Highly parallel transport recordings on a membrane-on-nanopore chip at single molecule resolution.

Membrane proteins are prime drug targets as they control the transit of information, ions, and solutes across membranes. Here, we present a membrane-on-nanopore platform to analyze nonelectrogenic channels and transporters that are typically not accessible by electrophysiological methods in a multiplexed manner. The silicon chip contains 250,000 femtoliter cavities, closed by a silicon dioxide top layer with defined nanopores. Lipid vesicles containing membrane proteins of interest are spread onto the nanopore-chip surface. Transport events of ligand-gated channels were recorded at single-molecule resolution by high-parallel fluorescence decoding.

Shiga toxin induces tubular membrane invaginations for its uptake into cells.

Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit-Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.

Complement activation by PEG-functionalized multi-walled carbon nanotubes is independent of PEG molecular mass and surface density.

Carboxylated (4%) multi-walled carbon nanotubes were covalently functionalized with poly(ethylene glycol)1000 (PEG1000), PEG1500 and PEG4000 with a PEG loading of approximately 11% in all cases. PEG loading generated non-uniform and heterogeneous higher surface structures and increased nanotube width considerably, but all PEGylated nanotube species activated the complement system in human serum equally. Increased PEG loading, through adsorption of methoxyPEG2000(or 5000)-phospholipid conjugates, generated fewer complement activation products; however, complement activation was never completely eliminated. Our observations address the difficulty in making carbon nanotubes more compatible with innate immunity through covalent PEG functionalization as well as double PEGylation strategies. FROM THE CLINICAL EDITOR: Complement-mediated toxicity is a major limiting factor in certain nanomedicine applications. This study clarifies that PEGylation of carbon nanotubes is unlikely to address this complication.

Permeation through phospholipid bilayers, skin-cell penetration, plasma stability, and CD spectra of α- and ß-oligoproline derivatives.

After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-ß(3)-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-ß(2)h- and oligo-ß(3)h-prolines without and with fluorescence labels (1-8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2-4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6-8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9-11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.

Cross-linked self-assembled micelle based nanosensor for intracellular pH measurements.

A micelle based nanosensor was synthesized and investigated as a ratiometric pH sensor for use in measurements in living cells by fluorescent microscopy. The nanosensor synthesis was based on self-assembly of an amphiphilic triblock copolymer, which was chemically cross-linked after micelle formation. The copolymer, poly(ethylene glycol)-b-poly(2-aminoethyl methacrylate)-b-poly(styrene) (PEG-b-PAEMA-b-PS), was synthesized by isolated macroinitiator atom transfer radical polymerization that forms micelles spontaneously in water. The PAEMA shell of the micelle was hereafter cross-linked by an amidation reaction using 3,6,9-trioxaundecandioic acid cross-linker. The cross-linked micelle was functionalized with two pH sensitive fluorophores and one reference fluorophore, which resulted in a highly uniform ratiometric pH nanosensor with a diameter of 29 nm. The use of two sensor fluorophores provided a sensor with a very broad measurement range that seems to be influenced by the chemical design of the sensor. Cell experiments show that the sensor is capable of monitoring the pH distributions in HeLa cells.

Lipid reorganization induced by Shiga toxin clustering on planar membranes.

The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb(3) in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb(3) clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb(3) lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol/Gb(3) (65:30:5) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb(3) (40:35:20:5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb(3), STxB-Gb(3) complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.

Influence of alpha-hydroxylation of glycolipids on domain formation in lipid monolayers.

By means of fluorescence and scanning force microscopy (SFM), we investigated the phase behavior of lipid monolayers composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin, and cholesterol (5/2/3) with either alpha-hydroxylated or nonhydroxylated galactocerebroside. Fluorescence images of lipid monolayers at the air-water interface demonstrate that, independent of the lipid mixture, phase separation occurs at low surface pressure up to 4-6 mN m(-1), while an almost homogeneous phase is observed at larger surface pressures. However, by means of SFM of lipid monolayers transferred by the Langmuir-Blodgett technique at around 30 mN m(-1), nanometer-sized domains became discernible in those lipid mixtures that contained galactocerebroside, while, in that without a glycolipid, no such domain formation was visible. Moreover, the alpha-hydroxy group of the galactocerebroside alters the size and the total area of the domains significantly.