Equivalent aqueous phase modulation of domain segregation in myelin monolayers and bilayer vesicles

Abstract

Purified myelin can be spread as monomolecular films at the air/aqueous interface. These films were visualized by fluorescence and Brewster angle microscopy, showing phase coexistence at low and medium surface pressures (<20-30 mN/m). Beyond this threshold, the film becomes homogeneous or not, depending on the aqueous subphase composition. Pure water as well as sucrose, glycerol, dimethylsulfoxide, and dimethylformamide solutions (20% in water) produced monolayers that become homogeneous at high surface pressures; on the other hand, the presence of salts (NaCl, CaCl(2)) in Ringer's and physiological solution leads to phase domain microheterogeneity over the whole compression isotherm. These results show that surface heterogeneity is favored by the ionic milieu. The modulation of the phase-mixing behavior in monolayers is paralleled by the behavior of multilamellar vesicles as determined by small-angle and wide-angle x-ray scattering. The correspondence of the behavior of monolayers and multilayers is achieved only at high surface pressures near the equilibrium adsorption surface pressure; at lower surface pressures, the correspondence breaks down. The equilibrium surface tension on all subphases corresponds to that of the air/alkane interface (27 mN/m), independently on the surface tension of the clean subphase.

Figure 1

Myelin monolayer equilibrium surface tension on different subphases. Typical surface tension curves over time are shown on different subphases. Note the convergence to the same equilibrium value, despite the original surface tension of the clean subphase before monolayer spreading. (Horizontal dotted lines) References at 27 mN/m.

Figure 2

Pattern I_film of myelin monolayers at the air/aqueous interface (non-ionic subphase) under three different lateral packing conditions (the numbers on the pictures indicate the surface pressures: π). The capital letters (from A to E) designate the subphase according to Materials and Methods. The patterns show fluid-to-fluid coexistence with the dark phase predominantly dispersed as round domains within the brilliant phase. Under compression, the patterns blur and become homogeneous. Under expansion, the phase coexistence reappears at approximately the same surface pressures (data not shown). The scale bar is 50 μm in length.

Figure 3

Pattern II of myelin monolayers at the air/water interface (ionic subphase) at three different lateral packing conditions (the numbers on the pictures indicate the surface pressures: π). The capital letters (from F to J) designate the subphase according to the description in the Materials and Methods. The patterns show fluid-to-fluid coexistence with the liquid-expanded phase (brilliant) predominantly dispersed within the dark phase. Under compression, the brilliant phase undergoes a percolation transition, forming fractal-like structures. The phase coexistence is present over the whole surface pressure range. The scale bar is 50 μm in length.

Figure 4

Brewster angle microscopy of myelin monolayer on pure water surface. The film is heterogeneous at low surface pressure (first image, 2 mN/m) with sharp domains edges; occasionally, myelin vesicles are observed as brilliant points at the lateral interfaces. At high surface pressure (∼30–35 mN/m), the domain borders become diffuse and the film becomes homogeneous in 1 h above 35 mN/m, similar to the fluorescence observations. The bar is 50-μm long.

Figure 5

Representative WAXS data at 20°C for hydrated myelin (water glucose 20%). (Inset) Dehydrated powder shows crystalline-like order centered at 4.15 Å (plus other minor peaks). Hydrated samples always show the broad peak shifted to lower q values, centered at ∼4.4 Å, characteristic of liquid-like acyl chains.

Figure 6

Raw SAXS data from the two-dimensional detector for myelin vesicles. The dominant signal is the one corresponding to the second-order and fourth-order harmonic (shown as 2 and 4, respectively). The samples A–E in the first column (see Materials and Methods) show single Bragg diffraction peaks corresponding to the normal spacing in CNS myelin (∼155 Å). The second column (F–J) shows the splitting of the Debye-Scherrer rings (labeled as 2-2′ and 4-4′) in salt-containing media; the ring-splitting points to the existence of two phases.

Figure 7

Selected SAXS data showing three conditions: (A) Pattern I_bulk for myelin vesicles in water. (B) Pattern II_bulk-e for vesicles in NaCl 100 mM. (C) Pattern II_bulk-c for vesicles in CaCl₂ 33 mM.