Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains

Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-1 and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.

Dynamics of GPI-anchored proteins on the surface of living cells

Françoise Gisou van der Goot Grunberg

Rather than being distributed homogeneously on the cell surface, proteins are probably aggregated in clusters or in specific domains. Some of these domains (lipid rafts) have lipid compositions, which differ from their surrounding membrane. They have been implicated in cell signaling, cell adhesion, and cholesterol homeostasis. Estimates of their size vary from 40 to 350 nm in diameter depending on the study and cell type used. Rafts are enriched in glycosphingolipids and cholesterol and appear to be in a more ordered lipid phase. Although there is some knowledge of their function in cell signaling, less is known about their assembly and dynamics in cells at various temperatures. We use image correlation spectroscopy and dynamic image correlation spectroscopy to study the clustering and diffusion of glycosylphosphatidylinositol (GPI)-anchored proteins within the plasma membrane of living cells at various temperatures. We find that GPI-anchored proteins occur both as monomers and in clusters at the cell surface. The propensities to cluster as well as the diffusion coefficient of these clusters are strongly temperature dependent. At 37 degrees C the GPI-anchored proteins are highly dynamic with a lower state of clustering than at lower temperatures.

2006

Association of Helicobacter pylori vacuolating toxin (VacA) with lipid rafts

Yiming Li, Françoise Gisou van der Goot Grunberg

A variety of extracellular ligands and pathogens interact with raft domains in the plasma membrane of eukaryotic cells. In this study, we examined the role of lipid rafts and raft-associated glycosylphosphatidylinositol (GPI)-anchored proteins in the process by which Helicobacter pylori vacuolating toxin (VacA) intoxicates cells. We first investigated whether GPI-anchored proteins are required for VacA toxicity by analyzing wild-type Chinese hamster ovary (CHO) cells and CHO-LA1 mutant cells that are defective in production of GPI-anchored proteins. Whereas wild-type and mutant cells differed markedly in susceptibility to aerolysin (a bacterial toxin that binds to GPI-anchored proteins), they were equally susceptible to VacA. We next determined whether VacA physically associates with lipid rafts. CHO or HeLa cells were incubated with VacA, and Triton-insoluble membranes then were separated by sucrose density gradient centrifugation. Immunoblot analysis revealed that a substantial proportion of cell-associated toxin was associated with detergent-resistant membranes (DRMs). DRM association required acid activation of the purified toxin prior to contact with cells, and acid activation also was required for VacA cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (a cholesterol-depleting agent) did not inhibit VacA-induced depolarization of the plasma membrane, but interfered with the internalization or intracellular localization of VacA and inhibited the capacity of the toxin to induce cell vacuolation. Treatment of cells with nystatin also inhibited VacA-induced cell vacuolation. These data indicate that VacA associates with lipid raft microdomains in the absence of GPI-anchored proteins and suggest that association of the toxin with lipid rafts is important for VacA cytotoxicity.

2002

Membrane elastic heterogeneity studied at nanometrical scale on living cells

Charles Roduit

The aim of this thesis was to explore the cell mechanical properties using the Atomic Force Microscope (AFM). The cell membrane contains lipids microdomains, called rafts, enriched in cholesterol and sphingolipids. The rafts are believed to play an important role in signal processing by acting as a "signaling platform". Indeed, membrane proteins involved in signal transduction concentrates into these rafts and are coupled with signaling pathways inside the cell. The mechanical properties of these rafts were characterized by targeting one of its component, the glycosylphosphatidylinositol-anchored protein (GPI-AP). During this work, we found these domains to be stiffer than the surrounding membrane. Several control experiments were performed to consolidate this finding. The extraction of cholesterol, one of the major component of raft, was shown to dramatically reduce the stiffness of the raft to reach the surrounding membrane value. The stiffness specificity of rafts may be related to the lower diffusion rate of proteins and can be, therefore, an important property for its role as a signaling platform. During this thesis, we also introduced a new AFM imaging mode, which we called "stiffness tomography". With this imaging mode, we were able to distinguish stiff materials inclusion located into the sample. Different control experiments were done to validate this imaging mode. A virtual experiment was performed with the help of the finite element modeling. This permitted us to validate our methodology, but also pointed us its limitations. The stiffness tomography was also used on living cells and showed significant differences between native and cytoskeleton depolimerized cells. Since no postprocessing tool was available at the beginning of this work, the software development was a very significant part of the project. Its development resulted in a toolbox (a collection of function), that is available for future software development. A non negligible part of the development consisted in the toolbox documentation that is reported in the appendix C. This software permitted to process force volume AFM les and to characterize the elastic properties of the cell membrane with a high precision and reliability.

EPFL2008

Membrane elastic heterogeneity studied at nanometrical scale on living cells

Charles Roduit

EPFL2008