Confocal laser scanning microscopy imaging of dynamic TMRE movement in the mitochondria of epithelial and superficial cortical fiber cells of bovine lenses.
OBJECTIVE
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 lens.


METHODS
Cultured bovine lenses were loaded with 5 microg/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 min using 488 nm Argon laser excitation and 505 nm long pass emission filter settings. The uncoupler of the electron transport chain potential, carbonyl cyanide m-chlorophenylhydrazone (CCCP, 32.5 microM), was used to demonstrate the fluorescent specificity of TMRE.


RESULTS
Multidirectional dynamic movement of TMRE was observed in epithelial cells and bidirectional dynamic movement was seen in the superficial cortical fiber cells of live bovine lenses. In the epithelium, the movement of TMRE fluorescence was up to 5 microm/min whereas in the superficial cortex the observed movement was up to 18.5 microm/min. The movement of TMRE fluorescence was abolished with treatment with the uncoupler, CCCP.


CONCLUSIONS
The observed dynamics of TMRE fluorescence movement may represent 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 fiber 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.
Bantseev
Vladimir
V
School of Optometry, University of Waterloo, Waterloo, Ontario, Canada. vbantsee@sciborg.uwaterloo.ca
Sivak
Jacob G
JG
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002417:Cattle; D004848:Epithelium / Q000378:metabolism; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D066298:In Vitro Techniques; D007904:Lens Cortex, Crystalline / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D012235:Rhodamines / Q000493:pharmacokinetics*; D014018:Tissue Distribution; D014475:Uncoupling Agents / Q000494:pharmacology
D005456:Fluorescent Dyes; D012235:Rhodamines; D014475:Uncoupling Agents; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C005358:tetramethylrhodamine