Fig. 6. γBnop-crystallin forms amyloid-like structures. (A and B) E18.5 lens from Crygbnop mice, stained with Congo red and haemotoxylin and viewed by bright field (A) and fluorescence microscopy (B). Only those nuclei near the centre of the lens are positive (arrows), but those in the outer lens fibre cells (fi), the lens epithelium (ep) and surrounding tissues (e.g. the cornea; co) are all negative (B, arrowheads). The lens capsule (B; c) is also negative. Adjacent sections were stained with γBnop-crystallin-specific antibodies to confirm that the Congo red-positive nuclear deposits contained γBnop-crystallin. Scale bar = 50 µm. (C) An example of a filamentous substructure of a nuclear inclusion found in lens fibre cells of E18.5 Crygbnop mice as seen by electron microscopy. The fibrillar-like substructure of the inclusions is indicated (arrow). Notice the clear fibrous substructure of this region of the inclusion in the insert (C; insert, arrows). This region was located at the edge of one of the inclusions and, as such individual filaments are not so obvious in the centre of the inclusion, it suggests that either the filamentous material ‘matures’ within the inclusion or that there is a degree of polymorphism in the aggregated forms of γBnop-crystallin. Scale bar = 500 nm. (D) Dialysis of recombinant γBnop-crystallin into 10 mM sodium phosphate pH 7.8, 1 mM EDTA results in the formation of both fibrils (arrows) and small rod-like particles (arrowheads), possibly due to morphological variation and/or the presence of intermediates in fibril formation. (E) Wild-type γ-crystallins do not form fibrils under these conditions. Scale bars = 200 nm. (F) X-ray diffraction of the fibrils assembled from γBnop-crystallin indicates formation of the typical ‘cross-β’ X-ray diffraction pattern characteristic of amyloid fibrils. Reflections can be observed at 4.7 Å (arrows) and two reflections at 10.7 and 12 Å (arrowheads).