Cooling induces phase separation in membranes derived from isolated CNS myelin

Abstract

Purified myelin membranes (PMMs) are the starting material for biochemical analyses such as the isolation of detergent-insoluble glycosphingolipid-rich domains (DIGs), which are believed to be representatives of functional lipid rafts. The normal DIGs isolation protocol involves the extraction of lipids under moderate cooling. Here, we thus address the influence of cooling on the structure of PMMs and its sub-fractions. Thermodynamic and structural aspects of periodic, multilamellar PMMs are examined between 4°C and 45°C and in various biologically relevant aqueous solutions. The phase behavior is investigated by small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC). Complementary neutron diffraction (ND) experiments with solid-supported myelin multilayers confirm that the phase behavior is unaffected by planar confinement. SAXS and ND consistently show that multilamellar PMMs in pure water become heterogeneous when cooled by more than 10-15°C below physiological temperature, as during the DIGs isolation procedure. The heterogeneous state of PMMs is stabilized in physiological solution, where phase coexistence persists up to near the physiological temperature. This result supports the general view that membranes under physiological conditions are close to critical points for phase separation. In presence of elevated Ca2+ concentrations (> 10 mM), phase coexistence is found even far above physiological temperatures. The relative fractions of the two phases, and thus presumably also their compositions, are found to vary with temperature. Depending on the conditions, an "expanded" phase with larger lamellar period or a "compacted" phase with smaller lamellar period coexists with the native phase. Both expanded and compacted periods are also observed in DIGs under the respective conditions. The observed subtle temperature-dependence of the phase behavior of PMMs suggests that the composition of DIGs is sensitive to the details of the isolation protocol.

Fig 1. X-ray diffraction pattern of isolated myelin as a function of temperature in Ringer’s solution.

At high temperature (37–46°C) two single rings Peaks 2 and 4 are observed. At 30°C the faint smallest peak (1) is observed near the beamstop. At 25°C beam splitting is more easily observed in the peak 4 and IV which is also evident in peaks 2 and II at 10°C.

Fig 2. Integrated signals of myelin at different temperatures and in different aqueous conditions.

Bi-distilled water (A), Ringer’s solution (B) and CaCl₂ 25 mM in Ringer’s solution (C). In the case of water dominates a simple pattern and only at low temperature the Bragg peaks splits and shifts to lower q. The same happens in the case of Ringer’s solution but at higher temperature. On the other hand, the response to Ca²⁺ goes in the opposite direction in q and it is as a jump (all or nothing) instead of a shift.

Fig 3. Quantification of the phases as a function of temperature in different conditions.

Bi-distilled water (black line), Ringer’s solution (red line) and CaCl₂ 25 mM in Ringer’s solution (blue line). Data is extracted from the ratio of diffracting power of the native phase to the total amount. Clearly, cooling (as in lipid raft isolation protocol) induces a decay of the native phase fraction and a concomitant increment of the non-native phase (not plotted).

Fig 4. DSC thermograms of myelin (20 Mm) under different aqueous media.

In the cases where homogenization takes place–bi-distilled water (black line) and near physiological medium (red line)–a maximum is reached after which a stabilization of the C_p is observed near physiological temperature. In the case of high [Ca²⁺] (blue line), a continuous variation of C_p according to a phase redistribution of components is observed.

Fig 5. Neutron diffraction signals of multilamellar planar arrays in different media and temperature.

The black curve is close to physiological condition. The red curve is the same sample after cooling at 5°C. Two phases are observed with expansion of the spacing. The blue curve is in the presence of high (Ca²⁺) at 37°C; a phase separation takes place in the opposite direction with a compaction.

Fig 6. SAXS of DIGs in different ionic conditions at T = 4°C.

In the presence of a physiological buffer (A) the first order peaks for DIGs (red dots) and non-native phase from PMMs (black dots) are respectively at 0.66 and 0.67 nm^-1 indicating lamellar spacings of 9.5 nm and 9.4 (expanded phase). In the presence of CaCl₂ 25 mM (B) the first order peaks for DIGs (blue dots) and non-native phase from PMMs (black dots) are respectively at 0.96 and 0.98 nm^-1 indicating lamellar spacings of 6.6 and 6.4 nm (compacted phase).

Fig 7. Number (n) of correlated membranes for the different phases as a function of temperature for whole myelin (empty) and DIGs (filled).

In black is marked the n for the native period, in red for the expanded period and in blue for the compact period. In the presence of physiological (diamonds) and high [Ca²⁺] (circles) media.