A ratiometric imaging method for mapping ion flux densities.

A heterogeneous distribution of ion channels on the cell surface is a prerequisite for several cellular functions. Thus, there has been considerable interest in methods allowing the mapping of ion channel distributions. Here we report on a novel ratiometric imaging technique appropriate to measure spatially resolved ion flux signals by using ion sensitive dyes. However, given that certain relevant cell properties like the surface to volume ratio may exhibit significant spatial heterogeneities, the local influx signal cannot be interpreted as a measure of the local open channel concentration or flux density. To overcome this problem, we suggest an internal normalization procedure, which, in analogy to, but clearly distinct from, well-established ratioing techniques, eliminates effects which would otherwise obscure the desired result. Ratioing is performed on flux signals from a given cell, triggered by two different, subsequent stimuli. If the two stimuli address different ion channels, the flux density distribution caused by two channel types can be determined relative to each other. In cases where one of the stimuli triggers a spatially homogeneous flux signal, ratioing yields an ion flux density map for a given channel type. Thus distribution patterns of ion channels active during a given stimulus may be derived.

Selective permeability of different connexin channels to the second messenger inositol 1,4,5-trisphosphate.

Intercellular propagation of signals through connexin32-containing gap junctions is of major importance in physiological processes like nerve activity-dependent glucose mobilization in liver parenchymal cells and enzyme secretion from pancreatic acinar cells. In these cells, as in other organs, more than one type of connexin is expressed. We hypothesized that different permeabilities towards second messenger molecules could be one of the reasons for connexin diversity. In order to investigate this, we analyzed transmission of inositol 1,4,5-trisphosphate-mediated calcium waves in FURA-2-loaded monolayers of human HeLa cells expressing murine connexin26, -32 or -43. Gap junction-mediated cell coupling in different connexin-transfected HeLa cells was standardized by measuring the spreading of microinjected Mn(2+) that led to local quenching of FURA-2 fluorescence. Microinjection of inositol 1,4,5-trisphosphate into confluently growing HeLa connexin32 transfectants induced propagation of a Ca(2+) wave from the injected cell to neighboring cells that was at least three- to fourfold more efficient than in HeLa Cx26 cells and about 2.5-fold more efficient than in HeLa Cx43 transfectants. Our results support the notion that diffusion of inositol 1,4,5-trisphosphate through connexin32-containing gap junctions is essential for the optimal physiological response, for example by recruiting liver parenchymal cells that contain subthreshold levels of this short lived second messenger.

Control of phobic behavioral responses by rhodopsin-induced photocurrents in Chlamydomonas.

Both phototactic and photophobic responses of Chlamydomonas are mediated by a visual system comprising a rhodopsin photoreceptor. Suction pipette recordings have revealed that flash stimulation causes calcium currents into the eyespot and the flagella. These photocurrents have been suggested to be the trigger for all behavioral light responses of the cell. But this has never been shown experimentally. Here we describe a detection technique that combines electrical and optical measurements from individual algae held in a suction pipette. Thus it is possible to record photocurrents and flagellar beating simultaneously and establish a direct link between the two. We demonstrate that in Chlamydomonas only the photoreceptor current in conjuction with a fast flagellar current constitutes the trigger for photophobic responses. Within the time of the action-potential-like flagellar current, the flagella switch from forward to backward swimming, which constitutes the beginning of the photoshock reaction. The switch is accompanied by a complex frequency change and beating pattern modulation. The results are interpreted in terms of a general model for phototransduction in green algae (Chlorophyceae).

Instrumentation for multiwavelengths excitation imaging.

We have developed a fluorescence ratio-imaging system which is based on a 12 bit, 2 MHz slow scan CCD camera and a patented (patent number P 42 28 366.3-52) polychromatic illumination system. The latter produces monochromatic light (12 nm bandwidth) of high intensity (> 3 mW between 300 and 500 nm) and allows one to switch to any wavelength between 260 and 680 nm in less than 3.5 ms in a computer-controlled fashion. The possibility to execute complex wavelength protocols facilitates multiple dye measurements with optimal exposure time for a given wavelength and the return to a dark phase in between exposures. Moreover, it allows sweeping over extended spectral regions in order to determine optimal experimental conditions for a given task. Wavelength selection is performed by a diffraction grating which is mounted onto a galvanometric scanner. The grating is illuminated by white light from a 75 W xenon lamp, using exclusively reflective optics, and the diffracted monochromatic light is coupled into the microscope by means of a single fibre quartz light guide. The epifluorescence optics, a special, achromatic, aplanatic UV condensor, image the exit face plate of the fibre into the specimen plane of an inverted microscope. This 'critical illumination' yields better homogeneity in the specimen plane than the classical Köhler illumination. Thus, with the Zeiss Fluar objective 40 x, NA = 1.3, fluence rates close to 10(23) photons m2 s-1 may be achieved at 340 nm. A DOS programme has been written in 'C' which controls both the monochromator and slow scan imaging system. It can acquire up to 13 full frames per s, and in its binning and skipping mode up to 100 subframes per s may be acquired. The frame-transfer structure of the chip allows one to acquire images at wavelength 'B' while simultaneously writing image data previously acquired at wavelength 'A' into the computer.

The nature of rhodopsin-triggered photocurrents in Chlamydomonas. I. Kinetics and influence of divalent ions.

In the green alga Chlamydomonas chlamyrhodopsin fulfills its role as a light sensor by absorbing light and activating photoreceptor channels within the eyespot area. At intense light stimuli, the photoreceptor (P) current triggers a fast and a slow flagellar current that finally leads to backward swimming (stop response). Here we report about probing the photoreceptor current directly at the eyespot. This allows the detection of the whole P current with a size of above 50 pA. The P current appears with a delay of less than 50 microseconds, suggesting that rhodopsin and the P channel are closely coupled or form one ion channel complex. The Ca2+ dependence of the P current has been demonstrated with the established suction technique in a capacitive mode. The P current shows the maximum amplitude at only 300 nM Ca2+, and it gradually declines at higher Ca2+. In addition to Ca2+, the photoreceptor and the fast flagellar current can be carried by Sr2+ and Ba2+. Mg2+ is conducted less efficiently and at high concentrations blocks the photoreceptor channel. A motion analysis of the cells shows that only Ca2+ and Sr2+ can induce physiological stop responses, whereas the large Ba2+ currents cause abnormal long-lasting cell spiraling.

The nature of rhodopsin-triggered photocurrents in Chlamydomonas. II. Influence of monovalent ions.

Chlamydomonas exhibits a sequence of a photoreceptor current and two flagellar currents upon stimulation with bright green flashes. The currents are thought to be a prerequisite for the well-known photophobic responses. In the preceding paper, we analyzed the kinetics of these currents and their dependence on extracellular divalent ions. Here, we show that the photoreceptor current can be carried by monovalent ions (K+ > NH4+ > Na+), provided that the driving force is high enough. The small residual photoreceptor current observed in the absence of Ca2+ is able to evoke flagellar currents at low extracellular pH. This demonstrates that signal transduction from the rhodopsin to the flagella is not inevitably dependent on extracellular Ca2+. Double-flash experiments exclude a contribution of intra-rhodopsin charge movements to the photoreceptor current signal. Evidence will be provided for the existence of nonlocalized K+ outward currents, which counterbalance the localized Ca2+ influx and repolarize the cell after a light flash. A model is presented that explains the different pathways for direction changes and phobic responses.

Isolation and separation of the glycan strands from murein of Escherichia coli by reversed-phase high-performance liquid chromatography.

The length distribution of the glycan strands in the murein (peptidoglycan) sacculus of Escherichia coli has been analyzed after solubilization of the murein by complete digestion with human serum amidase. The glycan strands released were separated according to length by reversed-phase HPLC on wide-pore Nucleosil 300 C18 material at 50 degrees C, employing a convex gradient from 5 to 11% acetonitrile. The length of the fractionated glycan strands, which carry a nonreducing 1,6-anhydromuramic acid as a natural end group, was calculated from the ratio of total to nonreducing terminal muramic acid residues. This was possible after complete hydrolysis of the isolated glycan strands by muramidase followed by separation of the released nonreducing and reducing di- and tetrasaccharides by reversed-phase HPLC on Hypersil C18. The method established allows the separation of the glycan strands of murein, a poly-GlcNAc(beta 1-4)MurNAc-polysaccharide, up to a degree of polymerization of approximately 60. The predominant lengths of the glycan strands were 5 to 10 GlcNAc(beta 1-4)MurNAc disaccharide units.