not enough time to do this during the 8- week
without hydroxyurea treatment. Controls left of border, HU to the
placement. However the result should not be
right. (A): probed with a WEE1 antibody; (B): probed with UBQ
dismissed because there is, perhaps, a faint band at
antibody
the approximate size of 54KDa that is likely to be
WEE1 protein. Furthermore there is a much stronger signal at G2/M in the hydroxyurea treatment, which could
be because hydroxyurea has induced increased ubiquitination of WEE1 than that induced in the control. Thus the
results gained have provided the ground work for optimization of this experiment to discover the levels of WEE1
ubiquitination during the cell cycle. This will be carried out by a current PhD student in the Cardiff lab.
/M
G1
S
G2
/M
G1
S
G2
S
G2
/M
G1
S
G2
/M
G1
Fig. (2A). HU treated BY-2 cells stained with inflorescence, 8 hours
after aphidicolin release. A cell in metaphase of mitosis can be seen in
the centre of the photograph.
Fig. (2B). The mean size of tobacco BY-2 cells in interphase and
mitosis with and without hydroxyurea treatment.
My experience: On completion of my placement I feel it is important to stress how much more confident I have
become in the lab environment, not only with the new techniques I have used such as extracting proteins and
western blotting but with basic skills that are crucial to any scientific research; calculating and making specific
quantities of stock solutions, along with the frequent usage of the micro pipettes are good examples. This will
give me a real advantage in any further practical based work I chose to do. In addition I found the problem
solving part of the placement invaluable, when a procedure did not go to plan (as was the case with many of my
western blots), I had to think logically and critically about why a problem might have occurred…was it the
concentration of blocking solution I used? Could it have been the contamination of one of my solutions? Had a
chemical component of the buffers I used expired? All questions I had to ask in order to get the results I wanted.
I have really loved the challenge this placement has offered me, and am now definitely considering staying on at
university to complete an MSc or even a PhD.
Value of studentship to the department: This Biochemical Society funded project has been extremely useful
to the group of Drs Rogers/Francis in that the work undertaken by the student on both pull downs and antibody
blots has been completed to a very high standard. Finding the right conditions for such blots is notoriously
difficult and time -consuming; the work achieved has been invaluable to us.