Ultraviolet confocal fluorescence microscopy of the in vitro cornea: redox metabolic imaging.

A laser scanning microscope was fitted with two argon-ion lasers that provided wavelengths in the regions of 364, 488, and 514 nm. A Zeiss water objective of 25 x , with a numerical aperture of 0.8, corrected for the UV, was used to measure the fluorescence from optical sections of freshly enucleated rabbit eyes. The confocal microscope was used in both the reflected and fluorescent modes to image in situ epithelial and endothelial cells. An excitation wavelength of 364 nm and emission at 400-500 nm were used to image the fluorescence from reduced pyridine nucleotides. We demonstrate the feasibility of two-dimensional fluorescent confocal imaging of reduced pyridine nucleotides in corneal epithelial and endothelial cells.

Working with the confocal scanning UV-laser microscope: specific DNA localization at high sensitivity and multiple-parameter fluorescence.

The use of fast-staining DNA-specific dyes such as DAPI or Hoechst 33342/33258 has been a major problem for confocal scanning laser microscopy (CSLM) studies of intranuclear chromatin organization. Moreover, the availability of a confocal ultraviolet scanning laser microscope configuration, which allows an excitation at wavelengths of 364 nm as well as 488, 514 and 543 nm, is a prerequisite for single as well as multiple fluorescence parameter studies, especially if these studies are concerned with the precise localization of intranuclear signals. Here we report the characteristics and application of a CSLM, which was adapted for UV-excitation and therefore enables comparison of the spatial distribution of several types of signals within one preparation. In addition to multiple-parameter studies, we have also investigated the sensitivity of the system with regard to the identification of the double-stranded DNA of lampbrush chromosome loops in germinal vesicles of amphibian oocytes.

Shuttling of the autoantigen La between nucleus and cell surface after uv irradiation of human keratinocytes.

During the past years we have established that the nuclear autoantigen La shuttles between the nucleus and the cytoplasm in tumor cells after inhibition of transcription or virus infection. We reinvestigated this shuttling using primary human keratinocytes from both healthy donors and patients with xeroderma pigmentosum. Ultraviolet irradiation resulted in both an inhibition of transcription and a translocation of La protein from the nucleus to the cytoplasm. After a prolonged inhibition of transcription La protein relocated into the nucleus and assembled with nuclear storage regions. The uv-induced shuttling included a translocation to the cell surface, where La protein colocalized with epidermal growth factor receptors.

Chemically induced changes in the morphology, dynamic activity and cytoskeletal organization of Physarum cell fragments.

Spherical cell fragments derived from Physarum polycephalum by caffeine-treatment were used as an experimental system to investigate the influence of 15 externally applied substances on the general morphology, motile behavior and cytoskeletal organization of the acellular slime mold. In comparison to controls, the most obvious changes observed after chemical stimulation proved to be cytokinetic activities, ameboid-like movement phenomena, intense cell surface dynamics and formation of cytoplasmic actin fibrils. The results demonstrate the high adaptability of the microfilament system in Physarum even when subjected to extreme conditions in the external environment.

Studies on microplasmodia of Physarum polycephalum : VI. Functional analysis of a cortical and fibrillar actin system by use of fluorescent-analog cytochemistry.

Fluorescently labeled actin (TRITC-G-actin) and heavy meromyosin (TRITC-HMM) derived from skeletal muscle and injected into microplasmodia of the acellular slime mold Physarum polycephalum were used to analyze the function of a cortical and fibrillar actin system in living specimens. The plasma membrane-attached cortical system can be labeled with TRITC-G-actin as well as with TRITC-HMM and visualized as a continuous sheath along the entire cell surface. Long-term experiments over time periods of several hours in conjunction with digital grey-value evaluations revealed that changes in the intensity of the fluorescent signal, as caused by alternative contraction and relaxation cycles of the cortical system, are distinctly correlated with periodic changes in the volume and shuttle streaming activity of the microplasmodia. The fibrillar actin system extending through the cytoplasmic matrix can be labeled only with TRITC-HMM. Formation and disappearance of fibrils were found to take place during relaxation and contraction of the cortical system, respectively. Results of the present paper indicate that the cortical actin system is mainly involved in motive force generation for alterations in cell surface morphology and locomotion activity, whereas the fibrillar actin system rather appears to maintain the mechanical stability of microplasmodia.

Distribution and dynamics of fluorochromed actin in living stages of Physarum polycephalum.

Isolated muscle and Physarum actins were labeled with various fluorochromes and microinjected into living stages of Physarum polycephalum (caffeine-droplets, endoplasmic drops, thin-spread macroplasmodia). Subsequent analysis of the intracellular redistribution by fluorescence microscopy, video-enhancement and digital image processing revealed RITC (rhodamineisothiocyanate) actin to be the most reliable molecular probe for the marking of microfilaments. In relaxed caffeine-droplets, the RITC-actin first diffuses randomly and then is locally incorporated into a thin cortical layer at the internal face of the plasma membrane. During Ca2+-induced contraction the fluorescent layer starts to detach from the plasma membrane, thus causing separation of central granuloplasm from peripheral hyaloplasm. Thin sections of both, relaxed and contracted specimens demonstrated that the RITC-actin layer in living droplets exactly coincides with a sheath of more or less oriented microfilaments. In contrast, RITC-bovine serum albumin (BSA) injected as control is excluded from those regions which show intense fluorescence with RITC-actin and the presence of an actin network with EM. Successful incorporation of the molecular probe into stages of Physarum polycephalum other than caffeine droplets was not yet achieved. The results obtained by fluorescent analog cytochemistry (FAC) are discussed with regard to the spatial organization of the actin system in acellular slime molds.

Lipid transport through the enterocytes of larval Aeshna cyanea (Insecta, Odonata).

The larval enterocytes of A. cyanea absorb lipid after luminal lipolysis in morphologically invisible form by direct membrane transport, presumably molecular diffusion. The lipolytic products are utilized for resynthesis of di- and triglyceride which become visible in the form of lipid droplets in the groundplasm. The putative site of lipid synthesis is the apical ER which locally forms highly ordered complexes. Lipid transport occurs in the form of matrix lipid so that the enterocytes of dragonfly larvae resemble in this respect the lipid-secreting mammocytes rather than the lipid-absorbing mammalian enterocytes. Lipid release involves partial lipolysis and direct membrane transport, possibly including membrane delamination, again in contrast to exocytosis in mammalian enterocytes and apocrine extrusion in mammocytes, which are both indirect membrane transport mechanisms.

Plasma membranes, cell junctions and cuticle of the rectal chloride epithelia of the larval dragonfly Aeshna cyanea.

The cell membranes and cell junctions of the rectal chloride epithelia of the larval dragonfly Aeshna cyanea were examined in thin sections and by freeze-fracture. These epithelia function in active ion absorption and maintain a high concentration gradient between the haemolymph and the fresh-water environment. Freeze-fracturing reveals fine-structural differences in the intramembraneous particles of the luminal and contraluminal plasma membranes of these epithelia, reflecting the functional diversity of the two membranes, which are separated by the junctional complex. The particle frequency of the basolateral plasma membranes is reduced after transfer of the larvae into high concentrations of environmental salinity. The junctional complex is located in the apical region and composed of three types of cell junctions: the zonula adhaerens, seen in freeze-fracture as a nearly particle-free zone; the extended and highly convoluted pleated septate junction and randomly interspersed gap junctions of the inverted type. Gap junctions also occur between the basolateral plasma membranes. They provide short-cuts in the diffusion pathway for direct and rapid co-ordination of the interconnected cell processes. Colloidal and ionic lanthanum tracer solutions applied in vivo from the luminal side penetrate through the cuticle via epicuticular depressions, but invade only the apical portion of the junctional complex. This indicates that the pleated septate junction constitutes a structural control of the paracellular pathway across the chloride epithelia, which are devoid of tight junctions. The structure of the pleated septate junctions is interpreted as a device for the extension of the diffusion distance, which is inversely related to the net diffusion. A conservative estimate of the total length of the junction, and the number and extension of septa reveals that the paracellular route exceeds the transcellular route by a factor of 50.