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G. J. Parfitt, C. Pinali, R. D. Young, A. J. Quantock, C. Knupp; Elucidation of Collagen-Proteoglycan Interactions in the Mouse Corneal Stroma by Electron Tomography. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3834.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal transparency is directly attributed to its organised collagen fibril architecture, which is regulated by the interfibrillar proteoglycan biopolymers. To understand how proteoglycans are organised and to interpret collagen-proteoglycan inter-relationships, we applied electron tomography techniques to visualise proteoglycan configurations in three-dimensions.
Adult mouse corneas were excised and fixed overnight in 2.5% glutaraldehyde in 25mM sodium acetate and 0.1M MgCl2, which also included cuprolinic blue. A -60o to +60o single axis tilt series of both longitudinal and transverse sections in the anterior, mid and posterior stroma were captured, in one degree increments at a magnification of x20k, with a JEOL 1010 transmission electron microscope. Alignment of the images was carried out using IMOD, whilst back-projection and segmentation of the reconstruction were performed using EM3D software.
Three-dimensional reconstructions of the three distinct stromal regions illustrated the regular organisation of collagen fibrils within the mouse cornea. No distinct ordered pattern or symmetry of proteoglycan orientation was observed. Three structurally distinct proteoglycan types are evident in the reconstructions. Large, electron-dense proteoglycans (possibly chondroitin sulphate/dermatan sulphate proteoglycans) interconnecting three adjacent collagen fibrils are present. These proteoglycans can also exist as a distinctly shorter population that bridge only two adjacent fibrils. Another sub-population of even shorter proteoglycans (possibly keratan sulphate proteoglycans) also interconnects neighbouring fibrils.
The reconstructions suggest a complex interaction between proteoglycans and collagen, which allows for the dynamic control of collagen fibril architecture in the cornea. Both keratan sulphate and chondroitin sulphate/dermatan sulphate are likely to utilise their electrostatic charges and self-aggregation as a means to optimise the spatial distribution of collagen fibrils. Morphological differences of proteoglycans suggest that they could form tetramers by lateral aggregation and in some cases they extend end-to-end connecting more than two fibrils. Ongoing studies of mouse strains with genetic alterations in proteoglycans and/or glycosaminoglycans will allow us to test these conclusions more fully.
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