Stefan Kaufmann

Native supported membranes on planar polymer supports and micro-particle supports

To bridge soft biological materials and hard inorganic materials is an interdisciplinary scientific challenge. Despite of experimental difficulties, the deposition of native biological membranes on supports is a straightforward strategy. This review provides an overview of advances in the fabrication and characterization of native biological membranes on planar polymer supports and micro-particles.

Covalent modification of chitin with silk-derivatives acts as an amphiphilic self-organizing template in nacre biomineralisation

Molluscs have a well-deserved reputation for being expert mineralizers of various shell types such as nacre. Nacre is defined as regularly arranged layers and stacks of approximately 0.5 microm thick aragonite platelets that are extracellularly formed within a complex mixture of organic matrix. The control of species-specific layer thickness by the animal is still enigmatic. Despite the recent findings on the periodic layer-by-layer structures of chitin layers and silk-like protein layers in nacre-type biominerals, little is known about how the interface is defined between two different layers. In this paper, we demonstrate the presence of covalently attached, hydrophobic amino acid side chains in the chitin matrix in the bivalve mollusc Mytilus galloprovincialis by the combination of infrared spectroscopy and mass spectroscopy. The accumulation of the modified chitin matrix at the interface is quantified by the critical aggregate concentration of the purified chitin matrix, which is approximately an order of magnitude smaller than that of pure chitin. Our finding suggests an active role of such chemically modified chito-oligosaccharides in the creation of a defined interface and guidance of the periodic matrix textures, which would result in unique material properties of natural mollusc shells.

Native supported membranes: Creation of two-dimensional cell membranes on polymer supports (Review)

In nature, membranes serve as fundamental platforms for many important biological processes, such as i hormone transduction and amplification through generation of second messengers, ii biosynthesis by membrane associated ribosomes at the endoplasmic reticulum, iii control of cell adhesion via protein-protein recognition, and iv lateral segregation of cell-surface receptors and the associated reorganization of the membrane-coupled cytoskeleton. Phospholipid bilayers deposited onto solid substrates called solid-supported membranes have been the simplest and most commonly used experimental model systems that allow us to gain insights into structure-function relationships in biological membranes. Supported membranes retain the intrinsic “fluid” nature to self-heal local defects and achieve an excellent mechanical stability. These properties offer distinct advantages over either freestanding black lipid membranes or spherical lipid vesicle suspensions: they can be subjected to various surface-sensitive physical characterization techniques such as ATR-FTIR, surface plasmon resonance, quartz crystal micro balance, and neutron reflectivity. However, in spite of great scientific achievements through solid-supported membranes, such systems have some fundamental drawbacks. Solid-supported membranes are confined at a deep potential minimum governed by van der Waals interactions and stay in close proximity of solid substrates. The water reservoir between the membrane and the underlying solid substrate, which is typically 5–20 A, is not thick enough to prevent proteins coming into direct contact with the bare substrate. In fact, this can cause a serious problem in dealing with transmembrane proteins, such as cell adhesion receptors, whose functional extracellular domains can extend to several tens of nanometers. In fact, the spreading of proteoliposomes doped with human platelet integrin IIb 3 and ATP synthase on quartz or glass substrates results in inhomogeneous patches of “pinned” proteins Fig. 1 a . Such problems can be overcome by separating the membrane from the solid substrate using soft polymeric spacer layers, whose thickness is less than 100 nm. Here, the membranes are separated from underlying solids via i polymer cushions or ii polymer/oligomer tethers. This

Dynamic Mechano-Regulation of Myoblast Cells on Supramolecular Hydrogels Cross-Linked by Reversible Host-Guest Interactions

A new class of supramolecular hydrogels, cross-linked by host-guest interactions between β-cyclodextrin (βCD) and adamantane, were designed for the dynamic regulation of cell-substrate interactions. The initial substrate elasticity can be optimized by selecting the molar fraction of host- and guest monomers for the target cells. Moreover, owing to the reversible nature of host-guest interactions, the magnitude of softening and stiffening of the substrate can be modulated by varying the concentrations of free, competing host molecules (βCD) in solutions. By changing the substrate elasticity at a desired time point, it is possible to switch the micromechanical environments of cells. We demonstrated that the Young’s modulus of our “host-guest gels”, 4–11 kPa, lies in an optimal range not only for static (ex situ) but also for dynamic (in situ) regulation of cell morphology and cytoskeletal ordering of myoblasts. Compared to other stimulus-responsive materials that can either change the elasticity only in one direction or rely on less biocompatible stimuli such as UV light and temperature change, our supramolecular hydrogel enables to reversibly apply mechanical cues to various cell types in vitro without interfering cell viability.

Lipid-coated mesoporous silica microparticles for the controlled delivery of β-galactosidase into intestines

β-Galactosidase has been drawing increasing attention for the treatment of lactose intolerance, but its delivery has been impeded by degradation under gastric conditions. We have demonstrated that the coating of mesoporous silica microparticles (diameter ≈ 9 µm, pore size ≈ 25 nm) with dioleoylphosphatidylcholine membranes significantly improved the loading capability and protected the enzymes from the loss of function under simulated gastric conditions. Once the particles are transferred to simulated intestinal conditions, the digestion of phosphatidylcholine with pancreatin led to the release of functional β-galactosidase. The coating of mesoporous silica nanoparticles with a single phospholipid bilayer opens up a large potential towards the controlled release of orally administrated drugs or enzymes to the intestines.

Functional expression of Ca2+ dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum

In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms.