roporated 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 treatment.
Two important caveats were noted during the course of these experiments. RAW cells exhibited a greater tendency than HeLa cells to accumulate exogenous proteins on the cellular surface irrespective of incubation (Figure 6A) or electroporation (Figure 6B). Despite this, all electroporated samples had increased internal protein (Figures 1, 2, 5B). Additionally, the membrane-associated protein disappeared over time. A second caveat was a small population of cells exhibiting a phenotype consisting of smaller, rounded cells loaded with high amounts of exogenous proteins in both control (incubated) and treated (electroporated) cells (Figure 6 C and D, incubation samples shown). The nuclei of these cells showed condensed chromatin based on DAPI staining, and filled the entirety of the cellular volume, suggestive of apoptosis. It is therefore possible that apoptotic cells (arising as a normal part of the cell cycle or due to harvest/treatment) have a propensity to absorb protein from the buffer.
To demonstrate that electroporated proteins were functional and could localize correctly inside the host cell, co-localization and protein-protein interaction between the Salmonella effector protein SspH1 and its known host target, protein kinase N1 (PKN1) 33 was shown. After electroporation, the physical interaction between SspH1 and PKN1 was verified by an affinity based immunoprecipitation followed by western blot (Figure 7A). Co-localization was also observed by confocal microscopy, which indicates the fluorophores are close enough spatially to overlap 34. After electroporation with 50 µg/ml SspH1, HeLa cells (Figure 7B) were fixed and stained for SspH1 (green) and PKN1 (red). At low laser power distinct foci (yellow) were observed in the nucleus indicating SspH1 and PKN1 were physically close enough to interact. This interaction has been established in the literature; however this study is the first to show co-localization in the nucleus.
Figure 1: Electroporation of GFP - HeLa or RAW 264.7 cells. Cells were incubated or