Osmotic Effects Induced by Pore-Forming Agent Nystatin: From Lipid Vesicles to the Cell

<div><p>The responses of Chinese hamster ovary epithelial cells, caused by the pore-forming agent nystatin, were investigated using brightfield and fluorescence microscopy. Different phenomena, i.e., the detachment of cells, the formation of blebs, the occurrence of “cell-vesicles” and cell ruptures, were observed. These phenomena were compared to those discovered in giant lipid vesicles. A theoretical model, based on the osmotic effects that occur due to the size-discriminating nystatin transmembrane pores in lipid vesicles, was extended with a term that considers the conservation of the electric charge density in order to describe the cell’s behavior. The increase of the cellular volume was predicted and correlated with the observed phenomena.</p></div

Concentration and osmolarity changes in the cell after the formation of 9 × 10⁷ nystatin pores with a 0.45-nm radius.

<p>The cellular osmolarity is depicted by full line and the sum of the cellular osmolarities (concentrations) of the most abundant ions by dashed line. The changes were predicted by the numerical procedure according to the model (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165098#pone.0165098.s001" target="_blank">S1 File</a>).</p

Sample images of calcein-loaded cells treated with 200 μmol/L nystatin.

<p>Images of calcein-loaded cells treated with 200 μmol/L nystatin. The images were taken using confocal microscopy at different time points after the nystatin addition (indicated on the images).</p

Maximal volumes of single cells, characteristic time behavior patterns of the cell volume changes and ratios between the cell volumes at different phases of the measurement.

<p>(a) Maximal volumes of single cells (<i>V</i>_max) relative to their volumes before the treatment (<i>V</i>(0)) at 150 (○, n = 11), 300 (□, n = 11), 400 (×, n = 13) and 600 (■, n = 11) μmol/L nystatin concentration. Average value (dashed line) of single cell measurements and standard deviation (dotted lines) are shown. (b) Three characteristic time behavior patterns of volume changes in cells treated by nystatin: “type A” (○), “type B” (×) and “type C” (■). The patterns for single cells with their maximum values closest to 2.9 are presented. As a control, a characteristic behaviour of “methanol only” treated cell at 6% volume fraction is shown (●). The lines are drawn only as a guide to the eye. (c) Ratios between the cell volume at the end of the measurement and in its middle phase (<i>V</i>₃/<i>V</i>₂) versus ratios between the cell volumes in the middle and in the beginning phase of the measurement (<i>V</i>₂/<i>V</i>₁) for the same cells as in (a). The volumes at different phases are defined through the expressions <i>V</i>₁ = [2<i>V</i>(5 min) + <i>V</i>(10 min)]/3, <i>V</i>₂ = [<i>V</i>(20 min) + 2<i>V</i>(30 min) + <i>V</i>(40 min)]/4 and <i>V</i>₃ = [<i>V</i>(50 min) + 2<i>V</i>(60 min)]/3. (d) Ratios between the increases of single cell volumes in the beginning of the measurement and their volumes before the treatment ((<i>V</i>₁-<i>V(0)</i>)/<i>V(0)</i>) versus their volumes before the treatment for the same cells as in (a).</p

Organization of the actin in blebs.

<p>In “living” cells the actin is organized in small (a) and bigger blebs (b), as depicted by fluorescent signal. Some representative blebs are indicated by arrows.</p

Predicted changes in the cell volume and in the tension-pore radius.

<p>The changes in the cell volume (a) and in the tension-pore radius (b) are shown for different numbers of nystatin pores 2.5 × 10⁷ (dash-dot line), 9 × 10⁷ (dashed line) and 3 × 10⁸ (full line), characteristic for type-I, type-II and type-III tension-pore behavior. The dotted line indicates the critical volume of the cell. The cell volume is normalized to its initial volume (<i>V</i>/<i>V</i>₀) and the tension pore radius to the radius of the cell (<i>R</i>_TP/<i>R</i>_c). The radius of the nystatin pores is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165098#pone.0165098.g007" target="_blank">Fig 7</a>. The numerical procedure is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165098#pone.0165098.s001" target="_blank">S1 File</a>.</p

The characteristic types of tension-pore behavior.

<p>The three types of tension-pore behavior as a function of the radius and the number of nystatin pores (<i>r</i>_NP and <i>N</i>_NP). The dashed line shows the border between the type-II and type-III tension-pore behavior for a ten-times-smaller lysis tension.</p

Organization of fluorescent actin structures in “living” cells as seen using confocal microscopy after the addition of 300 μmol/L of nystatin solution.

<p>Top view (in the middle) and side views (bottom and right) of the nystatin treated cells along the thin lines are presented at different time points as indicated (first row). As a control, the actin structures after the “methanol only” treatment at 3% volume fraction are shown (second row). The step size was equal to 0.5 μm, while the number of images in the stack was 86.</p

Characteristic behavior of GUVs at different nystatin concentrations.

<p>The GUVs show different behavior due to the pore-formation process in vesicles with smaller glucose molecules outside and larger sucrose molecules inside, as seen using phase-contrast microscopy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165098#pone.0165098.ref026" target="_blank">26</a>]. Development of typical membrane protrusions (indicated by arrows) due to the nystatin binding to the outer membrane monolayer at low nystatin concentrations (a). Loss of vesicle contrast due to transient tension pores at intermediate nystatin concentrations (b). Slow (c) and fast (d) vesicle ruptures at high nystatin concentrations: GUVs before the rupture (left), during the rupture (middle, tension pore indicated by arrow), and immediately after the rupture (right) are shown. The times after the GUV transfer into the nystatin solution are depicted on the images. The bars represent 20 μm. For more detailed descriptions see Refs. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165098#pone.0165098.ref026" target="_blank">26</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165098#pone.0165098.ref029" target="_blank">29</a>].</p

Slow (upper row) and fast (lower row) “cell-vesicle” ruptures as observed by the brightfield microscopy at a 400 μmol/L nystatin concentration.

<p>The left-hand-side images represent the “cell-vesicles” right before the rupture, the middle ones during the rupture and the right ones after the rupture. The times after the nystatin addition are indicated on the images. The white bars represent 20 μm.</p