CONFOCAL MICROSCOPIC OBSERVATION OF CYTOSKELETAL REORGANIZATIONS IN CULTURED SHARK RECTAL GLAND-CELLS FOLLOWING TREATMENT WITH HYPOTONIC SHOCK AND HIGH EXTERNAL K+

The dogfish shark (Squalus acanthias) rectal gland (SRG) cell has serv ed as a model experimental system for investigating the relationship b etween the actin cytoskeleton and cell volume regulation. Previous rep orts employing conventional fluorescence microscopy of tissue slices h ave shown that cells exposed to high external K+ and hypotonically-ind uced cell swelling displayed a fading of F-actin staining intensity, p articularly at the basolateral cell borders. However, spectroscopic me asurement of the F-actin present in similarly treated rectal gland sli ces failed to demonstrate a net change in F-actin amount. In an effort to resolve the structural reorganizations of F-actin which may be occ urring during high K+ and hypotonic shock treatments, we have used cul tured SRG cells in conjunction with confocal microscopic immunocytoche mical localization techniques to examine actin filament, microtubule, and cytokeratin filament dynamics under these two experimental conditi ons. The results reveal that F-actin in control cells exists in an arr ay of parallel linear bundles (which do not appear to be stress fiber- like given their lack of staining for myosin II or alpha-actinin) that is reorganized to a punctate pattern in hypotonic shock and a dense m eshwork in high K+. The linear bundle pattern of F-actin returns in ce lls undergoing regulatory volume decrease. Quantitative western blotti ng of F-actin in SRG cell detergent extracted cytoskeletons indicates no significant difference in the relative amounts of F-actin present i n control, hypotonic shocked, or high K+ cells. Anti-tubulin and anti- cytokeratin labeling of the treated SRG cells suggest that these other major cytoskeletal elements are not significantly altered by the trea tments. Taken together, our results reinforce the concept that there i s an association between the structural organization of the actin and cell volume regulation in the SRG epithelial cells. (C) 1997 Wiley-Lis s, Inc.