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.

Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation

“…In contrast, cholesterol transbilayer distribution remains unclear; several studies, however, reached the same conclusion that a significant part (50-75 %) is distributed in the inner leaflet [5,6]. Membrane cholesterol plays key regulatory roles including (1) membrane fluidity via lipid ordering; (2) membrane deformability by modulation of plasma membrane protein interactions at the interface with cortical cytoskeleton [7]; (3) formation and stabilization of nanometric lipid assemblies, rafts and caveolae [2,4], as important signaling platforms [8][9][10]; and (4) phase coexistence in artificial membranes [11][12][13]. Cholesterol is, thus, a key component of membrane biology and the concept of its clustering into membrane domains is attractive to explain its different functions.…”

Structure and Nanomechanics of Model Membranes by Atomic Force Microscopy and Spectroscopy: Insights into the Role of Cholesterol and Sphingolipids

“…This behavior is similar to that observed with SM, although the transition to the l o phase is well established even before reaching the 8 mol % Chol. In the case of Cer, the s o domains remain solid-like still with concentrations of Chol higher than 20 mol % [97], as previously commented.…”

Micrometric segregation of fluorescent membrane lipids: relevance for endogenous lipids and biogenesis in erythrocytes

“…Combination of alternative fl uorophore labeling is not without potential pitfalls. The impact of sphingolipid headgroups on lateral lipid organization has been demonstrated in artifi cial membranes ( 51 ). Moreover, we recently reported that BODIPY-GlcCer/GM1BODIPY and BODIPY-SM, built on the very same BODIPY-ceramide backbone and differing only by their respective polar head, segregated into distinct domains exhibiting different properties ( 20 ).…”