Towards a functional understanding of proteoglycan-collagen associations in the cornea by 3-dimensional electron microscopy of gene-targeted mutants

The cornea is the front clear part of the eye. It is essential for proper vision because it lets in light and focuses it on the retina at the back of the eye. Thus, a sharp image is formed and we can see properly. The cornea is a special tissue because it is transparent, and in this respect it is unlike other related tissues in the body -- the tendons that link our bones and muscles or the sclera (the white of the eye), for example -- which are made of similar components. Scientists believe that the cornea is transparent because the protein called collagen that forms much of the cornea is mostly in the form of long, thin rope-like structures called fibrils. Moreover, these collagen fibrils are formed into a very well defined arrangement that lets light through. If this arrangement breaks down the cornea looses its transparency and becomes cloudy. As a result, vision is severely compromised. Interestingly, scientists suspect that molecules called proteoglycans in the cornea influence the collagen fibrils and force them to take up the special arrangement that allows corneal transparency. Previous investigations have studied the structural relationship between collagen and proteoglycans using the corneas of mice that have been genetically modified so that they don't possess certain types of proteoglycan. This allows the structure of different proteoglycan sub-types to be understood. But, up to now it has only been possible to study the collagen and proteoglycan structures in 2 dimensions by examining very thin sections of cornea on an electron microscope, so the understanding that we can get is limited. Now, however, we are able to use a new modification of electron microscopy to produce images of the collagen and proteoglycans that allows us to see their structures in 3 dimensions. This project will thus discover the links between collagen and proteoglycans in the cornea in a detail not seen previously, and will help us to understand how the structure and transparency of the cornea is maintained.

The cornea is a pivotal model system to understand the physical basis of optical transmission in fibrous biomaterials and the molecular interactions in extracellular matrices. The cornea comprises a lamellar array of uniform diameter collagen fibrils with a high degree of spatial order, which makes it transparent due to the interference of scattered light. Significant deviations away from the norm will lead to increased light scattering and reduced transparency. Small extrafibrillar leucine-rich proteoglycans (PGs) are thought to govern the spatial arrangement of collagen, and recent research in our lab has generated 3D EM reconstructions of the cornea at high resolution. This information has started to provide a fundamental understanding of collagen-PG associations, and has led to a model whereby the major corneal PG, keratan sulphate (KS), prohibits the collagen fibrils from coming too close together. The other main PG in the cornea has glycosaminoglycan side chains that are a hybrid of chondroitin sulphate (CS) and dermatan sulphate (DS). In our hypothesised model these create a extended tethering network which keeps the corneal matrix intact. To establish our model fully we need to understand the 3D architecture of the collagen-PG associations and distinguish between the action of the PG sub-types. Thus, we will investigate the collagen-PG associations in the cornea of the mouse in 3D and at high resolution in the normal healthy situation. We will then characterise these relationships when KS PGs are absent, caused either by the genetic deletion of the lumican and keratocan core proteins or by the mutation of the sulphotransferase enzymes than synthesise KS. We will also investigate the corneal matrix architecture when CS and DS PGs are absent because of a double null mutation of decorin and biglycan. The resultant data will provide a high-resolution 3D understanding the corneal matrix and indicate respective roles for the stromal PGs.