Author(s): Xi JH, Bai F, Andley UP
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Abstract alphaA-Crystallin (alphaA) is a molecular chaperone expressed preferentially in the lens. alphaA transcripts are first detected during the early stages of lens development and its synthesis continues as the lens grows throughout life. alphaA(-/-) mouse lenses are smaller than controls, and lens epithelial cells derived from these mice have diminished growth in culture. In the current work, we tested the hypothesis thatalphaA prevents cell death at a specific stage of the cell cycle in vivo. Seven-day-old 129Sv (wild-type) and alphaA(-/-) mice were injected with 5-bromo-2'-deoxyuridine (BrdU) to label newly synthesized DNA in proliferating cells. To follow the fate of the labeled cells, wholemounts of the capsule epithelial explants were made at successive times after the BrdU pulse, and the labeling index was determined. Immunofluorescence and confocal microscopy showed that both wild-type and alphaA(-/-) cells had a 3-hour labeling index of 4.5\% in the central region of the wholemount, indicating that the number of cells in S phase was the same. Twenty-four hours after the pulse, individual cells labeled with BrdU had divided and BrdU-labeled cells were detected in pairs. The 24-hour labeling index in the wild-type lens was 8.6\%, but in the alphaA(-/-) lens it was significantly lower, suggesting that some of the cells failed to divide and/or that the daughter cells died during mitosis. TUNEL labeling was rarely detected in the wild-type lens, but was significant and always detected in pairs in the alphaA(-/-) wholemounts. Dual labeling with TUNEL and BrdU also suggested that the labeled cells were dying in pairs in the alphaA(-/-) lens epithelium. Immunolabeling of wholemounts with beta-tubulin antibodies indicated that the anaphase spindle in a significant proportion of alphaA(-/-) cells was not well organized. Examination of the cellular distribution of alphaA in cultured lens epithelial cells showed that it was concentrated in the intercellular microtubules of cells undergoing cytokinesis. These data suggest that alphaA expression in vivo protects against cell death during mitosis in the lens epithelium, and the smaller size of the alphaA(-/-) lens may be due to a decrease in the net production of epithelial cells.
This article was published in J Cell Sci
and referenced in Journal of Clinical & Experimental Ophthalmology