te antibodies Note: Here, anti-PKN1 primary antibody is used.
As an initial proof of concept, purified green fluorescent protein was successfully introduced into mammalian cells using electroporation. GFP, an approximate 27 kD a molecular weight protein is commonly introduced into mammalian cells (normally expressed from plasmid DNA) as a molecular biology tool without significant cellular toxicity. HeLa cells were incubated (Figure 1A) or electroporated (Figure 1B) with 25 µg/ml GFP, followed by immunofluorescence confocal microscopy to check for fluorescent GFP signal. To demonstrate that the GFP was inside the cellular cytosol, the cells were also stained with wheat germ agglutinin (WGA) to delineate the cytosolic boundary (red) as well as a nucleic acid stain, DAPI (blue) to indicate the nucleus. Intracellular GFP signal was observed only upon electroporation, while no intracellular protein was observed in cells incubated with GFP. Similar results were observed with RAW264.7 mouse macrophage-like cells (Figure 1C and 1D). Note that WGA is a lectin that preferentially binds to residues in the plasma membrane and thus can exhibit punctate staining based on the length of incubation. Compare Figure 1A and Figure 2A, where Figure 2A was incubated for a longer duration than Figure 1A.
Electroporation was then extended to a tagged, purified Salmonella effector, GtgE. GtgE, a known virulence factor 30, was discovered by our group to be secreted into host cells 31, and was recently shown to be a cysteine protease 32. HeLa cells were incubated or electroporated with 50 µg/ml GtgE. For immunofluorescence analysis, the cells were stained with an antibody against the streptavidin-binding peptide affinity tag on GtgE. The cells were also stained with WGA and DAPI. In incubated HeLa cells (Figure 2A), there is no intracellular GtgE as visualized by a lack of green fluorescent foci. In contrast, electroporated HeLa cells (Figure 2B) show significant fluorescent intracellular signal indicating effector protein had entered the cells due to the electroporation process. RAW cells showed a slightly increased propensity to accumulate protein on the cellular surface during incubation (Figure 2C), but intracellular signal was seen only upon electroporation (Figure 2D).
Determining the limits of detection for visualizing sub-cellular localization via confocal microscopy was important to ensure enough protein entered the cell, while avoiding overloading the cell with potentially toxic bacterial effectors. In Figure 3, GtgE was electroporated into RAW macrophage-like cells at 2.5, 25, and 50 μg/ml. The cells were then fixed and stained with an antibody against the tag on GtgE. Only a very small number of foci were observed at 2.5 μg/ml GtgE (Figure 3A), with increased numbers of foci apparent at 25 μg/ml, (Figure 3B), but the greatest number of distinct foci were seen at the maximum protein concentration tested, 50 μg/ml (Figure 3C). Similar staining patterns were observed between the samples indicating at these protein concentrations intracellular protein could be easily verified by confocal microscopy. Protein concentrations above 50 μg/ml were not tested because as the concentration of protein increased, so did the propensity for the target protein to become adsorbed to the surface of the cells. To avoid these aggregates of membrane associated protein, it became necessary to pool two electroporation cuvettes to obtain enough host interacting protein to visualize on a western blot (Figure 6, far right lane).
To further demonstrate that the introduced protein was not simply adsorbed to the surface of the cell, consecutive optical sections were imaged at focal planes every 0.35 to 0.43 micrometers (Z-stacks) using confocal microscopy. RAW cells were electroporated with 50 µg/ml GtgE and stained as described above (Figure 4, A and B). The Z-stacks showed that GtgE was indeed intracellular and the foci extend from the bottom (Figure 4A, plane 6 of 36) to the top of the cell (Figure 4B, plane 26 of 36). Similar results were obtained for all elect