We have investigated the organization, on the plasma membrane and in detergent-insoluble membrane vesicles, of two neuronal glycosylphosphatidylinositolanchored (GPI) proteins: Thy-1, a negative regulator of transmembrane signalling; and prion protein, whose rapid endocytosis and Cu 2ϩ binding suggest that it functions in metal ion uptake. Prion protein occurred on the neuronal surface at high density in domains, located primarily at the cell body, which were relatively soluble in detergent. Thy-1, although much more abundantly expressed on neurons, occurred at lower density over much of the surface of neurites (and in lower abundance at the cell body) in domains that were highly resistant to detergent solubilization. Detergentinsoluble membrane vesicles contained Thy-1 at a density similar to that on the neuronal surface. Vesicles containing each protein could be separated by immunoaffinity isolation; lectin binding showed that they were enriched in different glycoproteins. Our results demonstrate a structural diversity of the domains occupied by functionally different GPI proteins.

No abstract

It is generally assumed that rafts exist in both the external and internal leaflets of the membrane, and that they overlap so that they are coupled functionally and structurally. However, the two monolayers of the plasma membrane of eukaryotic cells have different chemical compositions. This out‐of‐equilibrium situation is maintained by the activity of lipid translocases, which compensate for the slow spontaneous transverse diffusion of lipids. Thus rafts in the outer leaflet, corresponding to domains enriched in sphingomyelin and cholesterol, cannot be mirrored in the inner cytoplasmic leaflet. The extent to which lipids contribute to raft properties can be conveniently studied in giant unilamellar vesicles. In these, cholesterol can be seen to condense with saturated sphingolipids or phosphatidylcholine to form μm scale domains. However, such rafts fail to model biological rafts because they are symmetric, and because their membranes lack the mechanism that establishes this asymmetry, namely proteins. Biological rafts are in general of nm scale, and almost certainly differ in size and stability in inner and outer monolayers. Any coupling between rafts in the two leaflets, should it occur, is probably transient and dependent not upon the properties of lipids, but on transmembrane proteins within the rafts.

Glycosylphosphatidylinositol-anchored prion protein and Thy-1, found in adjacent microdomains or "rafts" on the neuronal surface, traffic very differently and show distinctive differences in their resistance to detergent solubilization. Monovalent immunogold labeling showed that the two proteins were largely clustered in separate domains on the neuronal surface: 86% of prion protein was clustered in domains containing no Thy-1, although 40% of Thy-1 had a few molecules of prion protein associated with it. Only 1% of all clusters contained appreciable levels of both proteins (>3 immunogold label for both). In keeping with this distribution, immunoaffinity isolation of detergent-resistant membranes (DRMs) using the non-ionic detergent Brij 96 yielded prion protein DRMs with little Thy-1, whereas Thy-1 DRMs contained ϳ20% of prion protein. The lipid content of prion protein and Thy-1 DRMs was measured by quantitative nano-electrospray ionization tandem mass spectrometry. In four independent preparations, the lipid content was highly reproducible, with Thy-1 and prion protein DRMs differing markedly from each other and from the total DRM pool from which they were immunoprecipitated. Prion protein DRMs contained significantly more unsaturated, longer chain lipids than Thy-1 DRMs and had 5-fold higher levels of hexosylceramide. The different lipid compositions are in keeping with the different trafficking dynamics and solubility of the two proteins and show that, under the conditions used, DRMs can isolate individual membrane microenvironments. These results further identify unsaturation and glycosylation of lipids as major sources of diversity of raft structure.The separation of membrane lipids into different phases creates diverse microenvironments within a biological membrane (1, 2). In particular, cholesterol is believed to condense with saturated phosphatidylcholine (PC) 1 and sphingomyelin (SM) to form minute patches (40 -100 nm wide) of lipids in a liquid-ordered phase (3-7), creating specialized lipid microenvironments called "rafts" within the disordered fluid phase formed by unsaturated lipids (8). These ordered microdomains control the access and egress of subsets of membrane proteins, regulating signaling systems at the cell surface (9). liquidordered domains resist solubilization in non-ionic detergents (6, 7, 10 -13), enabling them to be isolated as detergent-resistant membranes (DRMs) that float at low density upon gradient centrifugation (14). Lipid-anchored proteins partition into both leaflets of these domains, the glycosylphosphatidylinositol (GPI)-anchored proteins into the outer (surface) layer and the diacylated cytoplasmic proteins into the inner layer (9, 15, 16). The membrane environment of GPI-anchored prion protein (PrP) is of particular interest since it is a candidate for chaperoning the conversion of PrP to the altered pathogenic conformation associated with prion disease (17-19). Immunolabeling shows PrP to be present on the neuronal surface in different, albeit often closely adjacent, d...

Banbury Conference on Genetic Background in Mice*identity of the genetic elements governing these other factors (modifiers) is usually unknown, it is important to keep them constant when evaluating the impact of a Mouse mutants derived by targeted mutagenesis in emmutation. Only if the same genetic background is used bryonic stem (ES) cells offer many advantages to the across experiments can differences between the phenostudy of the molecular and cellular mechanisms underlytypes obtained be ascribed to the mutations rather than ing behaviors such as learning and memory, circadian to different genetic backgrounds. Adoption of a comrhythms, motor coordination, and aggression, as well mon genetic background does not preclude comparison as other neuroscience research areas such as brain of the effects of a given mutation in different backdevelopment. The beginning of any new field, however, grounds. is often marked by a period in which key issues are Genetic background can be used as a tool in the debated, and as a consequence the approach is sharpanalysis of a mutation (e.g., quantitative trait loci analyened and focused. Theoretical and practical issues resis and enhancer/suppressor screens; Takahashi et al., lated to the impact of genetic background on the analy-1994). By placing the same mutation in different genetic sis of mutant mice have been a central topic of backgrounds, it is possible to study facets of gene funcdiscussion in this new field (Crawley, 1996(Crawley, , 1997 Crusio, tion that would elude studies in any single background. 1996;Gerlai, 1996;Lathe, 1996;Wehner and Silva, 1996).Additionally, powerful new mapping and cloning strate-Analysis of the literature reveals that there is no consengies may allow the identification of modifiers from differsus on the nature of appropriate controls for genetic ent backgrounds (Dietrich et al., 1993; Gould et al., background. This report summarizes a recent Banbury 1996). Genetic interactions between a mutation and the workshop held in Cold Spring Harbor, New York, on genetic background may account for the variable pene-December 8-11, 1996, to discuss these issues in an trance of human genetic diseases, and it is important effort to come to a consensus within the field. The recto study and understand the nature of these interactions. ommendations that follow reflect the need for rigorously Most targeting experiments to date have relied on controlling the genetic background of experimental anithe use of ES cells derived from substrain 129 mice. mals, and the practical issues surrounding the imple-However, the 129 substrains are a complex collection mentation of the appropriate controls.

We have exploited the structural homology, but different patterns of expression of the murine and human Thy‐1 genes to map a number of tissue‐specific enhancer elements in the genes. All of these are located downstream from the site of transcriptional initiation. The human gene contains separate elements which direct expression to the kidney or spleen epithelium. The murine gene lacks these elements but instead contains a thymocyte specific enhancer in the third intron. Developmentally‐regulated expression in nerve cells is directed (at least in part) by an atypical element in the first intron. The latter is active on heterologous promoters, but is position and distance dependent.

No abstract

Sphingomyelin is enriched within lipid microdomains of the cell membrane termed lipid rafts. These microdomains play a part in regulating a variety of cellular events. Computer simulations of the hydrogen-bonding properties of sphingolipids, believed to be central to the organization of these domains, can delineate the possible molecular interactions that underlie this lipid structure. We have therefore used molecular dynamics simulations to unravel the hydrogen-bonding behavior of palmitoylsphingomyelin (PSM). A series of eight simulations of 3 ns each of a single PSM molecule in water showed that the sphingosine OH and NH groups can form hydrogen bonds with the phosphate oxygens of their own polar head, in agreement with NMR data. Simulations of PSM in a bilayer assembly were carried out for 8 ns with three different force field parameterizations. The major physico-chemical parameters of the simulated bilayer agree with those established experimentally. The sphingosine OH group was mainly involved in intramolecular hydrogen bonds, in contrast to the almost exclusive intermolecular hydrogen bonds formed by the amide NH moiety. During the bilayer simulations the intermolecular hydrogen bonds among lipids formed a dynamic network characterized by the presence of hydrogen-bonded lipid clusters of up to nine PSM molecules.