Cell cultures in microsystems: biocompatibility aspects.

Bio-Micro-Electro-Mechanical Systems (BioMEMS) are a new tool in life sciences, supporting cell biology research by providing reproducible and miniaturized experimental platforms. In order to cultivate cells in such systems, appropriate microenvironmental conditions are required. Due to the multitude and variety of microbioreactors and cultivated cell types available, standardized cell handling methods and comprehensive biocompatibility data are sparse. The bioreactor developed at Ilmenau University of Technology features BioMEMS consisting of silicon, glass, and polymers, supplied by peripheral components. To verify the system's suitability for cell cultivation, it was necessary to prove whether materials and surfaces are biocompatible. Custom-tailored biocompatibility test procedures along with adequate cell seeding and handling methods had to be developed. According to this, proper positive and negative control samples had to be identified. The cultivation procedures were carried out using osteoblast-like murine fibroblasts (MC3T3-E1) and primary human osteoblasts (hOB). We could provide evidence that cultivation of these cells in our BioMEMS is feasible. In this context the relevant materials and the system's structure can be regarded as to be biocompatible. We could show that cell seeding and handling methods possess a strong impact on growth, development, and cellular activity of cell cultures in BioMEMS. Statistical biocompatibility data for the materials used is given.

Arboreal locomotion in small new-world monkeys.

The postural and locomotor activity and its relation to substrates was observed in 3 Saguinus oedipus, and 3 Saimiri sciureus for comparison, during a period of 10 h for each individual. The animals moved freely in cages of 3.40 m x 3.40 m x 2.40 m (height) on rather diverse substrates. Observations were made according to the focal-animal-method, with combined instantaneous and continuous sampling. They were protocoled in schematic form and video-recorded. In addition, 3 further Saguinus oedipus were subjected to an X-ray cinematographic study on a modified treadmill to unveil metric parameters of the locomotor pattern preferred on slender and compliant ("arboreal") substrates, the walk. Independent from the substrates, the postures of the two species differed in details, as do the preferred substrates. Horizontal, comfortable substrates are favored most. Walking ranked top in frequency, followed by jumping and galloping (in a strict sense). All other locomotor modes described for primates played a minor role or lacked entirely, like the trot. Average distance of leaps was only 0.60 m, landings were mainly on the same level as take-offs. In Saguinus, the movements of both limbs, including the shoulder blade, followed the pattern common to small mammals in general: At the end of the stance phase, humerus and tibia are nearly parallel to the substrate, while just before touchdown ulna and femur are in this position. The walk in both species was surprisingly fast (1 m/s), reaching the speed of much larger cursorial animals, like humans.

The proteasome: a macromolecular assembly designed for controlled proteolysis.

In eukaryotic cells, the vast majority of proteins in the cytosol and nucleus are degraded via the proteasome-ubiquitin pathway. The 26S proteasome is a huge protein degradation machine of 2.5 MDa, built of approximately 35 different subunits. It contains a proteolytic core complex, the 20S proteasome and one or two 19S regulatory complexes which associate with the termini of the barrel-shaped 20S core. The 19S regulatory complex serves to recognize ubiquitylated target proteins and is implicated to have a role in their unfolding and translocation into the interior of the 20S complex where they are degraded into oligopeptides. While much progress has been made in recent years in elucidating the structure, assembly and enzymatic mechanism of the 20S complex, our knowledge of the functional organization of the 19S regulator is rather limited. Most of its subunits have been identified, but specific functions can be assigned to only a few of them.

The 26S proteasome: a molecular machine designed for controlled proteolysis.

In eukaryotic cells, most proteins in the cytosol and nucleus are degraded via the ubiquitin-proteasome pathway. The 26S proteasome is a 2.5-MDa molecular machine built from approximately 31 different subunits, which catalyzes protein degradation. It contains a barrel-shaped proteolytic core complex (the 20S proteasome), capped at one or both ends by 19S regulatory complexes, which recognize ubiquitinated proteins. The regulatory complexes are also implicated in unfolding and translocation of ubiquitinated targets into the interior of the 20S complex, where they are degraded to oligopeptides. Structure, assembly and enzymatic mechanism of the 20S complex have been elucidated, but the functional organization of the 19S complex is less well understood. Most subunits of the 19S complex have been identified, however, specific functions have been assigned to only a few. A low-resolution structure of the 26S proteasome has been obtained by electron microscopy, but the precise arrangement of subunits in the 19S complex is unclear.

Recombinant expression, purification and characterization of Kch, a putative Escherichia coli potassium channel protein.

The Escherichia coli gene kch, similar in primary structure to eukaryotic voltage-gated potassium channels, was cloned and overexpressed in E. coli. The protein was solubilized from the plasma membrane with dodecylmaltopyranoside, lauryldimethylamine oxide or N-laurylsarcosine and was purified in milligram amounts by imidazole elution from a nickel-chelate column. The molecular mass of the purified protein in a number of detergents with 12 carbon atom chains suggests that rKch forms primarily tetramers of the 50 kDa monomers. CD spectroscopy of the purified protein indicates a significant alpha-helical content that is preserved upon addition of SDS.

The structure of recombinant human annexin VI in crystals and membrane-bound.

The crystal structure of calcium-free recombinant human annexin VI was solved at a resolution of 3.2 A by using the annexin I model for Patterson search and refined to an R-factor of 19.0%. The molecule consists of two similar halves closely resembling annexin I connected by an alpha-helical segment and arranged perpendicular to each other. The calcium and membrane binding sites assigned by structural homology are therefore not located in the same plane. Analysis of the membrane-bound form of annexin VI by electron microscopy shows the two halves of the molecule coplanar with the membrane, but oriented differently to the crystal structure and suggesting a flexible arrangement. Ion channel activity has been found for annexin VI and the half molecules by electrophysiological experiments.

Structural and functional characterisation of the voltage sensor in the ion channel human annexin V.

The ion channel properties of human annexin V, a calcium- and phospholipid-binding protein of the annexin family, have been structurally and functionally investigated by analysing the mutant Glu112 -->Gly. Glu112 forms a salt bridge with Arg271 located in the interior of the hydrophilic pore of the molecule which is conserved within the annexin family. The crystal structures of the mutant and wild-type proteins are very similar and show only marginal conformational changes around the mutation site. Electron microscopic images show a conserved four-domain structure upon membrane binding as in the wild-type annexin V. The channel properties of the mutant are drastically changed, as the mutant has lost the voltage-dependent channel gating and the selectivity for calcium ions over monovalent cations. These results strongly support the hypothesis that the central, hydrophilic pore is the ion-conducting pathway.

Annexin V: the key to understanding ion selectivity and voltage regulation?

Annexin V is a Ca(2+)-dependent membrane-binding protein that forms voltage-dependent Ca2+ channels in phospholipid bilayers and is the first ion channel to be structurally and functionally characterized. Data outlined here indicate that key amino acid residues act as selectivity filters and voltage sensors, thereby regulating the permeability of the channel pore to ions.

Three-dimensional structure of membrane-bound annexin V. A correlative electron microscopy-X-ray crystallography study.

We have used electron microscopy to analyse the structure of wild-type human annexin V (recombinant and placental) and of several mutants (single and double point mutants) bound to monolayers composed of DOPS, DOPE, or brain extract (Folch fraction III). On these phospholipids and on DOPS/DOPC (3:1, w/w) protein trimers, as also found in 3-D crystals, assemble to form a hexagonal lattice with a unit vector length of about 18 nm. The resolution obtained in projection is 1.7 to 2.2 nm for wild-type and mutants. There are no significant differences between the annexin V mutants and the wild-type protein at this resolution. All proteins bind as trimers with their convex side harbouring the Ca(2+)-binding sites facing the membrane. A comparison of the 3-D reconstruction of annexin V wild-type with the high resolution crystal structure shows that the domain structure is preserved but the relative orientation of the modules (II/III) and (I/IV) is slightly changed so that the Ca(2+)-binding sites in all four domains (including the recently observed binding site in domain III) become coplanar to the membrane. The thickness of the molecule obtained in the 3-D reconstruction corresponds well with the thickness of the high resolution crystal structure indicative of peripheral binding of annexin V without substantial penetration of the membrane.

Structural and electrophysiological analysis of annexin V mutants. Mutagenesis of human annexin V, an in vitro voltage-gated calcium channel, provides information about the structural features of the ion pathway, the voltage sensor and the ion selectivity filter.

Annexin V binds to phospholipids in a calcium-dependent manner and exhibits calcium channel activity in vitro. We prepared a variety of mutants yielding information about the structure-function relationship of the ion channel activity. All mutants were characterized by X-ray crystallography, electron microscopy and electrophysiological measurements. Their structures are insignificantly changed whereas their electrophysiological properties are drastically different. Glu95, located in the central hydrophilic pore of the molecule, is crucial for the ion selectivity filter as its exchange leads to reduced calcium and increased sodium conductance. The removal of Glu17, located on the protein surface and far from the ion conduction pathway, leads to the appearance of a second conductance level of 9 pS in addition to the conductance level of about 30 pS in the wild-type molecule. This was also the case for Glu78, which is part of a weak calcium binding site. The exchange of Glu17 and Glu78 produced a mutant retaining only the smaller conductance level. We conclude that these two residues influence the angle between the two halves of the molecule, which determines the diameter of the ion conduction pathway, thereby leading to the occurrence of a second conductance level.

Structure-function analysis of the ion channel selectivity filter in human annexin V.

Electrophysiology and structural studies were performed on an annexin V variant containing a mutation of glutamic acid-95 to serine in the center of the pore region. The mutation resulted in a lower single channel conductance for calcium and a strongly increased conductance for sodium and potassium, indicating that glutamic acid-95 is a crucial constituent of the ion selectivity filter. There were only minor differences in the crystal structures of mutant and wild-type annexin V around the mutation site; however, the mutant showed structural differences elsewhere, including the presence of a calcium binding site in domain III unrelated to the mutation. Analysis of the membrane-bound form of annexin V by electron microscopy revealed no differences between the wild type and mutant.

A rapid and efficient purification method for recombinant annexin V for biophysical studies.

Annexin V binds in a calcium-dependent manner to acidic phospholipids and exhibits ion channel activity in vitro. We are investigating mutants of annexin V by single channel measurements, X-ray crystallography and electron microscopy in order to understand the structure-function relationships of the ion channel activity. We describe here a method to obtain very pure recombinant annexin V required for such studies. The initial step is the mild opening of the bacterial cells by an osmotic shock. In the purification procedure, use is made of the reversible calcium-mediated binding of annexin V to liposomes. In the last purification step the protein is subjected to ion-exchange chromatography and elutes as a single peak free of any detectable contaminants.

Calcium influx through annexin V ion channels into large unilamellar vesicles measured with fura-2.

A new assay for the calcium channel activity of annexin V was developed. The calcium-sensitive fluorescence indicator, fura-2, was incorporated into large unilamellar vesicles (LUV). After establishing a calcium gradient across the liposomal membranes, native or mutated annexin V was added. The resulting calcium influx into the LUV detected through the fluorescence changes of fura-2 was used as a qualitative test for the electrophysiological properties of annexin V.