EM immunolocalization of α-crystallins: Association with the plasma membrane from normal and cataractous human lenses

Abstract

Purpose. To integrate past biochemical findings with past morphological observations of area insoluble material isolated from cataract and aged normal lenses, by determining the spatial distribution of α-crystallins associated with the plasma membrane (PM) of nuclear cataractous and age matched normal human lenses.

Methods. Lenses were homogenized, pelleted and washed several times in 0.05M Tris-C1 (pH 7.2) containing 100mM KC1, 1 mM MgCl₂ and 2mM (β-mercaptoethanol, followed by several washes in 8M urea. Urea insoluble pellets (UIP) were labeled before fixation and embedding with rabbit serum raised against α-crystallins, followed by goat anti-rabbit IgG conjugated to 5nm gold. Approximately 300 gold particles associated with the PM were counted, for each lens, on several electron microscopy (EM) micrographs. The number of gold particles/um of PM, number of individual vs clusters of gold particles were determined.

Results. Micrographs from both normal and cataractous human lenses clearly demonstrated the association of α-crystallins with the PM. Also apparent was the abundant labeling of the PM for cataractous lenses as compared to normal lenses. Quantification of the gold labeling revealed that not only was there an increase in the amount of labeling/um of PM in cataract lenses, but there was also an increased percentage of gold in clusters. These clusters were not only more numerous in cataractous lenses, but also contained a greater number of gold/cluster.

Conclusions. These findings provide morphological evidence that the PM in nuclear cataract lenses is associated with large aggregates of α-crystallin.

Key Words:

• α-crystallins
• cataract
• electron microscopy
• immunolocalization
• plasma membrane
• human