 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 electroporated proteins. The intrinsic fluorescent profile of control cell populations with and without effector protein or electroporation was examined by fluorescent confocal microscopy, and was found to be negligible.
Additionally, the electroporated protein was examined to see if it was targeted for degradation via endocytic pathways. Cells were stained for Ras-related protein 5A (Rab5), a marker for early endosomes (Figure 4C), or Lysosome-associated membrane protein 1 (Lamp1), a marker for late endosomes and lysosomes (Figure 4D). Co-localization between the electroporated protein and these cellular markers would indicate a close physical interaction between them (i.e. that the electroporated protein was inside the endosomes/lysosomes). Confocal microscopy showed that the electroporated protein (GFP or GtgE) did not co-localize with LAMP1 or Rab5. It was inferred from this that introduced protein was not directly endocytosed into the cells or targeted to the endocytotic pathway within four hours of treatment.
Exogenous protein introduction can have cellular effects over the course of several hours or days, and thus it is often beneficial to examine the persistence of introduced proteins. To determine how long an electroporated protein would persist without degradation after electroporation. Based on visualization of attachment and cell spreading, 4 hr was determined to be the approximate minimum recovery time for electroporated cells. To temporally observe protein persistence a time-course experiment was performed, staining the cells 4, 24, or 96 hr after electroporation. After 4 hr (Figure 5A), when cell morphology suggested recovery, there was an appreciable amount of intracellular protein. After 24 hr (Figure 5B), there was still substantial electroporated protein inside the cells. Even when the time after electroporation was extended to 96 hr before fixation and staining (Figure 5C) there were observable foci albeit at a reduced apparent abundance, demonstrating protein persistence days after initial treatme