[Show abstract][Hide abstract] ABSTRACT: Giant unilamellar vesicles (GUVs) represent a versatile in vitro system widely used to study properties of lipid membranes and their interaction with biomacromolecules and colloids. Electroformation with indium tin oxide (ITO) coated coverslips as electrodes is a standard approach to GUV production. In the case of cationic GUVs, however, application of this approach leads to notorious difficulties. We discover that this is related to aging of ITO-coated coverslips during their repeated use, which is reflected in their surface topography on the nanoscale. We find that mild annealing of the ITO-coated surface in air reverts the effects of aging and ensures efficient reproducible electroformation of supergiant (diameter > 100 μm) unilamellar vesicles containing cationic lipids.
[Show abstract][Hide abstract] ABSTRACT: We report on a minimal system to mimic intracellular transport of membrane-bounded, vesicular cargo. In a cell-free assay, purified kinesin-1 motor proteins were directly anchored to the membrane of giant unilamellar vesicles, and their movement studied along two-dimensional microtubule networks. Motion-tracking of vesicles with diameters of 1-3 μm revealed traveling distances up to the millimeter range. The transport velocities were identical to velocities of cargo-free motors. Using total internal reflection fluorescence (TIRF) microscopy, we were able to estimate the number of GFP-labeled motors involved in the transport of a single vesicle. We found that the vesicles were transported by the cooperative activity of typically 5-10 motor molecules. The presented assay is expected to open up further applications in the field of synthetic biology, aiming at the in vitro reconstitution of sub-cellular multi-motor transport systems. It may also find applications in bionanotechnology, where the controlled long-range transport of artificial cargo is a promising means to advance current lab-on-a-chip systems.
[Show abstract][Hide abstract] ABSTRACT: In Escherichia coli, the pole-to-pole oscillation of the Min proteins directs septum formation to midcell, which is required for symmetric cell division. In vitro, protein waves emerge from the self-organization of MinD, a membrane-binding ATPase, and its activator MinE. For wave propagation, the proteins need to cycle through states of collective membrane binding and unbinding. Although MinD presumably undergoes cooperative membrane attachment, it is unclear how synchronous detachment is coordinated. We used confocal and single-molecule microscopy to elucidate the order of events during Min wave propagation. We propose that protein detachment at the rear of the wave, and the formation of the E-ring, are accomplished by two complementary processes: first, local accumulation of MinE due to rapid rebinding, leading to dynamic instability; and second, a structural change induced by membrane-interaction of MinE in an equimolar MinD-MinE (MinDE) complex, which supports the robustness of pattern formation.
[Show abstract][Hide abstract] ABSTRACT: We describe a previously unreported coil-globule transition of DNA electrostatically bound to a freestanding fluid cationic lipid membrane. The collapse of a DNA coil into a compact globule takes place after the DNA molecule attaches in an extended conformation to the membrane. DNA condensation is favored at a higher cationic lipid content, while at lower membrane charge densities coexistence of DNA random coils, partially collapsed conformations, and globules is observed.
[Show abstract][Hide abstract] ABSTRACT: We describe a previously unreported coil-globule transition of DNA electrostatically bound to a freestanding fluid cationic lipid membrane. The collapse of a DNA coil into a compact globule takes place after the DNA molecule attaches in an extended conformation to the membrane. DNA condensation is favored at a higher cationic lipid content, while at lower membrane charge densities coexistence of DNA random coils, partially collapsed conformations, and globules is observed. Understanding the interaction of polyelectrolytes with oppositely charged lipid membranes is an important issue of soft matter physics, which provides an insight into mechanisms of interactions of biological macromolecules with cell membranes. Although the question has been addressed during the past decade both experimentally and theoretically [1,2], the understanding is far from com-plete, and some important unresolved questions, including the effects of the membrane local curvature and bending elasticity, remain to be addressed. A perfect model polymer to study electrostatic polyelectrolyte-membrane interactions is double-stranded DNA: it is a semiflexible polyelectrolyte carrying two negative charges per base pair (bp)  whose length and structure can be precisely controlled using the modern biotechnological methods; in addition, it allows for easy fluorescence labeling which facilitates single-molecule microscopy experiments. These advantages were used in a seminal work  where it was demonstrated that DNA molecules adsorbed at a fluid cationic lipid bilayer on a flat support assume a 2D random coil conformation and exercise translational Brownian motion. These results have since become a text-book example of polymer coil dynamics in 2D . A completely different picture is observed when double-stranded DNA interacts with small (20–100 nm) cationic liposomes: In this case DNA molecules wrap around lipo-somes and eventually form densely packed liquid crystal-line DNA-lipid globules  with the typical size of $100–200 nm [7,8]. Formation of DNA-lipid globules is an example of a more general phenomenon known as DNA condensation [9,10]. DNA condensation by cationic lipo-somes has attracted particular attention in view of its potential use in gene therapy  and importance for understanding the prebiotic chemistry . The striking contrast between the behavior of DNA at flat supported cationic lipid bilayers and at strongly curved small cationic liposomes naturally leads to the question of what kind of behavior can be expected upon interaction of DNA with freestanding (unsupported) cationic lipid bi-layers. The main differences between the supported and freestanding lipid bilayers are (i) the ability of the free-standing membrane to respond by an elastic deformation to an external mechanical force , which is strongly sup-pressed in the case of a supported lipid bilayer , and (ii) the high lipid mobility within the freestanding fluid lipid bilayer, which can be strongly inhibited by the solid support . Recent experiments on interaction of DNA with cationic membranes supported on structured surfaces demonstrated the importance of the local bilayer curvature in DNA-membrane interactions . Obviously, freestand-ing bilayers, capable of bending locally in response to an external perturbation, may show new unexpected ways of interaction with charged semiflexible DNA molecules. Surprisingly, very little is known about interaction of DNA with freestanding cationic lipid bilayers. To the best of the authors' knowledge, the only study in this direction was carried out in a series of works . The experimental approach used in these works could not, however, provide any information on conformation and dynamics of single DNA molecules. In this Letter we describe a previously unreported phe-nomenon of coil-globule transition of DNA molecules electrostatically bound to a freestanding fluid cationic lipid membrane. To model the flat freestanding fluid cationic lipid bilayer we used giant unilamellar vesicles (GUVs) with sizes in the range of 100–300 m (Fig. 1). At these sizes, the free membrane surface is essentially flat on the micrometer scale and can be directly confronted to the flat supported membrane. Both GUVs and DNA molecules were fluores-cently labeled at distinct spectral ranges, which allowed us to carry out single-molecule fluorescence microscopy experiments. Double-stranded DNA fragments with lengths of 5, 10, and 20 kbp, as well as -DNA (48.5 kbp), were obtained from Fermentas Life Sciences. DNA samples were fluo-rescently stained using the YOYO-1 dye (Molecular Probes) at the ratio of 0:2 dye=bp (reducing the staining ratio to 0:05 dye=bp did not affect our results within ex-perimental uncertainty). Giant unilamellar vesicles with the membrane in the fluid phase were produced from