Part:BBa_K1391112

pEXPR_Tret:Cofilin (Inactivated - pseudophosphorylated)

Cofilin is an actin depolymerizing factor. It is recruited when PirB/LilrB2 is activated by beta-amyloid. [1]

The 3rd amino acid in this construct has been mutated to make it pseudophophoralated and inactive. It was ordered from Adgene. In this construct, cofilin is under the inducible TreT promoter. It is activated in the presence of doxycyline.

[1] Taeho Kim, George S. Vidal, Maya Djurisic, et al. "Human LilrB2 is a beta-amyloid recptor and its murine homolog PirB regulates synaptic plasticity in an Alzheimer's Model". Science. 20 Sep 2013. <http://www.sciencemag.org/content/341/6152/1399>. 16 Oct 2014.

The characterization of the system this is part of can be found in the experience page.

Sequence and Features

Assembly Compatibility:

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1: Localization of receptors to the cell membrane

In the first preliminary experiment, we aimed to determine if the native receptors localized to the cell membrane. This is important to ascertain since the receptors would be used to detect beta-amyloid oligomers in the extracellular matrix of the brain. The system must therefore be able to detect the oligomers outside the cell and relate this information inside the cell.

To determine localization of the receptors, we used LilrB2 and PirB specific antibodies to immunostain for the receptors. We analyzed the immunostained samples in two ways. The first was through flow cytometry analysis which would allow us to determine if the antibodies, and in turn the receptors, were on the cell surface since the cells were not permeabilized. We also confocal microscopy to look at the immunostained samples in order to visualize membrane localization and determine subcellular localization, if any, in permeabilized cells.

For samples that were to be analyzed by flow-cytometry, we transiently transfected HEK293 cells with plasmids encoding constitutive expression of the LilrB2 and PirB under the hEF1a promoter along with hEF1a:mKate2 as a transfection marker. We then treated cells with primary antibodies specific to the receptors being expressed and secondary antibodies specific to the species of the primary antibody. The secondary antibodies were conjugated to a yellow AlexaFluor which allowed us to detect them using the flow cytometer.

LilrB2 Results

1a.	1b.	1c.	1d.
Figure 1: Flow cytometry data showing distribution of fluorescence for LilrB2 membrane localization experiment. Cells were transfected with plasmids encoding LilrB2 and mKate2 under constitutive promoter (hEF1a). Two days after transfection, cells were immunostained with a mouse primary antibody specific to LilrB2 and a donkey anti-mouse secondary conjugated to a yellow AlexaFluor 488. a. Untransfected, unstained HEK293 cells. b. Immunostained untransfected HEK293 cells. c. Immunostained HEK293 cells transfected with hEF1a:mKate2. d. Immunostained HEK293 transfected with hEF1a:mKate2 and hEF1a:LilrB2.

PirB Results

2a.	2b.	2c.	2d.
Figure 2: Flow cytometry data showing distribution of fluorescence for PirB membrane localization experiment. Cells were transfected with plasmids encoding PirB and mKate2 under constitutive promoter (hEF1a). Two days after transfection, cells were immunostained with a goat primary antibody specific to LilrB2 and a donkey anti-goat secondary conjugated to a yellow AlexaFluor 488. a. Untransfected, unstained HEK293 cells. b. Immunostained untransfected HEK293 cells. c. Immunostained HEK293 cells transfected with hEF1a:mKate2. d. Immunostained HEK293 transfected with hEF1a:mKate2 and hEF1a:PirB.

In our initial trial of this experiment, we saw an increase in yellow fluorescence between untransfected cell populations and transfected ones. The interesting result was that we saw similar amounts of yellow fluorescence between cells that were transfected with just hEF1a:mKate2 and those transfected with both hEF1a:mKAte2 and the receptor DNA and that the data showed a very strong one-to-one correlation between yellow and red fluorescence. This led us to believe that our results were actually stemming from bleedthrough of the mKate2 fluorescent protein into the FITC channel used to detect yellow fluorescence.
To address this problem, we decided to not use a transfection marker since all of the fluorescent proteins we had available to us would produce the same, if not a greater bleedthrough effect.

LilrB2 Results

3a(i).	3b(i).
3a(ii).	3b(ii).
Figure 3: Histogram showing distribution of yellow fluorescence in cell populations for the LilrB2 membrane localization experiment. Cells were transfected with plasmids encoding LilrB2 under a constitutive promoter (hEF1a). Two days after transfection, cells were immunostained with a mouse primary antibody specific to LilrB2 and a donkey anti-mouse secondary conjugated to a yellow AlexaFluor 488. Figures (i) and (ii) are biological replicates of the experiment. a. Untransfected, unstained HEK293 cells. b. Immunostained untransfected HEK293 cells. c. Immunostained HEK293 cells transfected with hEF1a:LilrB2.

PirB Results

4a(i).	4b(i).
4a(ii).	4b(ii).
Figure 4: Histogram showing distribution of yellow fluorescence in cell populations for the PirB membrane localization experiment. Cells were transfected with plasmids encoding PirB under a constitutive promoter (hEF1a). Two days after transfection, cells were immunostained with a goat primary antibody specific to PirB and a donkey anti-goat secondary conjugated to a yellow AlexaFluor 488. Figures (i) and (ii) are biological replicates of the experiment. a. Immunostained untransfected HEK293 cells. c. Immunostained HEK293 cells transfected with hEF1a:LilrB2.

By analyzing the mean yellow fluorescence for the stained cell populations, we saw an increase in yellow fluorescence between the cell populations that were not transfected and those that were expressing the receptor. This indicates that the receptors are getting expressed on the cell surface.

In our second method, we looked for membrane localization through confocal microscopy in order to visualize membrane localization and other, subcellular localization, if any. To do this we, again, transfected HEK293 cells with DNA encoding constitutive expression of the receptors and hEF1a:eYFP as a transfection marker. Our choice of transfection marker here was not important since any fluorescence would be quenched when the cells were fixed. We used the transfection marker to determine if the transfection efficiency was high enough before we proceeded with the immunostaining. After transfecting, we then fixed the samples and stained them with the same selection of antibodies we used for the flow cytometry analysis as well as DAPI to stain the nucleus for better visualization of the cells.

LilrB2 Results

5a.	5b.
Figure 5: Confocal microscope images of HEK293 cells expressing LilrB2 constitutively. Cells were fixed and immunostained with LilrB2 specific mouse primary antibody and a yellow AlexaFluor 488 conjugated donkey anti-mouse secondary antibody. a. Untransfected, immunostained HEK293 cells. b. Immunostained HEK293 cells transfected with hEF1a:LilrB2.

PirB Results

6a.	6b.
Figure 6: Confocal microscope images of HEK293 cells expressing PirB constitutively. Cells were fixed and immunostained with PirB specific mouse primary antibody and a yellow AlexaFluor 488 conjugated donkey anti-goat secondary antibody. a. Untransfected, immunostained HEK293 cells. b. Immunostained HEK293 cells transfected with hEF1a:PirB.

In the resulting images, we see a clear increase in yellow fluorescence between cells that were transfected with the receptors and those that were not. However, we do not see any clear localization to the membrane. Results of further experiments suggest that the receptors might be getting overexpressed, given the mass of receptor DNA we were transfecting and the fact that we were using a strong constitutive promoter to express the receptors.

Further experimentation: Microscopy on serial dilution of the receptor to determine at what transfected mass of the receptor, do we get membrane localization.

2: Beta-amyloid binding to receptors

In this experiment we aimed to determine whether or not the native receptors in our system were, in fact, binding to beta-amyloid oligomers. To do this, we transfected HEK293 cells with plasmids encoding our native receptors and hEF1a:eBFP2 as a transfection marker. We then treated the cells with biotinylated beta-amyloid oligomers and red AlexaFluor-conjugated streptavidin. If our receptors bound to the beta-amyloid, the streptavidin would, in turn, bind to the biotin on the beta-amyloid oligomers leading to a higher level of red fluorescence. Similar to the first experiment, we analyzed the cells using both flow cytometry and confocal microscopy, looking for increased red fluorescence in cell populations that were transfected with the receptors.

LilrB2 Results:

Figure 7: Fluorescence of HEK293 cells transfected with LilrB2 and eBFP2 under constitutive promoter (hEF1a) and stained with biotinylated beta amyloid and red AlexaFluor (594) conjugated streptavidin. Blue HEK293 cells transfected with hEF1a:eBFP2 and stained with beta amyloid and streptavidin. Red HEK293 cells transfected with hEF1a:eBFP2 and hEF1a:LilrB2 and stained with streptavidin only. Black HEK293 cells transfected with hEF1a:eBFP2 and hEF1a:LilrB2 and stained with beta amyloid and streptavidin.

PirB Results:

Figure 8: Fluorescence of HEK293 cells transfected with LilrB2 and eBFP2 under constitutive promoter (hEF1a) and stained with biotinylated beta amyloid and red AlexaFluor (594) conjugated streptavidin. Blue HEK293 cells transfected with hEF1a:eBFP2 and stained with beta amyloid and streptavidin. Red HEK293 cells transfected with hEF1a:eBFP2 and hEF1a:LPirB and stained with streptavidin only. Black HEK293 cells transfected with hEF1a:eBFP2 and hEF1a:PirB and stained with beta amyloid and streptavidin.

The cell population transfected with PirB shows a marked increase in red fluorescence which indicates that PirB is in fact binding abeta. Moreover, when the distribution of red fluorescence of the transfected population (gated using the blue fluorescence distribution) is plotted, you see a shift in the distribution towards more red fluorescence indicated by the increase in the mean from 171 in the PirB -ve population to 1069 in the PirB +ve population.

We also analyzed the cells through confocal microscopy to visualize where in the cell beta amyloid was binding. In a similar way to the experiment where we were tested localization, we were not concerned by bleedthrough of our transfection into our output channel because fluorescence would be quenched when the samples were fixed. We transfected plasmids encoding our native receptors and hEF1a:eYFP as a transfection marker. We then fixed the cells and treated them with biotinylated beta amyloid oligomers and red AlexaFluor conjugated streptavidin as well as DAPI staining the nuclei. Initially, we tried live staining the cells, i.e. without fixing them. However, we found that it was difficult to maintain the cells in a healthy condition through the staining process and that is why we switched to fixing the samples.

LilrB2 Results:

9a.	9a.	9a.
Figure 9:Confocal microscope images of HEK293 cells expressing LilrB2 constitutively and fixed and stained with biotinylated beta amyloid and red AlexaFluor (594) conjugated streptavidin.9a. Untransfected HEK293 cells stained with beta amyloid and streptavidin. 9b&c. HEK293 cells transfected with hEF1a:LilrB2 and stained with beta amyloid and streptavidin.

PirB Results:

10a.	10b.	10c.
Figure 10:Confocal microscope images of HEK293 cells expressing PirB constitutively and fixed and stained with biotinylated beta amyloid and red AlexaFluor (594) conjugated streptavidin.10a. Untransfected HEK293 cells stained with beta amyloid and streptavidin. 10b&c. HEK293 cells transfected with hEF1a:PirB and stained with beta amyloid and streptavidin.

The images show that there is a large amount of background binding of streptavidin indicated by the large amount of red fluorescence in the negative controls. There doesn’t seem to be a large increase in red fluorescence between the negative control with the cell population that isn’t expressing the LilrB2 and the cell population that is expressing the receptor. This might be due to the fact that only small subset of cells were shown to bind beta amyloid as was shown by the flow cytometry data. It is unlikely that the images we captured actually included one of the cells that was binding beta amyloid. However, in one of the images of the cell population expressing LilrB2, we can see a cell that shows more red fluorescence than others surrounding it. This may be a cell with beta amyloid bound to it.

PirB

Cells expressing PirB showed similar staining results as cells expressing LilrB2 in that there was little, if any, increase in fluorescence between cells expressing the receptor and those that weren’t expressing the receptor. Again, this may be due to the small proportion of cells that actually showed beta amyloid binding.

Further experimentation: FACS sort cells that showed a large red fluorescence in flow cytometry and stain that population of cells.

3: Evaluating relative quantities of endogenous cofilin and exogenous TEVp-cofilin

In this experiment, we want to compare the levels of endogenous cofilin and TEV protease (TEVp)-Cofilin expressed under an inducible promoter with different levels of dox induction. This will give us an indication as to what level of expression of cofilin would lead to the best signal:noise ratio of exogenous cofilin to endogenous cofilin. To do this we transfected HEK293 cells with DNA encoding inducible expression of our TEVp-Cofilin fusion construct and hEF1a:eYFP as a transfection marker as well as constitutive reverse tetracycline-controlled transactivator (rtTA) needed for doxycycline induction. We added different concentrations of doxycycline. We analyzed the cell lysates by probing for cofilin in a Western blot analysis.
We used an antibody specific to cofilin to probe for both cofilin and TEVp-Cofilin. The difference in size between the endogenous cofilin and the exogenous TEVp-Cofilin allow us to distinguish between the two on the Western blot, and hence compare their relative quantities. We also probed for GAPDH and eYFP in the Western blot. We used GAPDH as a loading control to allow us to normalize for the amount of protein loaded onto the protein gel. In a similar way, probing for eYFP allowed us to normalize for the transfection efficiency.

Protein	Size
Cofilin	18.5 kDa
TEVp-Cofilin	48.6 kDa
GAPDH	37 kDa
eYFP	27 kDa

For this Western blot analysis, we ran a standard SDS-Page, where we denatured the the proteins in the cell lysates. Based on the sizes of the proteins we were probing for, the expected results for the Western blot were as follows:

Results:

Trial 1

Because in the first trial, the antibody that we were using to probe for GAPDH resulted in a lot of nonspecific binding, we decided to not use it in the second trial. We also decided to only use the blocking buffer that gave us the least nonspecific binding of the antibodies

Trial 2

In both trials of this western blot, we don’t get any signal corresponding to to cofilin or TEVp-Cofilin. Our research indicates that HEK293 cells have a considerable amount of endogenous cofilin (cofilin is sometimes used as a loading control for Western blots). This may indicate that the antibody we chose to probe for cofilin may not be binding in sufficiently high quantities to give a signal under the conditions we ran the Western blot under.

Further experimentation: try running the western blot under different conditions; cofilin antibody positive control; look into another coffin antibody; look into another coffin antibody.

4: Inactive cleavage and release of transcription factor

In this experiment we wanted to investigate how much background cleavage of the TEV cleavage site (TCS) would occur under different levels of expression of the TEVp-Cofilin fusion protein, without activation of the receptors by beta-amyloid. Cleavage of the TCS would result in the release of the transcription factor, Gal4VP16, activating the Gal4 responsive promoter Gal4UAS driving the expression of mKate2, which acts as the reporter.

To do this we transfected TEVp-Cofilin under a TRE promoter, the receptor-TCS-Gal4VP16 fusion, eBFP as a transfection marker, Gal4UAS:mKate2 as a reporter and hEF1a:rtTA. To vary the level of expression of the TEV protease-Cofilin fusion, we treated the cell population with different concentrations of doxycycline inducing different levels of expression of TEVp-Cofilin. We measured the fluorescence of the different cell populations using flow-cytometry, such that red fluorescence indicated the extent of activation of the UAS promoter.

LilrB2 Results:

Figure 13: Fluorscence of HEK293 cells transfected with LilrB2-TCS-GAL4VP16, Gal4UAS:mKate2, TRE:TEVp-Cofilin and hEF1a:eBFP as a transfection marker. BlackHEK293 cells transfected with all components except the -TCS-Gal4Vp16. RedHEK293 cells transfected with all components except the TRE:TEVp-Cofilin. BlueHEK293 cells transfected with all components with maximum doxycycline induction.

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Figure 14: Fluorscence of HEK293 cells transfected with LilrB2-TCS-GAL4VP16, Gal4UAS:mKate2, TRE:TEVp-Cofilin and hEF1a:eBFP as a transfection marker. BlackHEK293 cells transfected with all components with no doxycycline induction. RedHEK293 cells transfected with all components with 1nM doxycycline. BlueHEK293 cells transfected with all components with 10nM doxycycline.GreenHEK293 cells transfected with all components with 1000nM doxycycline.

From these results we can see that there is a large amount of background cleavage of the TEV cleavage site. There also doesn’t seem to much increase in output as we increase the concentration of dox introduced to the system. This might be due to the high background which would make it difficult to notice a difference in output as TEVp-Cofilin expression increases.

PirB Results:
LilrB2 Results:

Figure 15: Fluorscence of HEK293 cells transfected with LilrB2-TCS-GAL4VP16, Gal4UAS:mKate2, TRE:TEVp-Cofilin and hEF1a:eBFP as a transfection marker. BlackHEK293 cells transfected with all components except the -TCS-Gal4Vp16. RedHEK293 cells transfected with all components except the TRE:TEVp-Cofilin. BlueHEK293 cells transfected with all components with maximum doxycycline induction.

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Figure 16: Fluorscence of HEK293 cells transfected with LilrB2-TCS-GAL4VP16, Gal4UAS:mKate2, TRE:TEVp-Cofilin and hEF1a:eBFP as a transfection marker. BlackHEK293 cells transfected with all components with no doxycycline induction. RedHEK293 cells transfected with all components with 1nM doxycycline. BlueHEK293 cells transfected with all components with 10nM doxycycline.GreenHEK293 cells transfected with all components with 1000nM doxycycline.

The controls for the inactive cleavage experiment for PirB, in a similar way to LilrB2, show that there is a large amount of background Gal4UAS:mKate2 activation in the absence of TEVp-cofilin. Here, we do see an increase in red output as we increase the concentration of doxycycline. It is interesting to note that the transfection efficiency in this experiment was a lot lower than that in the LilrB2 experiment. This means that the amount of receptor being expressed in the cells here is likely lower, enabling us to see the difference in output between cells with different levels of expression of TEVp-Cofilin.
In this experiment, we aimed to determine what levels of both TEV protease-cofilin fusion expression and native receptor transfection would result in the largest signal-to-background difference in output. We repeated Experiment 4, and added beta-amyloid to the cells to activate the receptors. The results of Experiment 4 indicated that we were transfecting an amount of receptor-TCS-gal4VP16 DNA that resulted in high levels of background UAS activation. To address this problem, we decided to transfect a range of amounts of receptor-TCS-gal4VP16 construct, while simultaneously varying expression of TEV protease-Cofilin construct. Ultimately, optimizing the signal to background ratio of a system is a time-intensive process that involves multiple iterations of experiments, but with this experiment we were able to make a start at characterization.Again, we measured the fluorescence of the cell populations using flow-cytometry. We looked for an increase in red fluorescence from populations that were not treated with beta-amyloid to those that were.

5: Active cleavage

In this experiment, we aimed to determine what levels of both TEV protease-cofilin fusion expression and native receptor transfection would result in the largest signal-to-background difference in output. We repeated Experiment 4, and added beta-amyloid to the cells to activate the receptors. The results of Experiment 4 indicated that we were transfecting an amount of receptor-TCS-gal4VP16 DNA that resulted in high levels of background UAS activation. To address this problem, we decided to transfect a range of amounts of receptor-TCS-gal4VP16 construct, while simultaneously varying expression of TEV protease-Cofilin construct. Ultimately, optimizing the signal to background ratio of a system is a time-intensive process that involves multiple iterations of experiments, but with this experiment we were able to make a start at characterization.
LilrB2 Results:

PirB Results:

In every case that we tested, the amount of system output was higher for the non-activated cells than for the activated cells, which was the opposite of what we were expecting. Though the exact mechanism behind this discrepancy remains unclear, it is possible that activation of our receptor causes secondary, unintended effects that affect the cell's ability to produce our output. Further investigation will be required to determine the mechanism behind this effect.

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