Günter Schwarzmann

Direct observation of the nanoscale dynamics of membrane lipids in a living cell

Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events. Although several experiments indicate their existence, lipid nanodomains (‘rafts’) remain controversial owing to the lack of suitable detection techniques in living cells. The controversy is reflected in their putative size of 5–200 nm, spanning the range between the extent of a protein complex and the resolution limit of optical microscopy. Here we demonstrate the ability of stimulated emission depletion (STED) far-field fluorescence nanoscopy to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes ∼70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently (∼10–20 ms) trapped in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas. The non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells.

GM1 structure determines SV40-induced membrane invagination and infection

Incoming simian virus 40 (SV40) particles enter tight-fitting plasma membrane invaginations after binding to the carbohydrate moiety of GM1 gangliosides in the host cell plasma membrane through pentameric VP1 capsid proteins. This is followed by activation of cellular signalling pathways, endocytic internalization and transport of the virus via the endoplasmic reticulum to the nucleus. Here we show that the association of SV40 (as well as isolated pentameric VP1) with GM1 is itself sufficient to induce dramatic membrane curvature that leads to the formation of deep invaginations and tubules not only in the plasma membrane of cells, but also in giant unilamellar vesicles (GUVs). Unlike native GM1 molecules with long acyl chains, GM1 molecular species with short hydrocarbon chains failed to support such invagination, and endocytosis and infection did not occur. To conceptualize the experimental data, a physical model was derived based on energetic considerations. Taken together, our analysis indicates that SV40, other polyoma viruses and some bacterial toxins (Shiga and cholera) use glycosphingolipids and a common pentameric protein scaffold to induce plasma membrane curvature, thus directly promoting their endocytic uptake into cells.

[26] Lysogangliosides: Synthesis and use in preparing labeled gangliosides

Publisher Summary Gangliosides are ubiquitous in vertebrate cells and are notably abundant in neuronal plasma membranes. The high concentration of gangliosides in neurons is conceivably related to membrane properties. Interactions of gangliosides with membrane proteins, other lipids, and with themselves have gained increasing interest in recent years. This chapter describes two efficient methods for the synthesis of lysogangliosides—that is, gangliosides lacking their fatty acyl residue, starting from tissue gangliosides. The first procedure begins with the free sialooligosaccharide of gangliosides GM1 and GM3, and makes possible the introduction of unnatural sphingoids or sphingoid analogs. In the second procedure, lysogangliosides are synthesized, which lack the fatty acyl residue, but contain the natural composition of sphingoids of their parent gangliosides. The chapter then describes radiochemical labeling of lysogangliosides. There is also a discussion on the various materials used in the general methods for the synthesis of gangliosides.

INTRACELLULAR DISTRIBUTION OF A BIOTIN-LABELED GANGLIOSIDE, GM1, BY IMMUNOELECTRON MICROSCOPY AFTER ENDOCYTOSIS IN FIBROBLASTS

A radioactive and biotin-labeled analogue of GM1 (biotin-GM1) was synthesized which enabled us to analyze its intracellular distribution in the compartments of the endocytic route by electron microscopic immunocytochemistry using thin sections of human skin fibroblasts labeled with gold-conjugated antibiotin antibodies. Metabolic studies with the biotin-GM1 showed its partial degradation to the corresponding GM2 and GM3 derivatives. Further degradation was inhibited by the biotin residue. The distribution of biotin-GM1 after uptake by cells was studied by postembedding labeling techniques. On the plasma membrane the biotin-GM1 was detectable in the form of patches (0.1 μm in diameter), in caveola-like structures and, to a much lesser extent, in coated pits or vesicles. During endocytic uptake, the biotin-GM1 became detectable in organelles identified as late endosomes and lysosomes. The intracellular distribution of the biotin-GM1 was compared to the localization of the EGF receptor in EGF-stimulated fibroblasts. Both the biotin-GM1 and the EGF receptor were transported to intraendosomal and intralysosomal membranes, indicating that both membrane constituents follow the same pathway of endocytosis. Our observations show that biotin-GM1 can be successfully incorporated into the plasma membrane and be used as a tool for morphological detection of its pathway to lysosomes. (J Histochem Cytochem 47:1005–1014, 1999)

Demonstration of Direct Glycosylation of Nondegradable Glucosylceramide Analogs in Cultured Cells

After uptake by various cells (human skin fibroblasts, rat neuroblastoma B 104, human neuroblastoma SHSY5Y, murine cerebellar cells), a radioactive and a fluorescent analog of a nondegradable glucosylceramide with sulfur in the glycosidic link were glycosylated to a cell-specific pattern of glycolipid analogs. These results, for the first time, show that a glucosylceramide analog can be conveyed from the plasma membrane of cultured cells to those Golgi compartments that function in the early glycosylation steps of glycolipids. This observation is further confirmed by the fact that the cationic ionophore monensin, known to impede membrane flow from proximal to distal Golgi cisternae, inhibited the formation of complex ganglioside analogs but not those of lactosylceramide, sialyl lactosylceramide (GM3), and disialyl lactosylceramide (GD3).

Identity of GA1, GM1a and GD1b synthase in Golgi vesicles from rat liver

Synthesis of ganglioside GDD1b from ganglioside GD2 was demonstrated using Golgi membranes isolated from rat liver. Competition experiments using gangliosides GA2, GM2 and GD2 as substrates, and as mutual inhibitors for ganglioside galactosyltransferase activity in preparations of Golgi vesicles derived from rat liver, suggested that galactosyl transfer to these three compounds, leading to gangliosides GA1, GM1a and GD1b respectively, is catalyzed by one enzyme. These results strengthen the hypothesis that the main site for the regulation of ganglioside biosynthesis occurs within the reaction sequence LacCer→GA3→GD3→GT3.

Synthesis of fluorescent and radioactive analogues of two lactosylceramides and glucosylceramide containing β-thioglycosidic bonds that are resistant to enzymatic degradation

Condensation of 2-S-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-2- thiopseudourea hydrobromide with 2,3,6-tri-O-benzoyl-4-O-trifluoromethylsulfonyl-beta-D-galactopyra nosyl- (1-->1)-(2S,3R,4E)-3-O-benzoyl-2-dichloroacetamido-4-octa decen-1,3-diol afforded S-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->4)-2,3,6-tri-O- benzoyl-4-thio-beta-D-glucopyranosyl-(1-->1)-(2S,3R,4E)-3-O-benzoy l-2- dichloroacetamido-4-octadecen-1,3-diol in good yield. Removal of the protecting groups, followed by selective N-acylation of the sphingosine amino group with either a fluorescent or a radioactive fatty acid, gave labeled lactosylceramide analogues in good yield. Since these products contained a beta-thioglycosidic bond between the two sugar moieties, they were totally resistant to the action of acid lysosomal glycosidases. Likewise, condensation of 2-S-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-2- thiopseudourea hydrobromide and 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->4)-2,3,6- tri-O-acetyl-1-S-acetyl-1-thio-beta-D-glucopyranose with (2R,3R,4E)-3-O-benzoyl-2-dichloroacetamido-1-iodo-4-octad ecen-3-ol in methanolic sodium acetate afforded the corresponding beta-thioglycosides 14 and 16, respectively, in good yield. These beta-thioglycosides were converted into glucosylceramide and lactosylceramide analogues following removal of the protecting groups and by subsequent selective N-acylation using either a fluorescent or adioactive fatty acid N-succinimidyl ester. Whereas the glucosylthioceramides thus obtained proved to be completely undegradable by lysosomal glucocerebrosidase, the lactosylceramides containing the beta-thioglycosidic bond between the lactose and the ceramide residues could be degraded by lysosomal GM1-beta-galactosidase to give the corresponding glucosylthioceramides. These compound did not yield to any further enzymatic degradation.

Gangliosides are transported from the plasma membrane to intralysosomal membranes as revealed by immuno-electron microscopy.

A biotin-labeled derivative of the ganglioside GM1 (biotin-GM1) was used to study its transport along the endocytic pathway of cultured fibroblasts by immuno-electron microscopy. Using electron dense endocytic tracers we could demonstrate that late endosomes and lysosomes of these cells are long living organelles with a high content of internal membranes. Our studies show that during endocytosis the biotin-GM1 was transported to these intraendosomal and intralysosomal membranes. These observations support the hypothesis that glycosphingolipids (GSL) are preferentially degraded in intralysosomal vesicles.

In Vitro Incorporation and Metabolism of Gangliosides

Gangliosides are ubiquitous in vertebrate tissue and are highly abundant in neuronal plasma membranes (for review see 1–3). The lipophilic ceramide moiety of gangliosides is anchored in the outer leaflet of the lipid bilayer and the hydrophilic sialooligosaccharide residue faces the extracellular space.

Labeled chemical biology tools for investigating sphingolipid metabolism, trafficking and interaction with lipids and proteins ☆

The unraveling of sphingolipid metabolism and function in the last 40 years relied on the extensive study of inherited human disease and specifically-tailored mouse models. However, only few of the achievements made so far would have been possible without chemical biology tools, such as fluorescent and/or radio-labeled and other artificial substrates, (mechanism-based) enzyme inhibitors, cross-linking probes or artificial membrane models. In this review we provide an overview over chemical biology tools that have been used to gain more insight into the molecular basis of sphingolipid-related biology. Many of these tools are still of high relevance for the investigation of current sphingolipid-related questions, others may stimulate the tailoring of novel probes suitable to address recent and future issues in the field. This article is part of a Special Issue entitled Tools to study lipid functions.