Abstract
Abstract: :
Purpose: Calpains are neutral proteases associated with both crystallin proteolysis occuring during normal lens development and in the abnormal proteolysis seen in many different types of cataract. In other tissues, calpains appear to regulate signal transduction cascades activated by integrin and growth factor signaling. This study investigates the expression pattern and subcellular localization of m-Calpain, calpain10 and the p94 splice variants Lp82 and Lp85 during murine lens development. Methods: Polyclonal antibodies against m-Calpain, calpain10, Lp82 and Lp85 were applied to murine eye sections from 9.5 dpc until adulthood. The bound antibodies were detected using fluorescent probes and confocal microscopy. Results: At 9.5-10.5 dpc, m-Calpain protein is detected in the nuclei of both the optic vesicle and lens placode/pit cells. From 12.5 dpc until 16.5 dpc, high levels of m-Calpain are associated with the nuclei of both epithelial and fiber cells, however after birth, m-Calpain is mostly found in the lens epithelium with much less protein detectable in fibers. Calpain 10 protein was also first detected in the lens placode and optic vesicle of 9.5 dpc mouse embryos and was distributed in both the cytoplasm and nucleus. From 12.5 dpc until adulthood, calpain 10 is predominately a nuclear protein located in the lens epithelium with much less protein detectable in fiber cells. Lp82 protein was essentially absent from the 9.5 dpc eye with the first expression detected in the cytoplasm of elongating primary fiber cells at 11.5 dpc. High levels of Lp82 are detected in the cytoplasm of lens fiber cells until two weeks postnatal while much less protein was detectable in the 12 week old lens. In contrast, Lp85, another p94 splice variant, was first detected in the lens placode of 9.5 dpc embryos and was detectable in the nuclei of both lens epithelial and fiber cells from 11.5 dpc until adulthood. Conclusion: Of the calpains known to be expressed in the rodent lens, only Lp82 was detected at high levels in the lens fiber cell cytoplasm throughout development suggesting that it is the major calpain involved in crystallin processing. The other calpains, which are either predominately found in the lens epithelium and/or are localized to cell nuclei are likely to be more important for the post translational processing of regulatory proteins such as transcription factors or cell signaling molecules.
Keywords: 417 gene/expression • 471 microscopy: confocal/tunneling • 530 proteolysis