Abstract. To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filtergrown Madin-Darby canine kidney (MDCK) cells from liposomes at 0~ After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20~ Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosylceramide. An analysis of the fluorescence pattern after 1 h at 20~ by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37~ for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction.Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37~ showed that the C6-NBD-glucosylceramide was two-to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.

No abstract

No abstract

No abstract

Confocal Laser Scanning Microscopy / Fluorescence / Methods and Systems / MembranesFluorescence confocal laser scanning microscopy (CLSM) is increasingly acknowledged as a suitable technique to study the 3-dimensional structure of animal and plant cells. The imaging process involves the collection of the emitted fluorescent light of a defined volume in the object. A powerful processing system is, however, required to store the single data and reconstruct the image of the object. A 3-Dimage of an object can be achieved either by the extended focus, the stereo image, or the shadowing mode'). -These procedures were applied to elaborate on the spatial arrangement of structural components and organells in fixed or living plant cells. Microtubule arrays in dividing onion root cells and in caulonema tip cells of moss protonemata are visualized by the indirect immunofluorescence technique.In addition, the endoplasmic reticulum is visualized through vital staining with the fluorochrome 3,3'-dihexyloxacarbocyanine-iodide (DiOC). For example in adaxial onion bulb scale epidermis cells and root hair cells. Also, several examples are shown which demonstrate the possibilities the CLSM offers with respect to magnification, image, and motility measurements of single organelles. Some of the measurements have been performed with a compact inverted confocal system which also includes a classical fluorescence microscope. Fluorescence confocal laser scanning microscopy turned out to be of great advantage for studying the 3-D-arrangement of plant cell constituents due to the property of the focal scanner which discriminates fluorescent light outside of the depth of focus.