Abstract
Abstract: :
Purpose: The morphological changes that take place during organelle loss are reminiscent of those that occur during programmed cell death, including chromatin condensation and the generation of fragmented DNA. Biochemical evidence also suggests that the cell death machinery may function during organelle loss because well–characterized apoptotic substrates are cleaved as lens fibers differentiate. Here we examine the role of executioner caspases (–3, –6 and –7) during fiber differentiation in the developing mouse lens. Methods: The distribution of mRNA, protein and enzymatic activity for each of the executioner caspases was measured in the mouse lens. Transcript levels were determined in fiber cells from adult mouse lenses by real time RT–PCR. Protein profiles were examined by western blot on whole and regionally dissected lenses. Caspase enzymatic activity was determined in lysates prepared from various regions of the lens using a fluorogenic assay. Lenses were also treated with staurosporine, an apoptosis inducing agent, to test whether caspase activity could be induced in the lens. Mice deficient for each of the caspases were included in all assays as controls. The cellular phenotypes of wildtype and knockout lenses were compared by confocal microscopy. Results: All three executioner caspases were expressed at the mRNA level in lens fiber cells. Caspases 6 and 7 were detected by western blot. Specific activity (defined as the difference in activity between wildtype and knockout lenses) was noted for caspases 3 and 7, but not 6. There was a marked induction of caspases 3 and 6 activity following staurosporine treatment. Deletion of any single executioner caspase had no effect on the programmed elimination of organelles although mice deficient in caspase 3 developed anterior polar cataracts. Conclusions: Executioner caspases are present and active in the mouse lens but no single caspase is absolutely required for organelle loss.
Keywords: cell death/apoptosis • transgenics/knock–outs • cataract