Influence of the cell wall on intracellular delivery to algal cells by electroporation and sonication

Abstract

To assess the cell wall's role as a barrier to intracellular delivery, wild-type Chlamydomonas reinhardtii algal cells and mutant cells lacking a cell wall were exposed to electroporation or sonication. Flow cytometry determined intracellular uptake of calcein and bovine serum albumin (BSA) and loss of cell viability as functions of electroporation transmembrane potential and acoustic energy. Electroporation of wild-type cells increased calcein uptake with increasing transmembrane potential, but delivered much less BSA. Electroporation of wall-deficient cells had similar effects on calcein uptake, but increased BSA uptake as much as 7.5-fold relative to wild-type cells, which indicated that the cell wall was a significant barrier to BSA delivery during electroporation. Sonication of wild-type cells caused calcein and BSA uptake at similar levels. This suggests that the cell wall barrier to BSA delivery can be overcome by sonication. Increased electroporation transmembrane potential or acoustic energy also caused increased loss of cell viability, where wall-deficient cells were especially susceptible to lysis. Overall, we believe this is the first study to compare directly the effects of electroporation and sonication in any cell type. Specifically, these findings suggest that electroporation primarily transports molecules across the plasma membrane because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell wall, because it nonspecifically disrupts cell-surface barriers.

Figure 1

Influence of the cell wall on intracellular uptake and cell viability as a function of transmembrane potential during electroporation. The normalized intracellular concentration of a small molecule, calcein (A), and a macromolecule, BSA (B), is shown versus the nominal, applied, maximum, transmembrane potential for two 1-ms electroporation pulses. For the same population of cells, the cell viability is also shown. The black and white symbols represent data from the wild-type and wall-deficient algal cells, respectively. The square and diamond symbols represent intracellular concentration and cell viability, respectively. These data show that although both cell strains took up similar amounts of calcein, wild-type cells took up significantly less BSA than wall-deficient cells.

Figure 2

Histograms of intracellular uptake on a per-cell basis measured by cell fluorescence representative of the data shown in Figure 1. Uptake of calcein (A, B) and BSA (C, D) is shown in wild-type (A, C) and wall-deficient (B, D) cells. In each graph, the gray curve represents fluorescence of control cells and the black curve represents fluorescence of electroporated cells. Intracellular fluorescence is reported in arbitrary flow cytometry units. Each histogram contains data from 20,000 cells.

Figure 3

Influence of the cell wall on intracellular uptake and cell viability as a function of transmembrane potential during electroporation. Uptake of calcein (A, B) and BSA (C, D) is shown in wild-type (A, C) and wall-deficient (B, D) cells. All cells exposed to electroporation have been categorized as (i) viable cells with uptake (black bar), (ii) viable cells without uptake (gray bar), (iii) non-viable cells (striped bar) and (iv) lysed cells (white bar). Note that the height of the black-plus-gray bars shows the overall level of cell viability. This figure was generated using data from Figure 1 that were reanalyzed using histograms like those shown in Figure 2. This analysis demonstrates that essentially all electroporated cells took up calcein, but only some cells took up BSA, where a larger fraction of wall-deficient cells took up BSA than wild-type cells.

Figure 4

Influence of the cell wall on intracellular uptake and cell viability as a function of energy exposure during sonication. The normalized intracellular concentration of a small molecule, calcein (A), and a macromolecule, BSA (B), is shown versus acoustic energy exposure. For the same population of cells, the cell viability is also shown. The black and white symbols represent data from the wild-type and wall-deficient algal cells, respectively. The square and diamond symbols represent intracellular concentration and cell viability, respectively. These data show that wild-type cells exposed to sonication take up large amounts of BSA, which contrasts with the lesser effects of electroporation, and that wall-deficient cells are easily killed by sonication and do not take up molecules at all. In (B), data are not shown for BSA uptake in wall-deficient cells, because (A) demonstrated that wall-deficient cells do not take up calcein and therefore are not expected to take up BSA either.

Figure 5

Histograms of intracellular uptake on a per-cell basis measured by cell fluorescence representative of the data shown in Figure 4. Uptake of calcein (A, B) and BSA (C) is shown in wild-type (A, C) and wall-deficient (B) cells. In each graph, the gray curve represents fluorescence of control cells and the black curve represents fluorescence of sonicated cells. Intracellular fluorescence is reported in arbitrary flow cytometry units. Each histogram contains data from 20,000 cells.

Figure 6

Influence of the cell wall on intracellular uptake and cell viability as a function of energy exposure during sonication. Uptake of calcein (A, B) and BSA (C) is shown in wild-type (A, C) and wall-deficient (B) cells. All cells exposed to sonication have been categorized as (i) viable cells with uptake (black bar), (ii) viable cells without uptake (gray bar), (iii) non-viable cells (striped bar) and (iv) lysed cells (white bar). Note that the height of the black-plus-gray bars shows the overall level of cell viability. This figure was generated using data from Figure 4 that were reanalyzed using histograms like those shown in Figure 5. This analysis shows that similar fractions of wild-type and wall-deficient cells took up calcein and BSA, but essentially no wall-deficient cells took up molecules.