Direct visualization of the lateral structure of porcine brain cerebrosides/POPC mixtures in presence and absence of cholesterol

Abstract

We studied the thermal behavior of membranes composed of mixtures of natural cerebrosides (from porcine brain) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with and without cholesterol, using differential scanning calorimetry, Fourier transform infrared spectroscopy, and confocal/multiphoton fluorescence microscopy. The POPC/cerebroside mixture display solid ordered/liquid disordered phase coexistence in a broad range of compositions and temperatures in agreement with previous results reported for POPC/(bovine brain)cerebrosides. The observed phase coexistence scenario consists of elongated, micrometer-sized cerebroside-rich solid ordered domains that span the bilayer, embedded in a POPC-rich liquid disordered phase. The data obtained from differential scanning calorimetry and Fourier transform infrared spectroscopy was in line with that obtained in the microscopy experiments for the binary mixture, except at very high cerebroside molar fractions (0.8-0.9) were some differences are observed. Cholesterol incorporation exerts strong changes on the lateral organization of POPC/porcine brain cerebroside membranes. At intermediate cholesterol concentrations (10-25 mol %) the solid ordered/liquid disordered phase coexistence scenario gradually transform to a solid ordered/liquid ordered one. Above 25 mol % of cholesterol two distinct regions with liquid ordered phase character are visualized in the membrane until a single liquid ordered phase forms at 40 mol % cholesterol. The observed cholesterol effect largely differs from that reported for POPC/porcine brain ceramide, reflecting the impact of the sphingolipids polar headgroup on the membrane lateral organization.

Figure 1

Representative two photon excitation fluorescence images showing the lateral pattern of GUVs composed of POPC with different amounts of pb-cerebrosides at 20°C. (Top row) LAURDAN fluorescence intensity images (false color representation) obtained with the two emission channel configuration (see Materials and Methods). Green color corresponds to liquid disordered phase domains, red color corresponds to solid ordered phase domains. Notice the strong photoselection effect in the solid ordered phase. (Bottom row) Computed LAURDAN GP images. The red areas correspond to high (solid ordered) GP values. Scale bar = 20 μM.

Figure 2

LAURDAN GP mean values of solid ordered and fluid disordered domains at different pb-cerebroside mol % in POPC/pb-pb-cerebrosides mixtures. The LAURDAN GP values are averages obtained from the individual domains (5 measurements per GUV) in 10–15 different GUVs.

Figure 3

(A) Comparison of DiI_C18 and LAURDAN fluorescence intensity from the same vesicle composed of POPC/pb-cerebrosides 3:2 mol. Left image showing DiI_C18 fluorescence (green), right image showing LAURDAN distinct fluorescence in the fluid disordered and solid ordered domains (green and red respectively). (B) Comparison of DiI_C18/Alexa 488 cholera toxin and LAURDAN fluorescence intensity in vesicles composed of POPC/pb-cerebrosides 3:2 mol, Left image showing DiI_C18 fluorescence (green) and Alexa 488 cholera toxin (red), right image showing LAURDAN distinct fluorescence in the fluid disordered and solid ordered domains (green and red, respectively). Scale bar = 20 μM.

Figure 4

DSC thermograms of multilamellar vesicles composed of POPC/pb-cerebroside mixtures at different pb-cerebroside molar ratios.

Figure 5

Thermal behavior of MLVs composed of dPOPC-d31/pb-cerebrosides 4:1 mol. The values were obtained from area changes of the FTIR spectrum peak at 2920 cm⁻¹ (undeuterated lipid chains) and at 2195 cm⁻¹ (deuterated lipid chains). The figure also shows their corresponding derivatives.

Figure 6

Two photon microscopy pictures of POPC/pb-cerebrosides (4:1 mol ratio) GUVs containing different amounts of cholesterol. Top row: LAURDAN fluorescence intensity images (false color representation) obtained with the two emission channel configuration (see Materials and Methods). Green and red color corresponds to disordered and ordered lipid phases. Bottom row: Computed LAURDAN GP images. Scale bar = 20 μM.

Figure 7

LAURDAN GP means values of the coexisting domains in POPC/pb-cerebrosides 4:1 mol mixture containing different amounts of cholesterol. The GP values are averages obtained from 10 to 15 GUVs per lipid mixing ratio.

Figure 8

DSC thermograms of multilamellar vesicles composed of POPC/pb-cerebroside 4:1 mol mixture containing different cholesterol concentrations. The two uppermost traces were multiplied by a factor of five for enhancement and clarity.

Figure 9

Thermal behavior of MLVs composed of dPOPC-d31/pb-cerebrosides 4:1 containing 26% cholesterol. The values were obtained from area changes of the FTIR spectrum peak at 2920 cm⁻¹ (undeuterated lipid chains) and at 2195 cm⁻¹ (deuterated lipid chains). The figure also shows their corresponding derivatives.

Figure 10

NBD-PC fluorescence intensity images of GUVs composed POPC/pb-cerebrosides 4:1 mol containing (A) 0 mol %, (B) 15 mol %, (C) 20 mol %, (D) 25 mol %, (E) 30 mol %, and (F) 40 mol % cholesterol. Scale bar = 20 μM.