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Note: Tables, appendix and figures
of the article can be accessed and seen in the PDF file.
Introduction
Autophagy is an intracellular degradation process
for worn out organelles or protein aggregates 4. It can
be activated by nutrient availability, hormones and
intracellular pathogens. Autophagy takes place by a
phagophore (double membrane structure) surrounding a
section of the cytoplasm, containing what is to be
degraded, and producing an autophagosome. The
autophagosome ultimately fuses with lysosomes containing
hydrolytic enzymes and acids, increasing the acidity
inside. This results in degradation of the proteins in
question (fig 1). A protein which is unique to the
phagophore and autophagosome membrane is LC3
(microtubule associated protein 1, light chain 3)5. LC3
binds, after post-translational modification, to the
inner and outer membrane of the autophagosome making it
a good candidate to be used as a marker for autophagy.
The marker LC3 is tagged with a red fluorescent protein
(mRFP) and a green fluorescent protein (EGFP). The mRFP
is stable under acidic conditions; i.e. when a lysosome
fuses with the autophagosome and releases its acidic
content. Whereas the EGFP is stable at the start of the
maturation process but loses its fluorescence under
acidic conditions caused when the lysosome fuses with
the autophagosome 3. A shift in colour from green to red
therefore should indicate autophagy progression. These
two colours can then be compared to assess autophagy
maturation and overall activity in a cell.
In neurodegnerarative diseases, such as Huntington’s,
neurotoxicity is brought about by expanded glutamine
repeats on the huntingtin protein within neurons 1.
There is debate over whether it is the large
polyglutamine aggregations which cause the toxicity or
whether they are just the end product of many more
soluble toxic proteins. Either way autophagy has been
shown to break down both types of proteins and so has an
important protective function in normal cells 2.
Upregulation of autophagy could also be beneficial in
patients with Huntington’s disease 7. All the research
done so far shows the importance of autophagy and the
need for further research to thoroughly characterise the
whole process both physiologically and
pathophysiologically.
Kimura has shown the functioning of the protein marker
mRFP-EGFP-rLC3 in mammalian cells and since there have
been strong effects of rapamycin in mice Huntington’s
models, it would be useful to create a system in vivo to
assess the different stages of the autophagy process. To
do this we wanted to create a Drosophila line able to
express the mRFP-EGFP-rLC3 in vivo. To achieve this we
decided to use the ФC31 based integration system. The
vector of which can be seen in figure 2 below. This new
system of transgenesis uses site specific recombination
by the use of the bacterial and plasmid attachment sites
to integrate at a specific site into the Drosophila
genome 8 (fig.2). It has also been adapted to have an
endogenously expressed integrase which increases the
transgenesis efficiency.
After the marker has been inserted correctly into the
vector ФC31 pUAST attB the DNA sequence can then be sent
for analysis to check for mutations, correct insertion
and orientation of the autophagy marker insert. Once
this has proved successful the construct can be tested
in Drosophila embryonic cells for green/red/yellow
expression of the construct.
Results
We wanted to clone the construct mRFP-EGFP-rLC3 into the
pUAST attB vector. The construct maker was originally in
a mammalian plasmid. The mRFP-EGFP-rLC3 was excised from
the plasmid by double digestion using EcoR1 and NheI and
inserted into the vector which was cut open by EcoRI.
The digested mammalian plasmid was then resolved by gel
electrophoresis and the mRFP-EGFP-rLC3 was then removed
and purified from the gel.
Out of 80 transformed E-Coli we found three inserts of
mRFP-EGFP-rLC3 including one insertion of the correct 5’
to 3’ orientation. A series of DNA digestions were
carried out throughout the protocol which were analysed
by gel electrophoresis. The vector’s original size was
approximately 8.5Kb and the insert was approximately
2Kb. Therefore the expected molecular size was 10.5Kb,
which can be seen in figure 3 below.
Once the bacterial E-Coli cells were transformed, plated
out and lysed as outlined in the methods section below,
their DNA needed to be analysed to check for the
presence of an insert and afterwards the correct
orientation.
The DNA content from different colonies was resolved
using gel electrophoresis. The results shown (fig.3)
indicate that there are two inserts present, as seen by
the slower running larger DNA fragments in the lanes
indicated. The difference in weight between the two
bands should be approximately 2Kb which is the
approximate size of the insert.
The orientation of the insert can then be determined by
using restriction enzymes to cut both the plasmid and
insert at specific sites and then by careful analysis.
We used BglII, as outlined in the methods below, which
cuts once inside the vector and also once in LC3. This
would result in two fragments of DNA being produced if
the insert was present, including a smaller and larger
fragment if the insert was correctly orientated 5’ to 3’
or more medium sized fragments if there was a 3’ to 5’
orientation as explained diagrammatically in figure 5
below:
Figure 5 shows the result of our DNA digest using gel
electrophoresis to separate out the fragments. It can be
seen that batch 23 contains the correctly inserted
fragment while 24 contains the incorrectly inserted
fragment.
After we had ascertained which colony contained the
correctly orientated insert we amplified and purified
the corresponding DNA, to send it to sequence and
eventually for microinjection. We sent the DNA off to be
sequenced by making a maxi DNA prep and sending in the
relevant primers as outlined in the methods. The
complete result of the DNA sequence can be seen in the
appendix. Figure 6 below shows a sample of the DNA
sequence showing the correct annealing, alignment and no
evidence of mutation. The presence and orientation of
the construct can also be checked by using BLAST
annealing program (www.ncbi.nlm.nih.gov/blast/) as seen
in the same figure below.
We confirmed by sequence that the colony number 23 was
5’-3’ orientated and there were no mutations.
The next step was to test for expression of the
construct using Drosophila DMel cells, an S2 derived
embryonic cell line. The cells were transfected with the
pUAST attB vector and the actin-Gal4 driver, to drive
the expression of the mRFP-EGFP-rLc3 construct. Before
harvesting, cells were treated with different compounds
to increase, or block autophagy. In particular,
bafilomycin (Baf) and rapamycin (Rap) as outlined in the
methods. The cells were then analysed using florescence
microscopy. Figure 7 below shows the expression of the
EGFP and mRFP in cells bathed in DMSO. As well as
testing for general expression of the insert we tried to
examine how the insert responds under different
conditions. The two extra conditions we used were with
bafilomycin and rapamycin. Rapamycin increases the
activity of autophagy and autophagosome maturation by
inhibiting mTOR. We made the prediction that due to the
increased acidity, because of the increased levels of
autophagy maturation, the cells should appear more red
than green. We also used bafilomycin which inhibits the
ATPase of the lysosome causing its acidity to decrease
by increasing the diffusion of H+ ions out of the
lysosome. The cells were then analysed looking at the
different fluorescence to check for expression of the
vector. In parallel we checked if we were able to
reproduce in Drosophila cells the data published using
mammalian cells 3.
The results shown in figure 7 shows that with rapamycin
there is a slight increase in the amount of red
fluorescence but with bafilomycin some cells showed
increased red fluorescence while some showed not much
change when compared to the basal levels with just DMSO.
However, these results are not conclusive.
Discussion
The results of the DNA digestions shown above indicated
that an insert was present and correctly orientated in
sample 23 (fig 4 & 5). On digestion with BglII the DNA
will be cut just after the insert in the vector and
between the EGFP and the rLC3 (fig.4). A prediction can
therefore be made that there should be two bands when an
insert is present in the vector. Furthermore, while in
the 5’-3’ orientation the bands produced should be 947
bases and 9951 base pairs in length. This can be seen on
the gel with sample 23, with a very fast running band
and a very slow running band. When the insert is
orientated 3’-5’ two fragments of 9475 bases and 1423
bases should be produced as seen by sample 24 (fig.5).
This was then proved by the DNA sequence analysis shown
above (fig.6). The sequence from BLAST in figure 6 shows
the sequence perfectly aligning at the beginning of mRFP
and ending with rLC3. It also shows the kozak sequence
just upstream of the mRFP. There is also a poly A tail
just downstream of the rLC3 ensuring that the mRFP, the
EGFP and the rLC3 will be translated as one unit. This
is important for the functioning of the marker as it
means the florescent tags will be incorporated with the
rLC3 within the autophagosome membrane.
When the construct was tested in Drosophila embryonic
cells, expression of the two colours red and green were
seen, showing that the construct was being expressed
correctly. The construct was also tested to see how it
performed under the influence of bafilomycin and
rapamycin. Bafilomycin is an ATPase inhibitor causing a
loss of H+ from the lysosome and ultimately inhibiting
autophagosome maturation. In contrast to this, rapamycin
inhibits mTOR resulting in activation of autophagy. The
application of these two drugs should therefore produce
different fluorescence. Both drugs should produce more
red due to increasing autophagy activity with Rap and
increasing acidic dispersal with Baf. However, on
analysis of the photos produced by the confocal
microscope as seen in figure 7, no conclusive remarks
can be made either way regarding the different levels of
autophagy induced by the applied drugs. This is because
of multiple reasons. Firstly, not enough magnification
was used to view individual puncta, representing
individual autophagocytic vesicles. Without this level
of magnification the precise localisation of the marker
cannot be observed resulting in more noise and less
contrast. Secondly, different cells treated with
bafilomycin produce conflicting results. Some were more
green when compared to just DMSO while some were more
red. This is probably due to not enough bafilomycin
reaching all of the observed cells and can be explained
by dose differences. Thirdly, there is a possibility of
saturation of some of the fluorescent makers in some of
the observed cells. Fourthly, the EGFP in the cells is
being expressed everywhere and especially in the
nucleus. This is due to the EGFP becoming incorporated
into the nucleus and bringing the rest of the autophagy
marker with it. This means that in order to analyse the
photos produced, the noise from the nucleus needs to be
ignored and discarded. In order to rectify these
difficulties and produce conclusive significant results
we could repeat the confocal microscopy with an
increased magnification to focus in on individual puncta
within each cell. Moreover it could be that the chosen
cells where highly fluorescence, but actually not that
appropriate to analyse the maturation of the autophagic
vesicles. A higher magnification would allow for
localisation of our marker signal and would be less
affected by the noise produced by expression of EGFP
from the nucleus. Another possibility could be to create
a dose response to the different treatments. It is
highly possible that the dosage of drugs used in
mammalian cells could not be appropriate for Drosophila
cells. A dose response curve should be organised to try
and ascertain what concentration of both rapamycin and
bafilomycin are required to produce conclusive results.
This could be undertaken by producing many more cell
cultures each bathed in increasing concentrations of the
drugs. Only once these improvements have been undertaken
can the suitability of the mRFP-EGFP-rLC3 marker in
Drosophila cells can be realised.
Once the suitability of this marker for autophagy is
confirmed, further research can also help to decipher
exactly how autophagy is involved in the pathology of
Huntington’s, especially concerning what type of
proteins are degraded and how are they targeted.
Methods and Materials
Vector insertion
The pUAST attB plasmid was kindly supplied by Konrad
Basler, while the mammalian plasmid containing the
mRFP-EGFP-rLC3 marker insert was kindly supplied by
Prof. Yoshimoni. The pUAST attB plasmid was linearised
by using EcoRI and the mRFP-EGFP-rLC3 insert was excised
by making a double digest of NHE1 and EcoRI as shown in
the diagram 9 below. All the restriction enzymes and
buffers were supplied by New England BioLabs (NEB) and
the manufacturer’s protocol was followed throughout.
Unfortunately corresponding sticky ends could not be
created by using restriction enzymes and so we proceeded
to blunt the ends by using T4 DNA pol (NEB). 100 µM
dNTPs were added and the solutions were left for 30
minutes at room temperature before adding 1 µl 10mM EDTA
to inhibit the T4 DNA pol. The solutions were then
heated to 70°C to denature the T4 DNA pol. Before the
DNA could be ligated the mRFP-EGFP-rLC3 marker had to be
separated from the rest of the digested mammalian
plasmid. This was done by resolving via gel
electrophoresis (8% agarose) and then extracting the
relevant band using QIAGEN MiniElute Gel Extraction kit
(catalogue number 28604). The manufacturer’s protocol
was followed. The insert and vector were then ligated
using T4 DNA ligase following the manufacturer’s
protocol. As explained above in the results, a ratio of
3:1 insert to plasmid was used to ensure efficient
ligation between the insert and vector. Before a
ligation could be carried out the relative quantities of
the insert and vector had to be ascertained. This was
done by running a selection of both on a gel and
comparing the relative intensities of the band produced.
It was estimated that the band for the insert is
10ng/µl. This knowledge enabled us to proceed with a
ligation in the ratio of 3:1 knowing that 10 µl of the
insert contained 100 ng of DNA. Due to the insert being
a third of the length of the vector, 100ng of each
provided the 3:1 ratio we were looking for. Buffer 2
(NEB) was used for both NheI, EcoRI and T4 DNA pol while
T4 DNA ligase buffer was supplied together with T4 DNA
ligase by NEB.
DNA Transformation
5 µl of the ligation was then added to 50µl of super
competent DH5 E-Coli and left to thaw for 30 minutes.
The bacteria were then heat shocked to open up pores on
the surface membrane to facilitate the transformation
for ten minutes. They were then placed in ice for two
minutes before being placed in nutrient broth for one
hour at 37°C. The bacteria were then plated out on an
agar medium infused with 100µg/ml ampicilin. They were
then left to form colonies overnight. A colony was then
taken by pipette and added to 2ml of broth which was
then left to culture at 37°C.
DNA Mini Prep
Half, i.e. 1 ml, of each colony in broth was then
microcentrifuged to return the bacteria, and the
supernatant as discarded. The E-Coli were then lysed
using the QIAprep Spin Miniprep kit from QIAGEN
(catalogue number 27106) for the first 40 cultures. For
the remaining 40, a mini DNA prep was undertaken using a
method produced by Zhou 10.
Insertion Analysis
The presence of an insertion was detected by running on
an 0.8% agarose gel by gel electrophoresis. 0.8% was
chosen to resolve the large fragments with enough
resolution and in a small enough time frame. Vectors
containing an insert were approximately 2Kb heavier and
this was detected by a slower moving band on the gel. A
DNA ladder was also used to allow quantification of the
weights of the different bands of DNA separated by the
electrophoresis. The ladder was purchased from (NEB)
The orientation was elucidated by digesting the insert
using BglII (NEB) with buffer 3 (NEB), following the
manufacturer’s protocol, and analysed as explained in
the results, discussion and diagram 4 above.
DNA Sequencing
The DNA was then prepared to be sent for sequencing, to
Geneservice Limited, by undertaking a Maxi DNA Prep.
This was undertaken by using a QIAGEN Plasmid Maxi kit
(catalogue number 12662) and the protocol was followed
as per the manufacturer’s guidelines. Relevant primers
were also sent which primed into the multiple cloning
site of the vector pUAST attB a forward primer pUASTF
(5’-GACTCTGATAGGGAATTG-3’) and a reverse primer Puastr
(5’-AATACACAAACATTATAC-3’) (MWG-Biotech). The following
programs were used in the analysis of the DNA sequence;
BLAST (www.ncbi.nlm.nih.gov/blast/), Workbench (http://workbench.sdsc.edu/)
and Chromas (http://www.technelysium.com.au/chromas.html).
Testing in Cell Culture
The pUAST attB construct containing the insert was
cotransfected with actin Gal4 into Drosophila D.Mel-2 S2
embryonic cells. 9 wells were used; 3 to be used for A
Western blot and 6 to be used for florescence
microscopy. 3 Million Drosophila cells were in each
well. Cellfectin from Invitrogen was used to help form
micelles with DNA to attach to the cell membranes. These
were then left to incubate at 37°C overnight. Before
harvesting the cells to be used for fluorescence
microscopy were treated with either Dimethyl Sulfoxide (DMSO),
a final concentration of 0.2µg/ml rapamycin, or a final
concentration of 400nM bafilomycin. The cells were then
rinsed with PBS and mounted using a drop of VECTASHIELD
(Vector), which is a mounting medium with DAPI to stain
nuclei.
Fluorescence Microscopy
The prepared slides were visualised using a camera
equipped fluorescent confocal microscope (Nikon Eclipse
E800) at 40x magnification. Red, green and yellow colour
analysis was performed using ImageJ software (http://rsb.info.nih.gov/ij/).
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