The epithelial to mesenchymal transition (EMT) is an important process that epithelial cells undergo in some conditions, in which epithelial cells lose their epithelial characteristics and change into mesenchymal cells. My work primarily consists of verifying the effect of TGF-β under different conditions using the immunofluorescence method. The most important point to consider is the possibility of cells being damaged by UV light.
In this project I aim to observe the effect on epithelial cells under different doses of UV exposure to identify toxicity of UV light. Keywords: Epithelial to mesenchymal transition (EMT), transforming growth factor beta (TGF-β), Optogenetics, UV light illumination.
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LIST OF COMMONLY USED ABBREVIATIONS
1 INTRODUCTION
Research Background and key question
The epithelial cells are classified according to the number of layers and the shape of the cells. More specifically, the cytoplasmic region of cadherin binds with β-catenin, and then β-catenin binds to α-catenin. Another class of intercellular junctions are tight junctions (TJ), which are located at the apical region of cells.
In other words, for a cell to be epithelial requires internal determinants (i.e. the ability to interact with other cell and create an epithelium) and external determinants given by the presence of neighbor to interact with. These EMT-TFs lead to repression of the epithelial genes and activation of the mesenchymal phenotype in EMT. Cytoskeleton makers including vimentin which are highly expressed in mesenchymal cells are alternative biomarkers to detect invasiveness of the cells [23].
2 THEORETICAL DEVELOPEMEMT
Sociological Perspectives
On the other hand, red nuclei are indicators to show that the cells are illuminated with UV light. One type of epithelial cells, Madin-Darby canine kidney (MDCK) cells, are modified as cells containing a SnailERT2 fusion protein called MDCK-Snail-ERT2. Snail proteins, as I mentioned in the previous section, are fused to a part of the estrogen receptor 2 (ERT2), which does not contain the DNA binding domain, but binds to the hormone and translocates to the nucleus when bound [35].
If there is no ligand to bind with ERT2, the fusion proteins are sequestered in the cytoplasm of the cell bound to hsp90. Next, using cage system regulated by UV illumination (365nm) is a useful way to control the EMT expression. In general, caged molecules are designed not to bind to their receptors, but when uncaged by UV light, they do.
In this case, a caged component is used that freely passes through the cell membrane, while the uncaged version does not, and is therefore trapped inside the cell. One method to pre-calibrate the effects of UV light is to check the photoconversion of a closed version of p-nitrophenol, which is known to follow similar photoconversion kinetics. By fusing the Dendra2 protein to histone H2B, photoconverted cells can be effectively tracked by screening for cells containing red nuclei [ 38 ].
Herein I want to validate the possible toxicity caused by different doses of UV lighting. Furthermore, I wanted to verify that the UV illumination conditions used in the induction of EMT via caged systems are not lethal to the cells. The EMT-induced cells with TGF-β and cyclofen show an elongated morphology (B-C) compared to normal cells (A).
The epithelial cells are represented by the expression level of the adherens junctions, E-cadherin (D-F) and tight junctions, ZO-1(J-L).
3 MATERIALS AND METHODS
- Cell culture
- Reconstituting TGF-β
- TGF-β induction
- Long-term imaging
- ImageJ analysis
- Immunofluorescence
- Antibodies
- Image acquisition
- Photoactivation
- Flow cytometry
The IncuCyte ZOOM® Live cell imaging system (Essen BioScience) is a well-known long-term modality for live cell imaging. Cells plated on a regular 24-well cell culture plate or ImageLock 24-well microplate (cat. #4365, Essence BioScience) were incubated in the incubator for 2-3 days. Cells were treated with blocking buffer (3% BSA and 0.2% Triton X-100 in PBS) on the shaking platform for one hour at room temperature.
Cells were incubated on the rocker at room temperature for one hour and then rinsed once rapidly with wash buffer (0.2% BSA and 0.05% Triton X-100 in PBS) and washed twice with wash buffer on the rocker for 5 min each. Finally, cells were fixed again with fixation buffer (3% PFA + 0.1% .GA) at room temperature for 5 min. Secondary antibodies: 1 mg of Alexa Fluor® 647 carboxylic acid (Cat. #A20106, Life Technologies) was dissolved in anhydrous dimethyl sulfoxide (DMSO) and dispensed into a sterile E-tube for storage at -20℃.
A 50 µl aliquot of unconjugated secondary antibodies was incubated with 8 µl 1 M sodium bicarbonate (NaHCO 3 ) and 0.8 µl Alexa Fluor® 647 for approximately 30 minutes at room temperature on the shaking platform. The reaction solution was passed through a Nap-5 gel filtration column (cat GE Healthcare), washed three times with 1X PBS. After the last droplet was drawn onto the top of the filter, the fastest moving colored band was collected in a new sterile E-tube.
A 365 nm collimated LED light source (cat#.M365L2-C4, Thorlabs) was used along with other optical components to construct a simple UV illumination platform. After illumination, cells were washed once with prewarmed PBS and trypsinized in an incubator (37°C, 5% CO2) for 7 min. The pellet was gently resuspended in a volume of 500 µl - 1000 µl FACS solution (PBS + 2% FBS) and washed/centrifuged three times with fresh FACS solution.
To achieve good compensation, three types of samples (Wild-type MDCK, only green MDCK-Snail-ERT2 H2B-Dendra2 and red MDCK-Snail-ERT2 H2B-Dendra2) were applied before flow cytometry analysis.
4 RESULTS
Validation of different EMT inducing conditions
- Correction of misaligned time-lapse images through ImageJ software
- Comparison study on the different EMT inducing conditions
- Optimazation of EMT markers through immunofluorescence
Compared to the normal state, cell area tends to shrink under starvation conditions (Figure 4.4 A and D). As shown in Figure 4.3 G and J, the effect of TGF-β does not show any major difference. The green fluorescent detection channel in IncuCyte is effectively used to detect the expression of the GFP-like signal, which is useful for counting the number of nuclei conjugated to Dendra2 photoswitchable proteins.
The IncuCyte software automatically measures confluence from the total surface area occupied and the number of nuclei on all images. Similar to confluency measurement, I verified that the IncuCyte software counts the number of nuclei correctly by comparing its performance with a manual quantification of counted nuclei. The plated cells with different initial concentration also show a slightly different trend as shown in Figure 4.6 and Figure 4.7.
Same as expected from phase images, cell confluence decreases for starved cells (Figure 4.6 C and D), but remains stable in normal condition (Figure 4.6 A and B). In addition, the average area of individual cells is quantified by calculating the ratio of confluency to the number of nuclei. The graphs in Figure 4.8 indicate the area of a single cell, calculated by the total cell area divided by the number of nuclei.
In Figure 4.8, the average single-cell surface area decreases over time under both starvation and normal conditions. After normal, the single-cell area is slightly expanded, regardless of TGF-β (Figure 4.8 A and B). The third row shows the results of confluency and number of nuclei under the IncuCyte analysis program.
The total cell area divided by the number of nuclei represents the average area of a single cell. Compared to F-actin, another mesenchymal maker of Vimentin expression (Figure 4.9 Q-T) is difficult to explain the effect of EMT. In addition to EMT marker antibodies, the normal cellular marker of Mitochondria (Figure 4.9 I-L) and Tubulin (Figure 4.9 M-P) help to understand the elongated morphology of cells.
Photoswitchable fluorescent protein in the photoactivation event
- Experimental distribution of homogeneous UV light
- Validation of photoconversion effect on fluorescent protein, Dendra2
- Quantification of photoconversion effect with flow cytometry
- Measurement of apoptosis in different UV illumination time
The bright field images (First row) help to find the same spot after continuous UV light exposure. Depending on increased exposure time, the GFP-like signal of Dendra2 disappears (Second row) and the RFP-like signal of Dendra2 appears (Third row). The GFP-like signal and the RFP-like signal of Dendra2 decrease and increase, respectively, as UV light illumination time passes.
Four different batches of cells irradiated with different doses (0 J/cm 2 , 13.5 J/cm 2 , 40.5 J/cm 2 and 81 J/cm 2 ) are prepared and each sample is analyzed via flow cytometry. As shown in Figure 4.13, the green-red pair of channels (FITC-A and PE-A channels) is used to analyze the different population patterns. There is no RFP-like signal before UV illumination (Figure 4.13 A) and most populations of cells have GFP staining.
When cells are exposed to UV light, flow cytometry analysis data reveal that the GFP-like signal shifts to RFP-like signal. As a result, it is interesting to note that the RFP-like signal is always accompanied by the GFP-like signal. In other words, a small fraction of cells do not undergo photoconversion even if all cells are illuminated.
The exact reason why the Dendra2 protein does not respond to UV light is unknown, so further study of Dendra2 is needed. Using the same UV illumination parameters described in the previous section 4.2.3., MDCK-Snail-ERT2 H2B-Dendra2 cells are irradiated with different doses of UV light and apoptosis is then observed. In Figure 4.14, each row shows different doses of UV light and each column shows the time course after UV light irradiation.
To better define the UV light dose threshold for apoptosis, a well-known experiment should be performed based on the fact that cells bind Annexin when they enter apoptosis.
5 DISCUSSION
The effect of TGF-β on cellular morphology using long-term imaging
Response to TGF-β of EMT related proteins
Unfortunately, overt E-cadherin activation and repression is hardly seen, probably due to the titration of the 2nd antibody. On the other hand, the expression of tight junction marker, ZO-1 appears in untreated cells and disappears upon TGF-β induction. In the case of Vimentin, I do not see variations with TGF-β and this can be explained by the fact that cells are only exposed to TGF-β for 24 hours.
In the present case, cells are incubated and observed here after 48 h, because the Dendra2 photoconversion signals used to track photo-induction fade after 48 h. Therefore, other photoconversion proteins should be considered not only to increase the incubation time of TGF-β but also to track the photoconversion signals.
Toxicity of time-dependent UV light illumination
6 CONCLUSION
7 REFERENCES
The Snail genes as inducers of cell movement and survival: implications in development and cancer. Depletion of E-Cadherin disrupts establishment but not maintenance of cell junctions in Madin-Darby canine kidney epithelial cells. Loss of E-cadherin is not a necessity for epithelial-to-mesenchymal transition in human breast cancer.
