Differential detection of phospholipid fluidity, order, and spacing by fluorescence spectroscopy of bis-pyrene, prodan, nystatin, and merocyanine 540

Abstract

The properties of liquid-ordered, solid-ordered, and liquid-disordered phases were investigated by steady-state fluorescence spectroscopy in liposomes composed of mixtures of dipalmitoylphosphatidylcholine and cholesterol (0-40 mol %) as a function of temperature (24-51 degrees C). The fluorescent probes used (bis-pyrene, nystatin, prodan, and merocyanine) were chosen because they differ in the location they occupy in the membrane and in the types of properties they sense. Comparison of phase diagrams with contour plots of the fluorescence data suggested that bis-pyrene is sensitive primarily to lipid order. In contrast, nystatin fluorescence intensity responded to changes in lipid fluidity. The shape of the prodan emission spectrum detected both liquid-solid and order-disorder transitions in the phase diagram. Merocyanine's behavior was more complex. First, it was more sensitive than any of the other probes to the membrane pretransition that occurs in the absence of cholesterol. Second, regardless of whether emission intensity, anisotropy, or spectral shape was observed, the probe appeared to distinguish two types of liquid-ordered phases, one with tightly packed lipids and one in which the apparent spacing among lipids was increased. The prodan data supported these results by displaying modest versions of these two observations. Together, the results identify eight regions within the phase diagram of distinguishable combinations of these physical properties. As an example of how this combined analysis can be applied to biological membranes, human erythrocytes were treated similarly. Temperature variation at constant cholesterol content revealed three of the eight combinations identified in our analysis of liposomes.

FIGURE 1

Effects of cholesterol content and temperature on bis-pyrene fluorescence. (A) The bis-pyrene emission spectrum in liposomes composed of 100% DPPC at 24°C (solid curve) and at 48°C (dashed curve). (B) Effect of temperature on the ratio of excimer (479–488 nm) to monomer (394–397 nm) fluorescence intensity in the same liposomes as seen in A. (C) Repeat of the experiment shown in B in DPPC liposomes containing 0 mol % (□), 25 mol % (•), 30 mol % (∇), or 40 mol % (▾) cholesterol.

FIGURE 2

Relationship between bis-pyrene excimer/monomer ratio and a theoretical DPPC/cholesterol phase diagram. The data from Fig. 1 C were used to generate a contour plot as explained in Materials and Methods (contour lines represent increments of 0.11 units of excimer/monomer ratio) superimposed on an idealized phase diagram of DPPC and cholesterol (23).

FIGURE 3

Effects of cholesterol and temperature on nystatin fluorescence intensity. Fluorescence emission spectra were obtained from liposomes composed of 0% (□), 10% (▴), 20% (○), or 40% (▾) cholesterol. Fluorescence intensity (404–412 nm) was normalized to the intensity measured at 50°C. The contour plot in B was generated as described in Materials and Methods, with lines representing 0.3-unit increments in normalized nystatin intensity.

FIGURE 4

Effects of temperature and cholesterol on prodan emission spectra. (A) Prodan emission spectrum in liposomes composed of 100% DPPC at 25°C (solid curve) and at 50°C (dashed curve). The same conditions were used with liposomes containing 10% (B) or 40% (C) cholesterol.

FIGURE 5

Relationship between prodan fluorescence the DPPC/cholesterol phase diagram. (A) 3WGP was calculated from spectra such as that in Fig. 4 for DPPC liposomes containing 0% (□), 5% (▪), 15% (▵), and 40% (▾) cholesterol. (B) Contour plot generated from the data in A as described in Materials and Methods, with lines representing increments of 0.11 units of prodan 3WGP.

FIGURE 6

Effects of cholesterol and temperature on MC540 fluorescence. (A) MC540 emission spectrum in liposomes composed of 100% DPPC at 25°C (solid curve) and at 50°C (dashed curve). (B) Ratio of emission intensity calculated at 583–587 nm to that at 619–623 nm in DPPC liposomes containing 0% (□), 10% (▴), 20% (○), 30% (∇), or 40% (▾) cholesterol.

FIGURE 7

Relationship between MC540 fluorescence and the DPPC/cholesterol phase diagram. Contour plots were generated using the data from Fig. 6 B (A) or anisotropy measurements (B) as explained in Materials and Methods. Lines represent increments of 0.13 units of MC540 intensity ratio (A) or anisotropy (B).

FIGURE 8

Effects of ergosterol and temperature on nystatin fluorescence intensity. The experiments of Fig. 3 were repeated using liposomes composed of DPPC and various concentrations of ergosterol: 0% (□), 10% (▴), 20% (○), and 30% (∇).

FIGURE 9

Combined contour plots revealing relative contributions of lipid order, fluidity, and spacing. The data from Figs. 2, 3, 5, and 7 A, together with results using the probe Laurdan from Harris et al. (2) were used to generate this figure. In each case, the data are represented by intensity of the color scaled to the fluorescence value from the corresponding figure at 24°C (black) to that at 48°C (full intensity) with pure DPPC. The ordinates in each panel represent temperatures ranging in 3°C increments from 24 to 48°C. The abscissas represent cholesterol contents in 5-mol % increments from 0 to 40 mol %. Green was used for probes that detect lipid order with increasing intensity, representing increasing disorder. The rows correspond to the various membrane depths represented by these probes; i.e., bis-pyrene (“chains”), Laurdan (“glycerol” backbone), and prodan (“heads”). Blue was used for nystatin “fluidity”. The top panel contains only the nystatin data. Each panel below in the same column contains the combined result of nystatin with the bis-pyrene, Laurdan, or prodan data. The same scheme applies to the third column for MC540 (red, “spacing”). The fourth column represents (top to bottom) nystatin and MC540 data combined with either bis-pyrene, Laurdan, or prodan.

FIGURE 10

Summary of phase diagram regions identified by combined fluorescence of prodan (green), nystatin (blue), and MC540 (red). The data are from the lower right panel of Fig. 9. L_α, liquid-disordered phase; P′_β, rippled phase; S_O, solid-ordered phase; L_O(I), liquid-ordered phase with decreased lipid spacing; L_O(II), liquid-ordered phase with increased lipid spacing, L_α∼L_O(II), undefined continuum between L_α and L_O phases.

FIGURE 11

Fluorescence of Laurdan, diphenylhexatriene, and MC540 in human erythrocytes as a function of temperature. Laurdan data are shown in green (“disorder”), and were obtained from Best et al. (21). Diphenylhexatriene (blue, “fluidity”) results were acquired as steady-state anisotropy, as described in Materials and Methods. The data were calibrated to the nystatin results using the S_O and L_α phases as standards (data for liposomes with 100% DPPC or 70% DPPC with 30% cholesterol are shown in the far right panel). MC540 data (red, “spacing”) were obtained from Jensen et al. (20). In the left panel, the data are scaled as in Fig. 9 for comparison to liposomes. In the center panel, the data are scaled to the maximum and minimum signals in erythrocytes specific to each probe.