Evaporation of water cannot be fully avoided when an unsupported thin vitrified film of an aqueous suspension is prepared for cryo-electron microscopy. This results in increasing concentration of ...solute which could affect the observed material. We have quantitatively studied this effect by measuring the contrast of polystyrene spheres in a metrizamide solution. The drying effect is generally negligible when specimens are prepared on a hydrophilic perforated support but it is frequently important when hydrophobic films are used instead. A flow of humid air, double blotting with minimal exposure of the thin liquid film to the atmosphere, or an automatic plunger optimizing the blotting conditions are simple methods for reducing drying effects. With this third device acting on a hydrophilic supporting film, the increase of solute concentration is limited to less than 20%.
The interaction of the 20-kDa pore-forming domain of colicin A with phospholipid vesicles was investigated by gel permeation chromatography, analytical centrifugation, and electron microscopy. Under ...the experimental conditions of this study, this peptide was found to interact only with vesicles containing negatively charged phospholipids. It forms a well-defined disklike complex with phosphatidylglycerols with a preference for those containing 12-14 atoms of carbon in their fatty acid chain. This complex has a diameter of 120 A and is about one bilayer thick. It contains nine molecules of peptide and is formed both at acidic pH (pH 5.0) and at neutral pH (pH 7.2).
Apicomplexans are curious single-celled organisms. Belonging to the group of chromalveolates, life for an apicomplexan can be parasitic and some species can cause diseases such as malaria or ...toxoplasmosis. No apicomplexan is alike, although they share some common features such as being highly polar cells with unique apical organelles. They often change the cells of their metazoan hosts. When they move, apicomplexans do not crawl but glide; when they divide, apicomplexans go through mechanisms matched in cell biological bizarreness only by their names. They undergo schizogony or endodyogeny, processes that are usually not part of a regular molecular cell biology textbook; but they should, as their uniqueness might lead to insights into what proteins and processes are truly essential to make progeny. Here we highlight some of our current knowledge of centrosome and microtubule biology of selected apicomplexan parasites for the yeast and metazoan cell biologist to contemplate.
The reaction center free light-harvesting core complex of Rp. marina was purified by DEAE 52 ion exchange chromatography in the presence of the detergent OG. The protein complex was crystallised by ...microdialysis yielding two-dimensional crystals with a diameter of up to 10 micrometers. The crystals were negatively stained with uranyl acetate or prepared in vitrified ice and electron micrographs were taken. They exhibited a hexagonal lattice with a lattice constant of 102 +/- 3 angstroms. The optical diffraction pattern of the best ordered areas of electron micrographs showed spots up to a resolution of 29 angstroms. Image processing revealed a six fold symmetry of the ring like B880-complex. The protein ring is hexagonal with one subunit in each corner of the hexagon and two subunits forming the connection site to the neighbouring B380-complex in the crystal. In freeze fracture preparations of whole cells the intra-cytoplasmic photosynthetic membranes are seen to be organised into large stacks that affect the organisation of the photosynthetic complexes. Most notably, the stacked membrane regions exhibit hexagonally packed photosynthetic complexes with a repeat of approximately 100 angstroms, which is very similar to the lattice of the artificial B880-complex crystals. The same quasi-crystalline structure appeared in the cytoplasmic membrane of the contact sites with the intra-cytoplasmic membrane stack, but was absent from the end membrane of the stack. Thus, membrane stacking appears to induce the formation of the crystalline arrays, presumably through interactions between the cytoplasmic surface domains of the photosynthetic complexes. Tight packing of the photosynthetic particles is not sufficient to induce the crystalline order. The intra cytoplasmic membranes form a continuum with the cytoplasmic membrane via their origins at the round invagination sites.
The most prominent vector-transmitted diseases in the first and third world are Lyme disease and malaria, respectively. In both cases the transmitted agents are introduced into the skin from where ...they eventually disseminate into the blood using active motility. We are interested in deciphering the molecular mechanisms underlying the motility of these pathogens and how they relate to the ultrastructure of the pathogens. Here we provide an overview of the microscopy techniques that we use to achieve these goals.
