Dynamics of membrane nanotubes coated with I-BAR.

Membrane deformation is a necessary step in a number of cellular processes such as filopodia and invadopodia formation and has been shown to involve membrane shaping proteins containing membrane binding domains from the IRSp53-MIM protein family. In reconstituted membranes the membrane shaping domains can efficiently deform negatively charged membranes into tubules without any other proteins present. Here, we show that the IM domain (also called I-BAR domain) from the protein ABBA, forms semi-flexible nanotubes protruding into Giant Unilamellar lipid Vesicles (GUVs). By simultaneous quantification of tube intensity and tubular shape we find both the diameter and stiffness of the nanotubes. I-BAR decorated tubes were quantified to have a diameter of ~50 nm and exhibit no stiffening relative to protein free tubes of the same diameter. At high protein density the tubes are immobile whereas at lower density the tubes diffuse freely on the surface of the GUV. Bleaching experiments of the fluorescently tagged I-BAR confirmed that the mobility of the tubes correlates with the mobility of the I-BAR on the GUV membrane. Finally, at low density of I-BAR the protein upconcentrates within tubes protruding into the GUVs. This implies that I-BAR exhibits strong preference for negatively curved membranes.

Vesicle Fusion Triggered by Optically Heated Gold Nanoparticles.

Membrane fusion can be accelerated by heating that causes membrane melting and expansion. We locally heated the membranes of two adjacent vesicles by laser irradiating gold nanoparticles, thus causing vesicle fusion with associated membrane and cargo mixing. The mixing time scales were consistent with diffusive mixing of the membrane dyes and the aqueous content. This method is useful for nanoscale reactions as demonstrated here by I-BAR protein-mediated membrane tubulation triggered by fusion.

Effect of salts, solvents and buffer on miRNA detection using DNA silver nanocluster (DNA/AgNCs) probes.

MicroRNAs (miRNAs) are small regulatory RNAs (size ~21 nt to ~25 nt) which regulate a variety of important cellular events in plants, animals and single cell eukaryotes. Especially because of their use in diagnostics of human diseases, efforts have been directed towards the invention of a rapid, simple and sequence selective detection method for miRNAs. Recently, we reported an innovative method for the determination of miRNA levels using the red fluorescent properties of DNA/silver nanoclusters (DNA/AgNCs). Our method is based on monitoring the emission drop of a DNA/AgNCs probe in the presence of its specific target miRNA. Accordingly, the accuracy and efficiency of the method relies on the sensitivity of hybridization between the probe and target. To gain specific and robust hybridization between probe and target, we investigated a range of diverse salts, organic solvents, and buffer to optimize target sensing conditions. Under the newly adjusted conditions, the target sensitivity and the formation of emissive DNA/AgNCs probes were significantly improved. Also, fortification of the Tris-acetate buffer with inorganic salts or organic solvents improved the sensitivity of the DNA/AgNC probes. On the basis of these optimizations, the versatility of the DNA/AgNCs-based miRNA detection method can be expanded.

Design aspects of bright red emissive silver nanoclusters/DNA probes for microRNA detection.

The influence of the nucleic acid secondary structure on the fast (1 h) formation of bright red emissive silver nanoclusters (AgNCs) in a DNA sequence (DNA-12nt-RED-160), designed for the detection of a microRNA sequence (RNA-miR160), was investigated. The findings show that especially the propensity for mismatch self-dimer formation of the DNA probes can be a good indicator for the creation and stabilization of red emissive AgNCs. Also, the role of the thermal stability of the secondary DNA structures (mismatch self-dimer and hairpin monomers) and the observed AgNC red emission intensity were investigated. These findings can form the basis for a rationale to design new red emissive AgNC-based probes. As an example, a bright red emissive AgNC-based DNA probe was designed for RNA-miR172 detection. The latter opens the possibility to create a variety of AgNC-based DNA probes for the specific detection of plant and animal miRNAs.