D L Boyle1, L Takemoto. 1. Division of Biology, Kansas State University, Manhattan, KS 66506, USA. dboyle@ksu.edu
Abstract
PURPOSE: To test the hypothesis that crystallin proteins may actively participate in the differentiation of lens epithelial cells into fiber cells. METHODS: Primary epithelial cells from adult bovine lenses were cultured at 37 degrees C until reaching 95-100% confluency in approximately 4-7 days. Using osmotic lysis of pinocytic vesicles, cells were then loaded with proteins labeled or unlabeled with the fluorescent marker Texas Red (TR). Fetal bovine proteins loaded into cells were lens water soluble fractions, HPLC-purified alpha-, beta-, and gamma-crystallin fractions, or bovine serum albumin (BSA). Cultures were then monitored for morphological changes over a 7 day period. RESULTS: Both TR-labeled and unlabeled water-soluble and a-crystallin fractions from bovine lenses resulted in morphological changes to epithelial cells during the first two h postloading. These changes included aggregation of epithelial cells into raised multilayered cell masses, as well as several cells losing attachment to the dish. The initial changes were subsequently followed by elongation of cells within the mass and an increase in size of the mass, so that by 4 days postloading the multilayered, multicellular structures could be visualized with the unaided eye. Differentiation was confirmed within these structures by expression of MIP 26, beta- and gamma-crystallin. These changes did not occur in cultures containing cells originally loaded with beta- or gamma-crystallin fractions, or with cells loaded with BSA. CONCLUSIONS: The results support the hypothesis that alpha-crystallins may actively participate in the differentiation of lens epithelial cells into fiber cells.
PURPOSE: To test the hypothesis that crystallin proteins may actively participate in the differentiation of lens epithelial cells into fiber cells. METHODS: Primary epithelial cells from adult bovine lenses were cultured at 37 degrees C until reaching 95-100% confluency in approximately 4-7 days. Using osmotic lysis of pinocytic vesicles, cells were then loaded with proteins labeled or unlabeled with the fluorescent marker Texas Red (TR). Fetal bovine proteins loaded into cells were lens water soluble fractions, HPLC-purified alpha-, beta-, and gamma-crystallin fractions, or bovineserum albumin (BSA). Cultures were then monitored for morphological changes over a 7 day period. RESULTS: Both TR-labeled and unlabeled water-soluble and a-crystallin fractions from bovine lenses resulted in morphological changes to epithelial cells during the first two h postloading. These changes included aggregation of epithelial cells into raised multilayered cell masses, as well as several cells losing attachment to the dish. The initial changes were subsequently followed by elongation of cells within the mass and an increase in size of the mass, so that by 4 days postloading the multilayered, multicellular structures could be visualized with the unaided eye. Differentiation was confirmed within these structures by expression of MIP 26, beta- and gamma-crystallin. These changes did not occur in cultures containing cells originally loaded with beta- or gamma-crystallin fractions, or with cells loaded with BSA. CONCLUSIONS: The results support the hypothesis that alpha-crystallins may actively participate in the differentiation of lens epithelial cells into fiber cells.