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U.P. Andley, F. Bai, J. Xi; Alpha–Crystallin Alters Microtubule Assembly in Mouse Lens . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1064.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: α–Crystallin stabilizes and regulates the microfilament and intermediate filament cytoskeleton. However, its effect on tubulin and microtubule assembly in the lens is unknown. We found that αA–crystallin colocalizes with centrosomes (microtubule organizing centers) and intercellular bridge microtubules in mitotic lens epithelial cells. Furthermore, lens epithelial cells derived from αA– or αB–crystallin knockout mice display aberrant mitotic profiles, and demonstrate increased cell death or hyperproliferation and genomic instability. Therefore, the goal of this study was to analyze the effect of α–crystallin expression on lens tubulin and the assembly of microtubules. We also analyzed lens microtubule–associated proteins. Methods: Adult mouse lenses were used in this study. αA–/– and αB–/– mice were kindly provided by Dr. Eric Wawrousek. Microtubules were isolated from dissected lens epithelial fractions by a procedure adapted from isolation of brain microtubules in the presence of Taxol and GTP. The nature of the microtubule pellet was analyzed by negative staining and electron microscopy (performed by Dr. Wandy Beatty). Microtubules were also isolated from lens outer cortical fractions. The expression of tubulin was analyzed by immunoblot analysis. Microtubule–associated proteins were isolated by washing the microtubule pellet with 0.35 M NaCl and analyzed by immunoblotting and mass spectrometry. Results: Lens epithelial microtubules had a characteristic protofilament structure when examined by electron microscopy. Peeling protofilaments from the ends of microtubules were readily visible. The length of microtubules was ∼4 µm and the width was 30 nm. In contrast αB–/– microtubules were 2.5 times longer than wild type. αA–/– microtubules were significantly thinner and slightly longer than wild type. When lens epithelial microtubules were washed with 0.35 M NaCl the extracted proteins consisted predominantly of the crystallins. In addition, transketolase was identified by mass spectrometry in the microtubule–associated protein fraction. The microtubule–associated protein tau was detected as a component of the lens cortical microtubules. In αB–/– microtubules, adducts of tau protein with tubulin were detected by immunoblot analysis. Conclusions: Our results suggest that expression of αA– and αB–crystallin has important and distinctive effects on microtubule assembly in lens epithelium. We propose that one of the functions of αA– and αB–crystallin is to enhance microtubule dynamics. The aberrant microtubule cytoskeleton of αA–/– and αB–/– cells may be due to defective assembly of microtubules in their absence.
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