Modulating vesicle adhesion by electric fields

We introduce an experimental setup for modulating adhesion of giant unilamellar vesicles to a planar substrate. Adhesion is induced by the application of an external potential to a transparent indium tin oxide-coated electrode (the substrate), which enables single-vesicle studies. We demonstrate tunable and reversible adhesion of negatively charged vesicles. The adhesion energy at different potentials is calculated from the vesicle shape assessed with confocal microscopy. Two approaches for these estimates are employed: one based on the whole contour of the vesicle and a second based on the contact curvature of the membrane in the vicinity of the substrate. Both approaches agree well with each other and show that the adhering vesicles are in the weak adhesion regime for the range of explored external potentials. Using fluorescence quenching assays, we detect that, in the adhering membrane segment, only the outer bilayer leaflet of the vesicle is depleted of negatively charged fluorescent lipids, while the inner leaflet remains unaffected. We show that depletion of negatively charged lipids is consistent Poisson-Boltzmann theory, taking into account charge regulation from lipid mobility. Finally, we also show that lipid diffusion is not significantly affected in the adhering membrane segment. We believe that the approaches introduced here for modulating and assessing vesicle adhesion have many potential applications in the field of single-vesicle studies and research on membrane adhesion

Binding of His-tagged fluorophores to lipid bilayers and giant vesicles

His-tagged molecules can be attached to lipid bilayers via certain anchor lipids, a method that has been widely used for the biofunctionalization of membranes and vesicles. To measure the coverage by the membrane-bound molecules, it is useful to study molecules that are fluorescent as well. Here, we use two such molecules, green fluorescence protein (GFP) and green-fluorescent fluorescin isothiocyanate (FITC), both of which are tagged with a chain of six histidines that bind to achor lipids within the bilayers. This His-tag is much smaller in size than the GFP molecule but somewhat larger than the FITC dye. The lipid bilayers form giant unilamellar vesicles (GUVs), the behavior of which can be directly observed in the optical microscope. Several protocols for the preparation of GUVs have been developed. We apply and compare three well-established protocols based on polyvinyl alcohol (PVA) hydrogel swelling, electroformation on platinum wires, and electroformation on indium tin oxide (ITO) glass. For the same nanomolar concentration in the exterior solution, the coverage by His-tagged FITC is much lower than the one by His-tagged GFP. However, for both GFP and FITC, we find that the binding of the His-tagged molecules to the anchor lipids depends strongly on the preparation method. The highest binding affinitiy is obtained for electroformation on platinum wires. PVA gel swelling gives rise to a somewhat smaller binding affinity whereas electroformation on ITO glass leads to essentially no binding. Furthermore, the binding affinitiy is also observed to depend on the pH of the aqueous solution, with a relatively weak and strong pH-dependence for His-tagged GFP and His-tagged FITC, respectively

Super-resolution imaging of highly curved membrane structures in giant vesicles encapsulating molecular condensates

Molecular crowding is an inherent feature of the cell interior. Synthetic cells as provided by giant unilamellar vesicles (GUVs) encapsulating macromolecules (polyethylene-glycol and dextran) represent an excellent mimetic system to study membrane transformations associated with molecular crowding and protein condensation. Similarly to cells, such GUVs loaded with macromolecules exhibit highly curved structures such as internal nanotubes. In addition, upon liquid-liquid phase separation as inside living cells, the membrane of GUVs encapsulating an aqueous two-phase system deforms to form apparent kinks at the contact line of the interface between the two aqueous phases. These structures, nanotubes and kinks, have dimensions below optical resolution and if resolved, can provide information about material properties such as membrane spontaneous curvature and intrinsic contact angle describing the wettability contrast of the encapsulated phases to the membrane. Previous experimental studies were based on conventional optical microscopy which cannot resolve these membrane and wetting properties. Here, we studied these structures with super-resolution microscopy, namely stimulated emission depletion (STED) microscopy, together with microfluidic manipulation. We demonstrate the cylindrical nature of the nanotubes with unprecedented detail based on the superior resolution of STED and automated data analysis. The spontaneous curvature deduced from the nanotube diameters is in excellent agreement with theoretical predictions. Furthermore, we were able to resolve the membrane “kink” structure as a smoothly curved membrane demonstrating the existence of the intrinsic contact angle. We find very good agreement between the directly measured values and the theoretically predicted ones based on the apparent contact angles on the micrometer scale. During different stages of cellular events, biomembranes undergo a variety of shape transformations such as the formation of buds and nanotubes regulated by membrane necks. We demonstrate that these highly curved membrane structures are amenable to STED imaging and show that such studies provide important insights in the membrane properties and interactions underlying cellular activities

Expanding Thurston Maps

We study the dynamics of Thurston maps under iteration. These are branched covering maps $f$ of 2-spheres $S^2$ with a finite set $\mathop{post}(f)$ of postcritical points. We also assume that the maps are expanding in a suitable sense. Every expanding Thurston map $f\: S^2 \to S^2$ gives rise to a type of fractal geometry on the underlying sphere $S^2$. This geometry is represented by a class of \emph{visual metrics} $\varrho$ that are associated with the map. Many dynamical properties of the map are encoded in the geometry of the corresponding {\em visual sphere}, meaning $S^2$ equipped with a visual metric $\varrho$. For example, we will see that an expanding Thurston map is topologically conjugate to a rational map if and only if $(S^2, \varrho)$ is quasisymmetrically equivalent to the Riemann sphere $\widehat{\mathbf{C}}$. We also obtain existence and uniqueness results for $f$-invariant Jordan curves $\mathcal{C}\subset S^2$ containing the set $\mathop{post}(f)$. Furthermore, we obtain several characterizations of Latt\`{e}s maps

Kooperation in der universitären Lehrerbildung :Der Einfluss von Lehrveranstaltungen auf die Einstellungen der Studierenden zur kollegialen Zusammenarbeit im Lehrberuf

Seit längerer Zeit sind kooperative Arbeitsstrukturen für die Schul- und Unterrichtsentwicklung sowie die Professionalisierung von Lehrkräften bedeutsam. Um diese in der universitären Lehrerbildung zu etablieren und die Lehramtsstudierenden auf kollegiale Kooperationen im zukünftigen Schulalltag vorzubereiten, wurde im Kontext der Qualitätsoffensive Lehrerbildung an der WWU Münster ein Seminar zur Kooperation im Lehrberuf in den Bildungswissenschaften entwickelt, welches sowohl eine theoretisch-konzeptionelle und empirische Auseinandersetzung mit dem Thema beinhaltet als auch die eigene Teamarbeit der angehenden Lehrkräfte nach dem Konzept der Professionellen Lerngemeinschaft fördert. Im Fokus der vorliegenden Arbeit steht (1) die Evaluation der Lehrveranstaltung hinsichtlich des Seminarprogramms und der Kooperationsbereitschaft der Studierenden sowie (2) die Analyse der Einstellungsveränderungen zur kollegialen Kooperation durch die Seminarteilnahme

Giant vesicles: a biomimetic tool for assessing membrane material properties and interactions

Giant unilamellar vesicles (GUVs) have sizes in the range of 10-100 μm, which defines their unique property: they are visible under a light microscope. GUVs provide a handy biomimetic tool for directly displaying the response of the membrane on the cellsize scale. They represent model biomembrane systems for systematic measurements of mechanical and rheological properties of lipid bilayers as a function of membrane composition and phase state, surrounding media, and temperature. Here, we first summarize methods for preparing GUVs and their observation. Then, we introduce different experimental techniques which can yield precise values of membrane material characteristics such as mechanical properties (bending rigidity, stretching elasticity, lysis tension, and spontaneous curvature) and rheology (fluidity and viscosity of the membrane). Design, setup, practical tips, and evaluation of such experiments are discussed. An example on vesicle immobilization facilitating such measurements is also introduced

A mental model for the solution of the direct and inverse kinematic problem

Steinkühler U, Deitert J, Cruse H. A mental model for the solution of the direct and inverse kinematic problem. In: Proceed. of the ESANN 93. 1993