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L. Li, H. Liu, K. Wang, C. Cheng, B. Chang, Q. Huang, J. Horwitz, X. Gong; Different Gamma–Crystallin Isoforms Uniquely Affect Lens Fiber Cell Maturation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1515.
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© ARVO (1962-2015); The Authors (2016-present)
To determine whether different γ–crystallin isoforms have additional functions besides acting as necessary structural components for maintaining lens transparency, we have investigated the molecular mechanisms for how several point mutations of different γ–crystallin isoforms lead to distinct lens phenotypes in mice.
Cataractous mouse line with point mutations of different γ–crystallin genes such as γB and γD were identified from ENU–mutagenized mice by a forward genetic approach. The lens phenotypes were characterized by morphological examination, including light and electron microscopic analysis, immunohistochemistry and biochemical assays. The properties of mutant γ–crystallin proteins were further examined in cultured cells in vitro by tagging with fluorescent proteins (GFP or RFP).
The γB–I4F point mutant mice showed lamellar cataracts associated with enlarged extracellular spaces between inner mature fiber cells and large aggregates inside mature fiber cells but had reduced lens opacity with age. The α–crystallins bind directly to γB–I4F mutant proteins in vivo and in vitro. By co–transfection of GFP–tagged αA–crystallins and RFP–tagged γB–I4F, we observed that αA–crystallins was sufficient to prevent the aggregation of γB–I4F mutant proteins in culture cells. In contrast, γD–V76D showed nuclear cataracts in heterozygous mutant mice but ruptured lenses in homozygous mice. Inner fiber cells of heterozygous and homozygous lenses had delayed denucleation process. In addition, γD–V76D mutant proteins also abolished protein aggregates induced in "cold cataracts" of wild–type lenses.
The γB–crystallin mutant uniquely affects appropriate packing of mature fiber cells while γD–crystallin mutant specifically perturbs the denucleation process of fiber cell maturation. This work suggests that γB play a role in appropriate assembly of the membrane–skeleton structures needed for the packing of mature fiber cells and that γD proteins play a role in regulating fiber cell denucleation required for establishing lens transparency. Thus, we hypothesize that different γ–crystallin isoforms are evolutionarily selected for their distinct functions in regulating fiber cell maturation besides their structural contributions in lens transparency.
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