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. 2018 Oct;24(5):553-563.
doi: 10.1017/S143192761801509X.

Ultrastructural Analysis of Vesicular Transport in Electrotransfection

Liangli Wang  1 , Sara E Miller  2 , Fan Yuan  1
Affiliations

Ultrastructural Analysis of Vesicular Transport in Electrotransfection

Liangli Wang et al. Microsc Microanal. 2018 Oct.

Abstract

Emerging evidence from various studies indicates that plasmid DNA (pDNA) is internalized by cells through an endocytosis-like process when it is used for electrotransfection. To provide morphological evidence of the process, we investigated ultrastructures in cells that were associated with the electrotransfected pDNA, using immunoelectron microscopy. The results demonstrate that four endocytic pathways are involved in the uptake of the pDNA, including caveolae- and clathrin-mediated endocytosis, macropinocytosis, and the clathrin-independent carrier/glycosylphosphatidylinositol-anchored protein-enriched early endosomal compartment (CLIC/GEEC) pathway. Among them, macropinocytosis is the most common pathway utilized by cells having various pDNA uptake capacities, and the CLIC/GEEC pathway is observed primarily in human umbilical vein endothelial cells. Quantitatively, the endocytic pathways are more active in easy-to-transfect cells than in hard-to-transfect ones. Taken together, our data provide ultrastructural evidence showing that endocytosis plays an important role in cellular uptake and intracellular transport of electrotransfected pDNA.

Keywords: clathrin-mediated endocytosis; electroporation; electrotransfection; intracellular trafficking; macropinocytosis.

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Figures

Figure 1.
Figure 1.. Distribution of dig-pDNA in different cell lines at 10 min after electrotransfection.
The digoxin labeled pDNA in cells was detected by immunostaining prior to TEM processing. All images in the figure have low contrast, because we purposefully chose not to post stain the samples with uranium and lead in order to improve visual contrast between the EDS from pDNA and subcellular structures. As a result, we could reduce false positive signals in image analysis. (A to C) These low magnification electron micrographs show unilateral distribution of the EDS (arrows), which indicates initial locations of electrotransfected pDNA in (A) COS7 cell, (B) HT29 cell, and (C) 4T1 cell. Bars = 2 μm. (D to F) The high magnification micrographs show associations of the EDS with the subcellular structures in (D) HCT116 cell, (E) HT29 cell, and (F) HCT116 cell. In D, the thick arrow indicates the formation of a macropinosome, and the thin arrow indicates an early endosome-like structure. Bar = 500 nm. In E, the arrow points to the EDS intermingled with vesicles of different sizes. Bar = 100 nm. In F, the thick arrow indicates a membrane protrusion around an electron dense particle. Bar = 500 nm.
Figure 2.
Figure 2.. Subcellular features associated with dig-pDNA at 40 min after electrotransfection.
All images were acquired on COS7 cells. (A) The low magnification image shows the distribution of the EDS in the cell; n, nucleus. Bar = 1 μm. (B) A train of electron dense caveolae-like structures is visible. Black arrows indicate caveolae with membranes associated with the EDS. White arrows indicate caveolae partially filled with the EDS. Bar = 100 nm. (C) Typical caveolae-like structures are filled with the EDS indicated by arrows. Bar = 100 nm. (D) Two membrane invaginations are associated with the EDS. Thin and thick arrows indicate pits with smooth and rough membranes, respectively. Bar = 100 nm. (E) An early endosome-like structure and two late endosome/lysosome- like structures are associated with the EDS, as indicated by the thin and the thick arrows, respectively. Bar = 500 nm.
Figure 3.
Figure 3.. Effects of cell treatment with electric pulses on numbers of small vesicles.
COS7 cells in the buffer containing non-labeled pDNA were treated with five electric pulses (400 V/cm, 5 msec, and 1 Hz). Cells in the control groups were prepared with the same procedures except that the electric pulses were not delivered. At 10 or 40 min post treatment, membranous structures in cells were post-stained with uranyl acetate and lead citrate to enhance their contrast under the electron microscope (see the Materials and Methods section), but no immunostaining was performed for these cells. Subcellular structures of pits and vesicles (50 – 100 nm in diameter) were grouped together. The structures with smooth and rough membranes were considered to be clathrin- and caveolin-coated, respectively. The total numbers of the structures were counted in 10 different cells (i.e., one section per cell); and the data in the pulsed groups were normalized by the corresponding controls. Bars and error bars represent the mean and the standard error of the mean, respectively. *p < 0.05, Ctrl vs Pulsed.
Figure 4.
Figure 4.. Representative electron micrographs of pDNA-positive structures.
Morphological criteria were developed in the study for the identification of subcellular structures associated with pDNA. Some morphological features are shown in Figs. 1 and 2, respectively. Others are shown in this figure. (A) A membrane protrusion/ruffle with a closure is associated with the EDS in a COS7 cell, indicating that macropinocytosis is involved in pDNA uptake. Bar = 500 nm. (B) An early endosome-like structure with an electron-translucent lumen contains a few internal vesicles that are positive for the EDS in an HCT116 cell. Bar = 500 nm. (C) A large and (D) a relatively smaller late endosome/lysosome-like structures are positive for the EDS in COS7 cells. Bar = 1 μm. (E) A few electron dense aggregates can be seen in an HT29 cell; and each aggregate is surrounded by multiple vesicles. Bar = 200 nm. e, extracellular space.
Figure 5.
Figure 5.. Relative numbers of subcellular structures associated with dig-pDNA.
COS7 and 4T1 cells were collected at 10 or 40 min post electrotransfection, and examined under the electron microscope. At each time point, we arbitrarily selected electron micrographs from 40 different cells that were electrotransfected with dig-labeled pDNA, and 40 from the control group (Ctrl, where unlabeled pDNA was for electrotransfection). The selection was performed without prior knowledge of whether the selected cells contained the EDS. The total numbers of the subcellular structures associated with the pDNA in these cells were counted manually for each group. The results are reported as the total number of pDNA-positive structures per group normalized by the data of COS7 at 10 min. The data show that the numbers of pDNA- positive structures are higher in easy-to-transfect cells (COS7) than in hard-to- transfect cells (4T1). The numbers of false positive structures in the control groups were negligible, compared to the corresponding data in the experimental groups. Bars and error bars represent the mean and the standard error of the mean, respectively. *p < 0.05.
Figure 6.
Figure 6.. Percentages of different subcellular structures associated with dig-pDNA.
The raw data for this figure are the same as those for Fig. 5. Instead of reporting the total numbers, the figure shows the percentages of each subcellular structures that were associated with the EDS. The structures include surface complex (SC), macropinosome-like compartment (ME), early endosome-like compartment (EE), intracellular complex (IC), and late endosome-, lysosome-, or multivesicular body-like compartment (LLM). The details of the definition of each structure are provided in the results section. The data demonstrate that most pDNA molecules were located near the plasma membrane at 10 min. (A) In easy-to-transfect cells (COS7), a large fraction of pDNA was transferred to the inner vesicles at 40 min. (B) The transfer was minimal in hard-to-transfect cells (4T1). Bars and error bars represent the mean and the standard error of the mean, respectively. *p < 0.05, 10 min vs 40 min for each structure.
Figure 7.
Figure 7.. Subcellular structures associated with dig-pDNA in HUVEC and COS cells after electrotransfection.
(A) A portion of HUVEC with unilateral distribution of the EDS is indicated by the arrow at 10 min after electrotransfection. Bar = 2 μm. (B) Electron dense aggregates near the plasma membrane are shown to be surrounded by vesicles of various sizes (50–300 nm), indicated by the arrows, at 10 min after electrotransfection. Bar = 500 nm. (C) A train of electron dense caveolae-like structures is seen in a HUVEC at 10 min after electrotransfection. Bar = 100 nm. (D) A macropinosome has formed to engulf an electron dense particle (arrow) in HUVEC at 20 min after electrotransfection. (E) A portion of a HUVEC contains clusters of intracellular caveolae-like structures (arrowheads), and membrane invaginations (arrows) with the EDS at 40 min after electrotransfection. Bar = 500 nm. (F) An elongated tubular structure from the plasma membrane of a HUVEC is seen to be associated with electron dense particles at 20 min after electrotransfection. Bar = 500 nm. The boxed area with a high magnification is shown in Panel (G). Bar = 100 nm. (H) An early endosome-like structure is present in a HUVEC at 40 min after electrotransfection. Bar = 500 nm. (I) Tubular vesicular structures associated with the EDS in the cytoplasm of a HUVEC at 20 min after electrotransfection. Bar = 100 nm. (J) A Golgi-like structure is associated with the EDS in caveolae rosette-like vesicles in a HUVEC at 40 min after electrotransfection; n, nucleus. Bar = 500 nm. (K) A tubulovesicular structure, similar to that observed in HUVEC, is associated with the EDS in a COS7 cell at 40 min after electrotransfection. Bar = 100 nm.
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