Mesoscopic structure in the chain-melting regime of anionic phospholipid vesicles: DMPG.

In a range of low ionic strength, aqueous dispersions of the anionic phospholipid DMPG (dimyristoylphosphatidylglycerol) have a transparent intermediate phase (IP, between T(m)(on) congruent with 20 degrees C and T(m)(off) congruent with 30 degrees C) between the turbid gel and fluid membrane phases, evidenced in turbidity data. Small angle x-ray scattering results on DMPG dispersions show that, besides the bilayer peak present in all phases, a peak corresponding to a mesoscopic structure at approximately 400 A is detected only in IP. The dependence of this peak position on DMPG concentration suggests a correlation in the bilayer plane, consistent with the stability of vesicles in IP. Moreover, observation of giant DMPG vesicles with phase contrast light microscopy show that vesicles "disappear" upon cooling below T(m)(off) and "reappear" after reheating. This further proves that although vesicles cannot be visualized in IP, their overall structure is maintained. We propose that the IP in the melting regime corresponds to unilamellar vesicles with perforations, a model which is consistent with all described experimental observations. Furthermore, the opening of pores across the membrane tuned by ionic strength, temperature, and lipid composition is likely to have biological relevance and could be used in applications for controlled release from nanocompartments.

Refined contour analysis of giant unilamellar vesicles.

The fluctuation spectrum of giant unilamellar vesicles is measured using a high-resolution contour detection technique. An analysis at higher q vectors than previously achievable is now possible due to technical improvements of the experimental setup and of the detection algorithm. The global fluctuation spectrum is directly fitted to deduce the membrane tension and the bending modulus of lipid membranes. Moreover, we show that the planar analysis of fluctuations is valid for spherical objects, even at low wave vectors. Corrections due to the integration time of the video camera and to the section of a 3D object by the observation plane are introduced. A precise calculation of the error bars has been done in order to provide reliable error estimate. Eventually, using this technique, we have measured bending moduli for EPC, SOPC and SOPC: CHOL membranes confirming previously published values. An interesting application of this technique can be the measurement of the fluctuation spectra for non-equilibrium membranes, such as "active membranes".

Amplitude hierarchy of vesicle shapes.

Shapes of fluid lipid vesicles are governed by the bending elasticity of their membrane as described by the Area-Difference-Elasticity (ADE) model. These shapes can be quantified using a suitable modal representation of the vesicle contour. Prolate vesicles are characterized by a hierarchy in their shape amplitudes. Experimentally, we find an ordering of the amplitudes with mode number both in large (100 nm) as well as giant (10 µm) unilamellar vesicles. Mean shapes are found only within the small energetically stable region of the prolate phase. Our study demonstrates that bending energy concepts may be quantitatively used on cellular length scales ranging from the size of organelles to the plasma membrane.

Influence of transbilayer area asymmetry on the morphology of large unilamellar vesicles.

The morphological consequences of differences in the monolayer surface areas of large unilamellar vesicles (LUVs) have been examined employing cryoelectron microscopy techniques. Surface area was varied by inducing net transbilayer transport of dioleoylphosphatidylglycerol (DOPG) in dioleoylphosphatidylcholine (DOPC):DOPG (9:1, mol:mol) LUVs in response to transmembrane pH gradients. It is shown that when DOPG is transported from the inner to the outer monolayer, initially invaginated LUVs are transformed to long narrow tubular structures, or spherical structures with one or more protrusions. Tubular structures are also seen in response to outward DOPG transport in DOPC:DOPG:Chol (6:1:3, mol:mol:mol) LUV systems, and when lyso-PC is allowed to partition into the exterior monolayer of DOPC:DOPG (9:1, mol:mol) LUVs in the absence of DOPG transport. Conversely, when the inner monolayer area is expanded by the transport of DOPG from the outer monolayer to the inner monolayer of non-invaginated LUVs, a reversion to invaginated structures is observed. The morphological changes are well described by an elastic bending theory of the bilayer. Identification of the difference in relaxed monolayer areas and of the volume-to-area ratio of the LUVs as the shape-determining factors allows a quantitative classification of the observed morphologies. The morphology seen in LUVs supports the possibility that factors leading to differences in monolayer surface areas could play important roles in intracellular membrane transport processes.

Budding and fission of vesicles.

We report on budding and fission of protein-free vesicles swollen from a natural lipid mixture of bovine brain sphingomyelins. Budding was induced by increasing the area-to-volume ratio through heating. Morphological changes were monitored by phase contrast microscopy and correlated with the thermal behavior of the bilayer by differential scanning calorimetry. Freeze fracture electron microscopy revealed that budding and fission are not restricted to giant vesicles but also occur on length scales relevant for cellular processes. We also observed osmotically induced budding and fission in mixtures of dimyristoyl phosphatidylcholine with cholesterol. We find that these shape transitions are driven by liquid/gel domain formation and/or coupling of the spontaneous curvature of the membrane to the local lipid composition. Our results provide evidence that coat proteins are not necessary for budding and fission of vesicles. The physics of the lipid bilayer is rich enough to explain the observed behavior.