Generation of nanopores down to 10 nm for use in deep-nulling interferometry.

Scanning electron microscope images show that it is easy to generate nanopores on polycarbonate membranes with well-defined pore diameters by ion-track perforation and subsequent magnetron sputtering with metal. The size reduction of the nanopores during sputtering with gold is a linear function of time. Images of different angles and from the bottom side of the membrane show that the channels are the smallest very close to the surface of the metal layer, have a conelike shape, and reach about half as much into the polymer membranes as the metal-layer thickness. This topographical pore shape is ideal for use as optically coherent near-field sources in deep-nulling microscopy. We present the first results of significantly improved nulling stabilization in the presence (<2 nm optical pathway difference) and the absence (<0.6 nm optical pathway difference) of the nanoapertures in the focal region of a deep-nulling microscope.

Dynamic and flexible H3K9me3 bridging via HP1ß dimerization establishes a plastic state of condensed chromatin.

Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1ß is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1ß bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1ß genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 proteins, which might contribute to the plastic nature of condensed chromatin.

Single-particle identification of encoded nanospheres.

Recently, it has been shown that 2-photon fluorescence correlation spectroscopy of single glycosylated 20-nm fluorescent spheres allows measurement of the relative carbohydrate binding affinities of unlabeled proteins and that these modified spheres can mimic the glycocalix of cell or virus surfaces. An especially useful extension would be the analysis of mixtures of nanospheres that each contain different fluorescent labels and are thus differentially "encoded." If the surfaces of these encoded nanospheres are modified with various receptors, many different biomolecule-surface interactions and concurrent reactions can be measured quickly and simultaneously in a single-reaction vessel. An essential prerequisite for this general assay principle is the ability to identify with an accuracy of nearly 100% any encoded nanosphere present in a mixture on a single-particle level. Here the authors present a method that indeed allows certain identification of differently encoded nanospheres during single transits through the focal volume of a microscope objective (ø approximately 200-500 nm) in aqueous solution. This opens the way for using the encoded nanospheres in 1-well measurements of a large variety of biomolecular receptor-ligand interactions, inhibition and concurrent reactions, and thus either for testing the behavior of ligands in a mimicked complex biomolecular environment or for a fast simultaneous measurement of a multitude of receptor-ligand interactions.

Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes with multiple topologies.

SNARE proteins mediate membrane fusion in eukaryotic cells. They contain conserved SNARE motifs that are usually located adjacent to a C-terminal transmembrane domain. SNARE motifs spontaneously assemble into four helix bundles, with each helix belonging to a different subfamily. Liposomes containing SNAREs spontaneously fuse with each other, but it is debated how the SNAREs are distributed between the membranes. Here, we report that the SNAREs mediating homotypic fusion of early endosomes fuse liposomes in five out of seven possible combinations, in contrast to previously studied SNAREs involved in heterotypic fusion events. The crystal structure of the early endosomal SNARE complex resembles that of the neuronal and late endosomal complexes, but differs in surface side-chain interactions. We conclude that homotypic fusion reactions may proceed with multiple SNARE topologies, suggesting that the conserved SNARE structure allows for flexibility in the initial interactions needed for fusion.

A two-photon fluorescence-correlation study of lectins interacting with carbohydrated 20 nm beads.

We present results of a two-photon fluorescence-correlation study carried out with glycosylated and untreated 20 nm fluorescing spheres that interacted with the carbohydrate-binding proteins soybean agglutinin (SBA) and concanavalin A (Con A). The assay principle allows protein-carbohydrate binding interactions to be determined without protein labeling. This assay might serve as a simple model system for studying physical and chemical interactions between proteins and carbohydrates, for example, at cell or virus surfaces. In experiments with galactosylated 20 nm beads and SBA, several stages of protein-carbohydrate interactions could be clearly distinguished. Initially, only a few lectins bound to the nanospheres. At higher lectin concentrations polymerization occurred, and aggregates consisting of about 2.6 x 10(5) glycosylated nanospheres were formed. At very high lectin concentrations, the degree of polymerization dropped, and the size of single SBA-covered nanospheres increased to approximately 40 nm. When Con A was used instead of SBA, a significantly smaller degree of aggregation (4 x 10(4) spheres) was obtained. Treatment of unglycosylated 20 nm beads with SBA as a negative control sample resulted in a much lower unspecific aggregation (5 x 10(3) spheres). The assay principle can thus help to elucidate relative binding affinities.