Effect of osmotic stress on live cell plasma membranes, probed via Laurdan general polarization measurements

Abstract

Here we seek to gain insight into changes in the plasma membrane of live cells upon the application of osmotic stress using Laurdan, a fluorescent probe that reports on membrane organization, hydration, and dynamics. It is known that the application of osmotic stress to lipid vesicles causes a decrease in Laurdan's generalized polarization (GP), which has been interpreted as an indication of membrane stretching. In cells, we see the opposite effects, as GP increases when the osmolarity of the solution is decreased. This increase in GP is associated with the presence of caveolae, which are known to disassemble and flatten in response to osmotic stress.

Figure 1

(A) CHO cells at 90% media dilution with diH₂O. A region of interest from a cell (yellow) is analyzed, and the average Laurdan spectrum is calculated and shown in Fig 3A. (B) Laurdan spectrum from a single pixel within the yellow region. To see this figure in color, go online.

Figure 2

(A) Integrated fluorescence images of cells labeled with Laurdan, at different media dilutions with diH₂O. (B) Osmolarity of the solutions as a function of diH₂O content.

Figure 3

(A) Emission spectra of Laurdan in CHO cells at 0% and 90% media dilution. (B) Laurdan GP in CHO cells as a function of diH₂O media dilution. GP was calculated using Eq. 1. The notched box line represents the mean; the top and bottom represent the 75^th and 25^th percentiles, respectively, and the bars represent the standard deviations. To see this figure in color, go online.

Figure 4

(A) Emission spectra of Laurdan in CHO cells in normal media, as a function of temperature; RT in blue, 37°C in red, and 42°C in dark gray. (B) Laurdan GP as a function of temperature in CHO cells. (C) Emission spectra in CHO cells at 90% media dilution, as a function of temperature; RT in cyan, 37° in orange. (D) Laurdan GP as a function of temperature at 90% media dilution. The notched box line represents the mean; the top and bottom represent the 75^th and 25^th percentiles, respectively, and the bars represent the standard deviations. To see this figure in color, go online.

Figure 5

(A) Emission spectra of Laurdan in MEF (−/−) cells at 0% media dilution (dark gray) and 90% media dilution (dark green). (B) Emission spectra of Laurdan in MEF (+/+) cells at 0% media dilution (light gray) and 90% media dilution (mint green). (C) Comparison of Laurdan’s GP for MEFs (+/+) and MEFs (−/−) at 0% media dilution and 90% media dilution. The notched box line represents the mean; the top and bottom represent the 75^th and 25^th percentiles, respectively, and the bars represent the standard deviations. To see this figure in color, go online.

Figure 6

(A) Emission spectra of Laurdan in CHO cells at 90% media dilution (cyan) and in plasma membrane-derived vesicles (light gray). (B) Comparison of Laurdan GP for CHO cells at 90% media dilution and membrane-derived vesicles. The notched box line represents the mean; the top and bottom represent the 75^th and 25^th percentiles, respectively, and the bars represent the standard deviations. To see this figure in color, go online.