Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the C-terminal end that serves as an anchor to the plasma membrane and could be responsible for the presence of GPI proteins in rafts, a type of functionally important membrane microdomain enriched in sphingolipids and cholesterol. In order to understand better how GPI proteins partition into rafts, the insertion of the GPI-anchored alkaline phosphatase (AP) was studied in real-time using atomic force microscopy. Supported phospholipid bilayers made of a mixture of sphingomyelindioleoylphosphatidylcholine containing cholesterol (Chl+) or not (Chl-) were used to mimic the fluid-ordered lipid phase separation in biological membranes. Spontaneous insertion of AP through its GPI anchor was observed inside both Chl+ and Chl-lipid ordered domains, but AP insertion was markedly increased by the presence of cholesterol.

The time-dependent topology of domains in supported phospholipid bilayers of a binary mixture of dioleoylphosphatidylcholine and dipalmitoylphosphatidylcholine under a buffer solution has been studied by atomic force microscopy. We observe a transient regime of the phase separation until 45 min after a temperature quench from a miscible state of the system into the gel−liquid crystal coexistence region with the earliest observation after 20 min showing large gel-phase domains (containing ∼104−106 molecules) of irregular shapes. The transient regime is characterized by a power law for the growth rate of the domain size (A) with n = 3.0 ± 0.4 in A ∝ t 2/ n . After 45 min, an asymptotic power law with n = 20 ± 10 is observed and is linked to an inhibited domain growth. The evolution of individual domains suggests that domain growth in the transient regime is governed by a ripening mechanism. The growth inhibition is linked to the observation that the gel domains in each leaflet of the bilayer must grow simultaneously at the same sites as they remain superimposed on each other throughout the phase separation process.

Elucidating origin, composition, size, and lifetime of microdomains in biological membranes remains a major issue for the understanding of cell biology. For lipid domains, the lack of a direct access to the behaviour of samples at the mesoscopic scale has constituted for long a major obstacle to their characterization, even in simple model systems made of immiscible binary mixtures. By its capacity to image soft surfaces with a resolution that extends from the molecular to the microscopic level, in air as well as under liquid, atomic force microscopy (AFM) has filled this gap and has become an inescapable tool in the study of the surface topography of model membrane domains, the first essential step for the understanding of biomembranes organization. In this review we mainly focus on the type of information on lipid microdomains in model systems that only AFM can provide. We will also examine how AFM can contribute to understand data acquired by a variety of other techniques and present recent developments which might open new avenues in model and biomembrane AFM applications.

The lipid rafts membrane microdomains, enriched in sphingolipids and cholesterol, are implicated in numerous functions of biological membranes. Using atomic force microscopy, we have examined the effects of cholesterol-loaded methyl-beta-cyclodextrin (MbetaCD-Chl) addition to liquid disordered (l(d))-gel phase separated dioleoylphosphatidylcholine (DOPC)/sphingomyelin (SM) and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SM supported bilayers. We observed that incubation with MbetaCD-Chl led to the disappearance of domains with the formation of a homogeneously flat bilayer, most likely in the liquid-ordered (l(o)) state. However, intermediate stages differed with the passage through the coexistence of l(o)-l(d) phases for DOPC/SM samples and of l(o)-gel phases for POPC/SM bilayers. Thus, gel phase SM domains surrounded by a l(o) matrix rich in cholesterol and POPC could be observed just before reaching the uniform l(o) state. This suggests that raft formation in biological membranes could occur not only via liquid-liquid but also via gel-liquid immiscibility. The data also demonstrate that MbetaCD-Chl as well as the unloaded cyclodextrin MbetaCD make holes and preferentially extract SM in supported bilayers. This strongly suggests that interpretation of MbetaCD and MbetaCD-Chl effects on cell membranes only in terms of cholesterol movements have to be treated with caution.

The membrane surface of living CV-1 kidney cells in culture was imaged by contact-mode atomic force microscopy using scanning forces in the piconewton range. A simple procedure was developed for imaging of the cell surface with forces as low as 20-50 pN, i.e., two orders of magnitude below those commonly used for cell imaging. Under these conditions, the indentation of the cells by the tip could be reduced to less than l0 nm, even at the cell center, which gave access to the topographic image of the cell surface. This surface appeared heterogeneous with very few villosities and revealed, only in distinct areas, the submembrane cytoskeleton. At intermediate magnifications, corresponding to 20-5 microm scan sizes, the surface topography likely reflected the organization of submembrane and intracellular structures on which the plasma membrane lay. By decreasing the scan size, a lateral resolution better than 20 nm was routinely obtained for the cell surface, and a lateral resolution better than 10 nm was obtained occasionally. The cell surface appeared granular, with packed particles, likely corresponding to proteins or protein-lipid complexes, between approximately 5 and 30 nm xy size.

The external membrane leaflet plays a key role in the organization of the cell plasma membrane as a mosaic of ordered microdomains enriched in sphingolipids and cholesterol and of fluid domains. In this study, the thermotropic behavior and the topology of bilayers made of a phosphatidylcholine/sphingomyelin mixture, which mimicks the lipid composition of the external leaflet of renal brush-border membranes, were examined by differential scanning calorimetry and atomic force microscopy. In the absence of cholesterol, a broad phase separation process occurred where ordered gel phase domains of size varying from the mesoscopic to the microscopic scale, enriched in sphingomyelin, occupied half of the bilayer surface at room temperature. Increasing amounts of cholesterol progressively decreased the enthalpy of the transition and modified the topology of membranes domains up to a concentration of 33 mol % for which no membrane domains were detected. These results strongly suggest that, in membranes highly enriched in sphingolipids like renal and intestinal brush borders, there is a threshold close to the physiological concentration above which cholesterol acts as a suppressor rather than as a promoter of membrane domains. They also suggest that cholesterol depletion does not abolish the lateral heterogenity in brush-border membranes.According to the current view, the plasma membrane of eucaryotic cells is organized in an in-plane mosaic of microdomains (1, 2). Rafts correspond to a category of microdomains, enriched in sphingolipids (SPL) 1 and cholesterol (Chl), which play a key role in the expression and regulation of the plasma membrane functions (3, 4). This conclusion was reached essentially through the use of two experimental procedures, the low temperature non-ionic detergent extraction (2) and the Chl depletion of cells (5, 6). The resistance to low temperature, non-ionic detergent extraction of numerous membrane proteins is associated to a liquid ordered (L o ) or to a gel ordered (L ␤ ) state of membrane lipids, which strongly suggests that the physical state of these membrane lipids is of primary importance in the formation of the membrane microdomains mosaic (7,8). Formation of the L o phase, or more precisely of the fluid liquid ordered L o␣ and gel liquid ordered L o␤ phases (9), depends on the presence of Chl (10, 11). SPL also appear to be determinant for the existence of eucaryotic plasma membrane rafts (3, 4), and this could be explained by the preferential interaction of Chl with SPL rather than with the other phospholipid species in natural phospholipid-Chl mixtures (10, 12, 13). Because SPL are essentially localized on the external leaflet of the plasma membrane (14), this strongly suggests that this membrane leaflet plays a crucial role in the existence of microdomains.Renal brush-border membranes (BBM), which constitute the apical membrane of the proximal tubule epithelial cells, are highly ordered structures, as shown by fluorescence polarization and ESR data (15). Their glycerophospholipid GPL...

The plasma membrane outer leaflet plays a key role in determining the existence of rafts and detergent-resistant membrane domains. Monolayers with lipid composition mimicking that of the outer leaflet of renal brush border membranes (BBM) have been deposited on mica and studied by atomic force microscopy. Sphingomyelin (SM) and palmitoyloleoyl phosphatidylcholine (POPC) mixtures, at molar ratios varying from 2:1 to 4:1, were phase-separated into liquid condensed (LC) SM-enriched phase and liquid expanded (LE) POPC-enriched phase. The LC phase accounted for 33 and 58% of the monolayers surface for 2:1 and 4:1 mixtures, respectively. Addition of 20-50 mol % cholesterol (Chl) to the SM/POPC (3:1) mixtures induced marked changes in the topology of monolayers. Whereas Chl promoted the connection between SM domains at 20 mol %, increasing Chl concentration progressively reduced the size of domains and the height differences between the phases. Lateral heterogeneity was, however, still present at 33 mol % Chl. The results indicate that the lipid composition of the outer leaflet is most likely responsible for the BBM thermotropic transition properties. They also strongly suggest that the common maneuver that consists of depleting membrane cholesterol to suppress rafts does not abolish the lateral heterogeneity of BBM membranes.

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