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.

Phase Diagram of Purified CNS Myelin Reveals Continuous Transformation between Expanded and Compacted Lamellar States

“…PMMs retain the biomolecular composition of myelin [16] as well as the characteristic patterns of isolated myelin in TEM [3,17,18]. PMMs also exhibit an electron density profile [3] similar to that of myelin [19].…”

“…The change in the interlamellar water amount upon variation of the lamellar period by ∆d can be roughly estimated when assuming a constant membrane thickness. In this case, ∆d directly translated into a change in the equivalent number ∆n l = ∆d/l w of "water layers", where l w = (v w ) 1/3 = 0.31 nm is the linear dimension of a water molecule of partial molecular volume, v w = 0.030 nm 3 . This procedure yielded ∆n l = 10 between the lamellae in the more extreme calculation; this was considering full expansion in NaCl at 4 • C and taking the most compacted state (CaCl 2 , at the same temperature).…”

“…Alternatively, the new peak can appear at q > 0.8 nm −1 , more precisely around 1.0 nm −1 , in which case the phase is called (over)compacted and this is clearly a lipid-enriched phase [25], with only d = 6.6 ± 0.4 nm, depleted of proteins [2]. These different non-native phases are well known and observations of them are found overall in the myelin literature [2,3]. [13].…”

“…Apart from the native period, myelin can also exhibit a so-called "expanded" and a so-called "(over)compacted" phase [1,2]. In our previous studies, we described the phase separation of purified myelin membranes (PMMs) induced by cooling [3] and its relationship to detergent-insoluble glycosphingolipid (DIGs) [4][5][6][7]. In the present work, we performed a systematic study on the influence of the ionic strength (IS) on PMMs and established temperature/IS phase diagrams revealing the identity of the phases involved in the phase separation.…”

“…In the present work, we explored in a systematic way the combined response of CNS PMMs to temperature and ionic milieu and establish 2D phase diagrams. We selected NaCl and CaCl 2 , as they are physiologically very relevant, their interactions with myelin are mainly unspecific, and at the same time their effects are well studied for certain conditions of concentration and temperature in the classical literature [1,2] and by ourselves as researchers [3,9], showing that they can modulate the structure of myelin. Small angle X-ray diffraction was employed to identify different membrane phases from their characteristic lamellar periods [2].…”