Previous work indicates that inhibitors of actin, myosin, and myosin light chain kinase are associated with a softening of the lens. The softening of lenses did not affect the optical quality, despite the application of inhibitors that, based on Western blot analysis, affect the cytoskeleton of lens fibre cells. Inhibitors can impart their effects biochemically or structurally, therefore, in the present study, the cytoskeletal distribution of inhibitor-treated lenses was examined using confocal microscopy.Methods
Lenses of 7-day-old White Leghorn chickens were treated (15 mins) with 10 μM of either a vehicle (dimethyl sulfoxide) or an inhibitor (1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride [ML-7], a myosin light chain kinase inhibitor; 1-phenyl-1,2,3,4-tetrahydro-4-hydroxypyrrolo[2.3-b]-7-methylquinolin-4-one [blebbistatin], a myosin II inhibitor; latrunculin, an actin inhibitor). After treatment, lenses were incubated in antibodies against the appropriate cytoskeletal proteins, and mounted onto slides in toto for examination under confocal microscopy. Images taken from the confocal microscope were analyzed using nearest neighbour analysis for irregularity.Results
Myosin distributions were highly regular, with a nearest neighbour value (Rn) of 2.02. Blebbistatin treatment resulted in a less regular arrangement (Rn=1.65), approaching a random distribution. Actin distributions in the latrunculin-treated lenses were disorganized, showing irregular spatial distributions between vertices (Rn=1.53) compared to those in vehicle treated lenses (Rn=1.91).Conclusion
Inhibitors were shown to affect the distributions of actin and myosin, which indicates that these inhibitors impart their effects by altering cytoskeletal ultrastructure.