Surface topography of membrane domains

金沢大学理工研究域数物科学系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. © 2009 Elsevier B.V. All rights reserved

Temperature Dependence of the Surface Topography in Dimyristoylphosphatidylcholine/Distearoylphosphatidylcholine Multibilayers

Simple lipid binary systems are intensively used to understand the formation of domains in biological membranes. The size of individual domains present in the gel/fluid coexistence region of single supported bilayers, determined by atomic force microscopy (AFM), generally exceeds by two to three orders of magnitude that estimated from multibilayers membranes by indirect spectroscopic methods. In this article, the topography of equimolar dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) multibilayers, made of two superimposed bilayers supported on mica surface, was studied by AFM in a temperature range from room temperature to 45°C. In the gel/fluid phase coexistence region the size of domains, between ∼100 nm and a few micrometers, was of the same order for the first bilayer facing the mica and the top bilayer facing the buffer. The gel to fluid phase separation temperature of the first bilayer, however, could be increased by up to 8°C, most likely as a function of the buffer layer thickness that separated it from the mica. Topography of the top bilayer revealed the presence of lipids in ripple phase up to 38–40°C. Above this temperature, a pattern characteristic of the coexistence of fluid and gel domains was observed. These data show that difference in the size of lipid domains given by AFM and spectroscopy can hardly be attributed to the use of multibilayers models in spectroscopy experiments. They also provide a direct evidence for metastable ripple phase transformation into a gel/fluid phase separated structure upon heating

Microscopie à force atomique : de l’imagerie cellulaire à la manipulation moléculaire

Le microscope à force atomique explore la surface d’échantillons biologiques à l’aide d’une pointe effilée portée par un ressort très souple. La précision des déplacements de cette pointe, dans les trois plans de l’espace, couplée à une utilisation dans des solutions physiologiques, permet de visualiser aussi bien des structures biologiques complexes que des molécules uniques, et cela dans leur état fonctionnel. Les résolutions latérale et verticale peuvent atteindre quelques angströms. Outil de dissection et de manipulation à l’échelle moléculaire, le microscope à force atomique offre également une nouvelle approche pour la détermination, sur molécules uniques, des forces intra- et intermoléculaires.Using a sharp tip attached at the end of a soft cantilever as a probe, the atomic force microscope (AFM) explores the surface topography of biological samples bathed in physiological solutions. In the last few years, the AFM has gained popularity among biologists. This has been obtained through the improvement of the equipment and imaging techniques as well as through the development of new non-imaging applications. Biological imaging has to face a main difficulty that is the softness and the dynamics of most biological materials. Progress in understanding the AFM tip-biological samples interactions provided spectacular results in different biological fields. Recent examples of the possibilities offered by the AFM in the imaging of intact cells, isolated membranes, membrane model systems and single molecules at work are discussed in this review. Applications where the AFM tip is used as a nanotool to manipulate biomolecules and to determine intra- and intermolecular forces from single molecules are also presented

Use of Cyclodextrin for AFM Monitoring of Model Raft Formation

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-β-cyclodextrin (MβCD-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 MβCD-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 MβCD-Chl as well as the unloaded cyclodextrin MβCD make holes and preferentially extract SM in supported bilayers. This strongly suggests that interpretation of MβCD and MβCD-Chl effects on cell membranes only in terms of cholesterol movements have to be treated with caution

La microscopie à force atomique, une technique de pointe appliquée à la biologie

Le microscope à force atomique (AFM) permet l’observation de la surface d’échantillons biologiques dans un tampon physiologique avec des résolutions latérale et verticale qui peuvent atteindre quelques angströms. Il permet d’observer aussi bien des structures biologiques complexes que des molécules uniques. Il s’est avéré être un outil très performant dans l’étude des membranes isolées, biologiques ou artificielles, et de leurs constituants lipidiques et protéiques et il permet également de traduire en image, dans leur état fonctionnel, d’autres structures biologiques comme des complexes nucléoprotéiques. Il peut servir d’instrument de dissection et de manipulation à l’échelle moléculaire et est utile pour évaluer les forces d’interactions intra ou intermoléculaires