Abstract
Abstract: :
Purpose:Recent confocal laser scanning microscopy studies of the mitochondria of vertebrate lenses show a striking difference in the distribution and morphology of the mitochondria of lens epithelial and superficial cortical cells. This study, using confocal microscopy, was undertaken to image the movement of the mitochondria–specific dye tetramethylrhodamine ethyl ester (TMRE) in the epithelium and superficial cortex of whole live bovine lenses. To evaluate mitochondrial connectivity, fluorescent recovery after photobleaching (FRAP) was used. Methods: Cultured bovine lenses were loaded with 5µg/ml TMRE for 15 min. at room temperature. TMRE fluorescence was acquired with a Zeiss 510 (configuration META 18) confocal laser scanning microscope for 10 to 15 minutes using 488nm Argon laser excitation and 505nm Long Pass emission filter settings. The uncoupler of the electron transport chain potential, carbonyl cyanide m–chlorophenylhydrazone (CCCP, 32.5µM), was used to simulate lens damage. Results: Dynamic movement of TMRE was observed in both epithelial and superficial cortical fiber cells of live bovine lenses. In the epithelium, the movement of TMRE fluorescence was up to 5µm/min whereas in the superficial cortex the observed movement was up to 18.5µm/min. The movement of TMRE fluorescence was abolished with treatment with the uncoupler. The fluorescence of TMRE in both epithelial and superficial fibers was abolished after brief single or repeated bleaching exposures, but the recovery of fluorescence was noted after 4 second in epithelial cells and 3 seconds in the superficial cortical fiber cells. Conclusions: The observed dynamics of TMRE fluorescence movement may represent the actual mitochondrial movement, indicating the dynamic state of the mitochondria in both lens epithelium and superficial cortex. That this activity is found not only in the epithelium but also in the superficial cortex indicates that the superficial cortical fibre cells play a much more active role in lens metabolism than previously suspected. Alternatively, the observed movement of TMRE across a mitochondrial network could represent change in the distribution of potential across the inner membrane, presumably allowing energy transmission across the cell from regions of low to regions of high ATP demand. In addition, the results of the FRAP experiments suggest that the mitochondria of the lens may exist in the form of a functional syncytium.
Keywords: mitochondria • microscopy: confocal/tunneling • motion-3D