Sabine Dieluweit

Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification

Efficient delivery of biomolecules into membranes of living cells as well as cell surface modifications are major biotechnological challenges. Here, novel liposome systems based on neutral and cationic lipids in combination with lipids modified by aromatic groups are introduced for such applications. The fusion efficiency of these liposome systems was tested on single cells in culture like HEK293, myofibroblasts, cortical neurons, human macrophages, smooth muscle cells, and even on tissue. Fusogenic liposomes enabled highly efficient incorporation of molecules into mammalian cell membranes within 1 to 30 min at fully unchanged cell growth conditions and did not affect cell behavior. We hypothesize that membrane fusions were induced in all cases by the interaction of the positively charged lipids and the delocalized electron system of the aromatic group generating local dipoles and membrane instabilities. Selected applications ranging from basic research to biotechnology are envisaged here.

Mechanical Properties of Bare and Protein-Coated Giant Unilamellar Phospholipid Vesicles. A Comparative Study of Micropipet Aspiration and Atomic Force Microscopy

In this study, protein-coated giant phospholipid vesicles were used to model cell plasma membranes coated by surface protein layers that increase membrane stiffness under mechanical or osmotic stress. These changed mechanical properties like bending stiffness, membrane area compressibility modulus, and effective Youngs modulus were determined by micropipet aspiration, while bending stiffness, effective Youngs modulus, and effective spring constant of vesicles were analyzed by AFM. The experimental setups, the applied models, and the results using both methods were compared here. As demonstrated before, we found that bare vesicles were best probed by micropipet aspiration due to its high sensitivity. The mechanical properties of vesicles with protein surface layers were, however, better determined by AFM because it enables very local deformations of the membrane with barely any structural damage to the protein layer. Mechanical properties of different species of coating proteins, here streptavidin and avidin, could be clearly distinguished using this technique.

Orientation dependence of the Cu(001) surface step stiffness: Failure of solid-on-solid and Ising models to describe experimental data

We have investigated the step stiffness on Cu(001) surfaces as a function of step orientation by two independent methods at several temperatures near 300 K. Both sets of data agree well and show a substantial dependence of the stiffness on the angle of orientation. With the exception of steps oriented along , the experimental stiffness is significantly larger than the stiffness calculated within the solid-on-solid model and the Ising model, even if next-nearest-neighbor interactions are taken into account. Our results have considerable consequences for the understanding and for the theoretical modeling of equilibrium and growth phenomena, such as step meandering instabilities.

On Pattern Transfer in Replica Molding

Nano- and micromolding of elastic materials produces smoothed replicas of the mold structures. This limits the techniques resolution. Here we identified surface tension as the cause of smoothing and derived explicit equations for calculating molded feature shapes. The characteristic length scale for smoothing is given by the ratio of the interface tension to Youngs modulus of the molded material. This approach offers the possibility to correct for the smoothing caused by surface tension during mold design. Moreover, it can be exploited to measure interface tension.

STM studies on the island dynamics on Au(100) electrodes in sulfuric acid

We have studied the dynamics of monolayer Au islands on a Au(100) electrode in sulfuric acid using electrochemical scanning tunnelling microscopy. We present quantitative studies of the equilibrium shape of Au islands on Au(100) electrodes and of the thermal fluctuations around the equilibrium shape. As was recently demonstrated for islands on metal surfaces under ultra-high vacuum (UHV), step and kink energies may be determined. Furthermore, we present data on the decay of Au islands on Au(100) electrodes due to Ostwald ripening and due to the reformation of the potential-induced reconstruction on the gold electrode. In both cases we observe that different dominant mass transport processes control the island decay. In the case of island decay during potential-induced reformation of the reconstruction, we find furthermore evidence for rapid island decay processes as were recently reported for metal surfaces in UHV studies.

Potential dependence of step and kink energies on Au(100) electrodes in sulfuric acid

Using electrochemical STM we studied monolayer high Au islands on Au(100) electrodes in sulfuric acid as a function of the electrode potential. We made use of theoretical and experimental methods recently developed for UHV experiments on metal islands. It is demonstrated that these models are likewise applicable to islands on metal electrodes in a liquid environment. From a quantitative analysis of the equilibrium island shape and of the island shape fluctuations we determined the step free energy (line tension) as a function of orientation and the kink energy, and the dependence of these quantities on the electrode potential. In a first approach to a theoretical understanding the electrostatic contributions to the line tension are considered. It is concluded that these contributions should add significantly to the observed variation with the potential. This fails however to provide essential features of the experimental result.

Determination of step and kink energies on Au(100) electrodes in sulfuric acid solutions by island studies with electrochemical STM

We have studied monolayer Au islands on a Au(100) electrode in sulfuric acid solution using electrochemical scanning tunneling microscopy. We present quantitative studies of the equilibrium shape of Au islands on Au(100) electrodes and of island shape fluctuations. As was recently demonstrated for islands on metal surfaces under ultra-high vacuum, step and kink energies may be determined. Furthermore, we have measured the angle dependence of the step energy and the curvature of the quasi-straight island segments as a function of the electrode potential. We find that the step and the kink energies are potential dependent.

Identification of Key Enzymes for Pectin Synthesis in Seed Mucilage

Mutations in two glycosyltransferase-encoding genes severely impair the elongation of pectic rhamnogalacturonan I, resulting in hydrophobic seeds that do not release mucilage polymers. Pectin is a vital component of the plant cell wall and provides the molecular glue that maintains cell-cell adhesion, among other functions. As the most complex wall polysaccharide, pectin is composed of several covalently linked domains, such as homogalacturonan (HG) and rhamnogalacturonan I (RG I). Pectin has widespread uses in the food industry and has emerging biomedical applications, but its synthesis remains poorly understood. For instance, the enzymes that catalyze RG I elongation remain unknown. Recently, a coexpression- and sequence-based MUCILAGE-RELATED (MUCI) reverse genetic screen uncovered hemicellulose biosynthetic enzymes in the Arabidopsis (Arabidopsis thaliana) seed coat. Here, we use an extension of this strategy to identify MUCI70 as the founding member of a glycosyltransferase family essential for the accumulation of seed mucilage, a gelatinous wall rich in unbranched RG I. Detailed biochemical and histological characterization of two muci70 mutants and two galacturonosyltransferase11 (gaut11) mutants identified MUCI70 and GAUT11 as required for two distinct RG I domains in seed mucilage. We demonstrate that, unlike MUCI70, GAUT11 catalyzes HG elongation in vitro and, thus, likely is required for the synthesis of an HG region important for RG I elongation. Analysis of a muci70 gaut11 double mutant confirmed that MUCI70 and GAUT11 are indispensable for the production and release of the bulk of mucilage RG I and for shaping the surface morphology of seeds. In addition, we uncover relationships between pectin and hemicelluloses and show that xylan is essential for the elongation of at least one RG I domain.

Chemically defined, ultrasoft PDMS elastomers with selectable elasticity for mechanobiology

Living animal cells are strongly influenced by the mechanical properties of their environment. To model physiological conditions ultrasoft cell culture substrates, in some instances with elasticity (Youngs modulus) of only 1 kPa, are mandatory. Due to their long shelf life PDMS-based elastomers are a popular choice. However, uncertainty about additives in commercial formulations and difficulties to reach very soft materials limit their use. Here, we produced silicone elastomers from few, chemically defined and commercially available substances. Elastomers exhibited elasticities in the range from 1 kPa to 55 kPa. In detail, a high molecular weight (155 kg/mol), vinyl-terminated linear silicone was crosslinked with a multifunctional (f = 51) crosslinker (a copolymer of dimethyl siloxane and hydrosilane) by a platinum catalyst. The following different strategies towards ultrasoft materials were explored: sparse crosslinking, swelling with inert silicone polymers, and, finally, deliberate introduction of dangling ends into the network (inhibition). Rheological experiments with very low frequencies led to precise viscoelastic characterizations. All strategies enabled tuning of stiffness with the lowest stiffness of ~1 kPa reached by inhibition. This system was also most practical to use. Biocompatibility of materials was tested using primary cortical neurons from rats. Even after several days of cultivation no adverse effects were found.

TRM4 is essential for cellulose deposition in Arabidopsis seed mucilage by maintaining cortical microtubule organization and interacting with CESA3

Summary The differentiation of the seed coat epidermal (SCE) cells in Arabidopsis thaliana leads to the production of a large amount of pectin‐rich mucilage and a thick cellulosic secondary cell wall. The mechanisms by which cortical microtubules are involved in the formation of these pectinaceous and cellulosic cell walls are still largely unknown. Using a reverse genetic approach, we found that TONNEAU1 (TON1) recruiting motif 4 (TRM4) is implicated in cortical microtubule organization in SCE cells, and functions as a novel player in the establishment of mucilage structure. TRM4 is preferentially accumulated in the SCE cells at the stage of mucilage biosynthesis. The loss of TRM4 results in compact seed mucilage capsules, aberrant mucilage cellulosic structure, short cellulosic rays and disorganized cellulose microfibrils in mucilage. The defects could be rescued by transgene complementation of trm4 alleles. Probably, this is a consequence of a disrupted organization of cortical microtubules, observed using fluorescently tagged tubulin proteins in trm4 SCE cells. Furthermore, TRM4 proteins co‐aligned with microtubules and interacted directly with CELLULOSE SYNTHASE 3 in two independent assays. Together, the results indicate that TRM4 is essential for microtubule array organization and therefore correct cellulose orientation in the SCE cells, as well as the establishment of the subsequent mucilage architecture.