STRUCTURAL PROPERTIES AND INTERACTIONS AT THE SURFACE OF BIOMIMETIC AGGREGATES.
Creators: name: RONDELLI, VALERIA MARIA
Publisher: name: Universit� degli Studi di Milano
Year: 2012

description: The compositional complexity of biological membranes results in a high organization degree, giving rise to properties far from average. Besides proteins, lipids strongly contribute to membrane structure and functionality, a concept that has been highlighted in connection with membrane microdomains, patches of variable size and composition, involved in different cell functions. Among them, GEMs (Glycosphingolipids Enriched membrane Microdomains) include components such as gangliosides and cholesterol, that have an exotic and tunable packing shape with respect to the glycerophospholipid matrix, as individuals and as a pair. Both glycolipids and cholesterol play an important role in membrane functionality and their disposition is well defined, both across and along the membrane.
We focused on the study of the surface structuring of membranes in connection to the presence and disposition of components and as a result of their interactions, side-to-side, back-to-back and with the external world. Moreover, their interplay gives rise to a dynamic response of the structure of a membrane, induced by interactions taking place at its surface. 
To access these fine structures and dynamics, we started from simple models with controlled composition and environmental conditions. Model systems of different geometries were chosen, where a membrane patch is embedded with different constraints, in order to enhance different properties, so giving peculiar information. For example, micelles were chosen as they are extremely responsive to molecular packing changes, while single membranes deposited over large areas are appropriate for the study of cholesterol transverse disposition inside the leaflets. We applied different spectroscopic and thermodynamic techniques to study many model systems getting complementary information. 

1) Micelles and Vesicles

We started our study from model membranes constituted by lipid micelles and vesicles in water solution. We focused on asymmetry, an aspect found in natural membranes, where gangliosides reside only in the external layer. 
We built up an asymmetric model membrane by incubating GM1 and GD1a gangliosides micelles on preformed DMPC vesicles, in appropriate proportions. In few-hours times and via monomer exchange, ganglioside molecules enter the phospholipid vesicles and, being the flip-flop process prevented by their huge hydrophilic headgroup, they remain confined into the outer layer of the vesicle. Together with phospholipids and gangliosides, we added cholesterol alternatively premixed with the preformed micelles or with the preformed vesicle. We studied the structure of the resulting systems by the Small Angle X-ray Scattering (SAXS) technique. The final aggregates were found to be different if cholesterol was in the starting micelles or in the starting vesicles.
Moreover, thanks to the slow exchange process and to the fast and powerful technique, we have been able to follow on the way the structural rearrangement due to gangliosides entry in DMPC vesicles. The typical mixing times have been extracted and cholesterol has been found to fasten the incubation process, the more if originally embedded in the ganglioside micelles. 

Then we observed the structural changes induced in model aggregates, pure or mixed micelles or vesicles containing ganglioside GD1a, following the action of the enzyme sialidase.
Sialidase detaches the external sialic acid from GD1a sugar head, turning it into GM1. We applied Small-Angle X-ray and Neutron scattering techniques to follow the time evolution of the aggregate structures while chemically undergoing the enzymatic action. We found that the aggregates could be either very stable, in single component micellar systems such as GD1a micelles, or structurally responsive, in mixed model systems, as GD1a+C12PC and GD1a+DMPC. Moreover, while in progress, the sialidase?ganglioside interaction seems to define a time lag where the system is structurally off the smooth route between the initial and the final states.

2) Extended single membranes deposited on a macroscopic support.

In the last few years a considerable effort has been put in developing protocols to prepare model systems that, although still extremely simple to allow for structural studies, could reproduce some clue features of natural ones. One of them is asymmetry. 
Asymmetric model membranes are rare, due to the difficulty of realizing hydrated artificial membranes with well defined heterogeneous composition and applicable for non-average structural investigation. Reflectivity technique is currently being developed to respond to this challenge. In collaboration with the Institut Laue Langevin (ILL) in Grenoble, we developed and tested an experimental model bearing forced membrane asymmetry in a macroscopic single bilayer, to be investigated by X-ray and neutron reflectivity. The sample preparation protocol involves the ?Langmuir-Blodgett film deposition? technique to build asymmetric floating single or double bilayers. The LB-technique is one of the most promising techniques for preparing thin films as it enables the precise control of the monolayer thickness, homogeneous deposition of the monolayer over large areas and the possibility to make multilayer structures with varying composition of the layers. The sample preparation takes at least 4 hours and enables the preparation of 2500mm2 big membranes. 
Then, the Angstrom scale of X-rays and neutrons accessibility is able to scan the membrane in the cross direction and describe its structural properties. The incoming beam crosses the membrane as a stack of semireflecting mirrors: the water layers, the polar heads, the hydrophobic chains. Due to the different composition and thickness of the layers, the intensity reflected or diffracted by each of them is different. The measured intensity spectrum is the sum of the radiation waves diffracted from each layer of the sample. The technique, then, gives information about the transverse structure of the sample, layer by layer: thickness, composition, compactness, surface roughness.
 
Before starting our studies on single or double model membranes, we studied monolayers on water surface.
Pure lipids or lipid mixtures were spread on a water surface, where they float, headgroups dipped in, to form a film. We compressed the film until its compression limit, meanwhile performing isotherm measurements in the form of surface pressure (mN/m) versus area per lipid molecule (A2/molecule). This technique provides information about the phase behaviour of each monolayer as a function of its packing density. First, we verified the effect brought about by addition of cholesterol to DPPC, by recording the (?-A) curves of the mixed Langmuir monolayers at the air-water interface as a function of different molar ratios between DPPC-d75 and cholesterol. Progressive addition of cholesterol to DPPC-d75 results in progressively decreasing the occupied area per average molecule. We assessed that for all of the investigated mixed systems, at a given pressure, the measured area per molecule is lower than expected for ideal mixing. In fact, the effect of cholesterol is to rigidify and order the lipid chains, with a consequent reduction of the occupied area per average molecule at the air-water interface. Moreover, by surface study we could establish the optimal conditions to deposit the layers, in order to have compact and stable samples to be investigated by reflectivity.

X-rays and neutrons reflectivity measurements were performed on single and double bilayers systems to get the main structural properties of model membranes formed by DPPC, cholesterol and gangliosides. 
The molar ratio glycerolipid:ganglioside:cholesterol 10:1:2.5 was biosimilar. The use of fully deuterated (D-isotope) phospholipids makes the lipid matrix ?transparent? for neutrons, exalting the contrast profile of cholesterol and gangliosides, while X-rays reflectivity experiments have been carried on normal (H-isotope) lipid membranes and are more sensible to gangliosides polar heads. Experiments suggest new guidelines for protocols to be used with asymmetric samples, both for lipid redistribution and for macroscopic bilayer integrity. We carried the measurements both in water and in physiological 156 mM salt (NaCl, RbCl) solution. Salts act in slightly increasing the membrane thickness but do not destabilize it, opening the way to the study of membranes in biosignificant solvents.
We found that in an originally asymmetric only-DPPC-cholesterol membrane, submitted to annealing, cholesterol migrates between layers, assuming a symmetric disposition in the two leaflets of the membrane.
On the contrary, in a DPPC-(one-side-GM1)-cholesterol membrane, redistribution results in a preferred asymmetric disposition of cholesterol: the majority resides in the ganglioside-free leaflet, and the minority (about 20%) in the ganglioside-rich leaflet. The same preferred distribution of cholesterol is realized whatever the initial distribution of cholesterol and the modality of GM1 insertion, either spread with the film during the deposition procedure or incubated onto an existing DPPC+cholesterol symmetric membrane. This is an important finding, as ganglioside-cholesterol structural coupling has been widely indicated to be determinant for membrane GEMs structure, but never experimentally confirmed before. 
Actually this experimental proof was made possible thanks to the cutting-edge neutron reflection technique applied to the asymmetric LB deposition protocol, newly developed in this thesis work. This combination of techniques is extremely promising, as it gives the opportunity to create biologically interesting model membranes with distinctive features such as asymmetry, and allow a fine study of their internal structure through a unique detailed insight on the nanometric scale.