Hunting for the Adhesion Molecule, Retinoschisin, in Retina using Cemovis

Abstract

In studying biologically relevant molecules, the ultimate aim is to visualize them in their native environment. One technique that has the potential to resolve individual molecules in cells and tissues is cryo-electron microscopy of vitreous sections (CEMOVIS) combined with tomography. We are interested in revealing the disposition of the retinal adhesion protein, retinoschisin (RS1), within the retina. Immuno-fluorescence and immuno-electron microscopy showed that RS1 is abundant in the intercellular spaces between the inner segments of photoreceptors, and between bipolar cells. Loss-of-function mutations in RS1 cause X-linked retinoschisis (XLRS), a debilitating disease associated with the formation of retinal cavities or cysts (schisis) and vision loss in young males. We solved the structure to 4.1 Å by cryo-electron microscopy (cryoEM), a 16-mer composed of two octameric rings with a total weight of ∼380 kDa. From this structure it is clear that many mutations in the subunit interfaces would preclude assembly of the rings. However, other mutation sites are exposed at the periphery and it was not evident how they would cause disease. In further cryoEM experiments, we observed that RS1 double rings self-assemble into extensive branched networks, suggesting a mechanism for its adhesive function. To ascertain whether this assembly also occurs in situ, we are examining mouse retinal tissue using CEMOVIS and cryo-electron tomography. We obtained images of 50 nm cryosections that show clear distinction of microtubular protofilaments separated by ∼5 nm. This resolution is clearly sufficient to see the RS1 double ring and whether it assembles into networks in the intercellular space between photoreceptors and bipolar cells.