Karl-Otto Greulich

Endosymbiont-Dependent Host Reproduction Maintains Bacterial-Fungal Mutualism

Bacterial endosymbionts play essential roles for many organisms, and thus specialized mechanisms have evolved during evolution that guarantee the persistence of the symbiosis during or after host reproduction. The rice seedling blight fungus Rhizopus microsporus represents a unique example of a mutualistic life form in which a fungus harbors endobacteria (Burkholderia sp.) for the production of a phytotoxin. Here we report the unexpected observation that in the absence of endosymbionts, the host is not capable of vegetative reproduction. Formation of sporangia and spores is restored only upon reintroduction of endobacteria. To monitor this process, we succeeded in GFP labeling cultured endosymbionts. We also established a laserbeam transformation technique for the first controlled introduction of bacteria into fungi to observe their migration to the tips of the aseptate hyphae. The persistence of this fungal-bacterial mutualism through symbiont-dependent sporulation is intriguing from an evolutionary point of view and implies that the symbiont produces factors that are essential for the fungal life cycle. Reproduction of the host has become totally dependent on endofungal bacteria, which in return provide a highly potent toxin for defending the habitat and accessing nutrients from decaying plants. This scenario clearly highlights the significance for a controlled maintenance of this fungal-bacterial symbiotic relationship.

Repair of Sparfloxacin-Induced Photochemical DNA Damage In Vivo

The induction and subsequent repair of photochemically induced DNA damage by sparfloxacin was assessed in different tissues of juvenile Wistar rats. The animals were treated once orally with 500 mg kg(-1) of sparfloxacin and irradiated 3 hours later with 7 J cm(-2) UVA. Induction and repair of DNA damage was studied in the skin, retina and cornea using the alkaline comet assay. After a tissue-specific increase in the initial DNA damage (higher in the cornea than in skin and retina), an exponential decrease was found in the skin and retina, whereas in cornea a further increase of the DNA damage after 1 hour followed by an exponential decrease was observed. The half-lives for DNA repair were approximately 3 hours for skin and retina and 1 hour for cornea. After a recovery time of 6 hours, the majority of the induced DNA damage detectable with the comet assay had been removed. In conclusion, the data indicate that (1) photochemically induced DNA damage by sparfloxacin is efficiently removed in skin, retina and cornea, (2) repair of these DNA lesions follows an exponential decrease, (3) the induction and repair of sparfloxacin-mediated photochemical DNA damage might be tissue specific.

Scanning near-field optical microscopy of cell surfaces after structure conserving air drying

Scanning near-field optical microscopy (SNOM) can simultaneously map topographic and optical properties of surfaces with a spatial resolution between that of far-field light microscopy and electron microscopy, i.e. in the range of 100 nm. Since commercially available SNOM instruments came on the market, this technique has become interesting for the routine biological research laboratory especially in combination with far-field light imaging. However, due to the usually applied shear-force feedback controlling the SNOM tip, this technique still poses several challenges for biological applications. In our experiments for instance imaging of soft samples, large topographical changes on structurally conserved cell surfaces, and in particular the requirement for completely dried specimen had to be considered. To visualize surfaces of cells fixed on standard glass slides by SNOM, an easy to handle, optimized protocol using dehydration and hexamethyldisilazane exposure before air drying was developed. Using the commercially available instrument SNOM 210 with micro-fabricated silicon nitride tips, it was shown for several cell systems that the cellular morphology and surface structures were well preserved after this procedure of drying.

Trapping of dielectric particles and cells by a fiber-coupled laser trap

The laser for an optical trap can be coupled into the microscope via a sort of telescope (conventional coupling) or via optical fibers. The latter coupling results in high mechanical vibration resistance of the system and allows easy exchange of lasers. At a given laser power, coupling via fibers with a diameter below a critical value allows trapping of polystyrene beads even when trapping after conventional coupling is not possible. This is particularly the case when no cover slide can be used. With biological cells such as erythrocytes and NC 37 lymphoblastoids the differences between fiber coupling and conventional coupling are less pronounced.

Single-cell and single-molecule laser biotechnology

While lasers have found a wide field of application in the analysis of cells and biomolecules, their use in manipulation is less common. Now, new applications of lasers are emerging, which aim at cells and even molecules as biotechnological individuals: For example, in single cell gel electrophoresis individual cells are irradiated by UV laser pulses which cause radiation damage to DNA. When the whole cell is positioned in an electric field and the UV induced damages are converted into DNA strand breaks, the resulting DNA fragments are eluted out of the cell nucleus. Small fragments are running further than large ones. After staining of the DNA fragments, the cell has the appearance like a comet (therefore comet assay). The tail moment, a parameter quantifying the shape of the tail, gives information on the degree of DNA damage. The kinetics of DNA damage induction can be described by a type of exponential law with parameters which are related to radiation sensitivity of the DNA. A further emerging technique aims at DNA as a molecular individuum. One pivotal step for single molecule DNA analysis is single molecule handling. For that purpose, a DNA molecule is coupled to a micrometer sized polystyrene bead, either via an avidin-biotin bridge or, more specifically, by strand recognition, and labeled with fluorescence dyes such as DAPI. In order to visualize the dynamics of individual DNA molecules, highly sensitive video processing and single photon counting is required. Moving the polystyrene bead using optical tweezers, the molecule can be deformed, i.e., bent, turned or stretched. Using a laser microbeam, the same individual molecule can be cut into smaller portions.

Comet assay, cloning assay, and light and electron microscopy on one preselected cell

In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular redox state, impaired cell division, formation of giant cells and cell shrinking, swelling of mitochondria and loss of cristae as well as DNA damage.

Taking light pressure serious: light as a quasimechanical microtool

Light pressure may arise from absorption and can then be calculated as pressure equals intensity / vacuum velocity of light. Alternatively, it may result from scattering and is then called gradient force. In that case a quality factor Q has to be introduced, which has to be determined by calibration. Its numerical value is between 0.05 and 0.3. By coupling a NdYAG laser into a microscope with a high numerical aperture objective scattering light pressure can be used to move micrometer-sized dielectric objects. Such optical tweezers can be calibrated and have been used to measure forces needed to stretch individual DNA molecules, and to measure forces exerted by the motor proteins myosin, kinesin and dynein non-calibrated optical tweezers are used to handle individual DNA molecules after their coupling to micrometer-sized microbeads. Using enzymes which cut DNA molecules in a sequence specific fingerprint-like pattern, it is possible to analyze DNA on a single molecule basis.

FISH analysis of the arrangement of chromosomes in interphase nuclei using telomeric, centromeric, and DNA painting probes

Fluorescence in situ hybridization is used to study the arrangement of chromosomes in interphase nuclei of unsynchronized human lymphocytes. DNA probes specific for telomeric DNA, centromeric (alpha) -satellite DNA and whole chromosomes 2, 7, 9 and X are employed. It is demonstrated that the shape of the chromosome territories is variable in cycling cells, for example, close to the metaphase chromosome homologues are arranged pairwise. Furthermore, the relative arrangement of chromosome homologues to each other is not spatially defined. Also, the relative orientation of centromeres and telomeres within a chromosome domain is variable.

UVA-induced oxidative stress in single cells probed by autofluorescence modifications, cloning assay, and comet assay

Cell damage by low-power 365 nm radiation of a 50 W high-pressure mercury microscopy lamp was studied. UVA exposure to CHO cells resulted for radiant exposures greater than 10 kJ/m2 in significant modifications of NADH-attributed autofluorescence and in inhibition of cell division. Single cell gel electrophoresis (comet assay) revealed UVA-induced single strand DNA breaks. According to these results, UVA excitation radiation in fluorescence microscopy may damage cells. This has to be considered in vital cell microscopy, e.g. in calcium measurements.

Combined optical, electrostatic, and enzymatic handling of single DNA molecules

Complete handling of single DNA molecules and their enzymatic restriction are described. For that purpose a microsphere was bound to a DNA molecule and trapped by optical tweezers. It could be moved in any direction. For stretching or rotating the molecule an electrical field was applied while the bead was fixed by the optical tweezers. In a near-equilibrium state of the resulting forces, the DNA remained stretched. Subsequently, a restriction endonuclease was activated by liberating Mg2+ from a caged compound. The enzymatic reaction could be directly observed in the light microscope. While the bead remained in the focus of the laser trap the other restricted part of the DNA molecule is pulled away in the electrical field. The reaction was directly monitored and recorded on videotape.