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Gah-Jone Won, Vivian Choh; The Optical Quality of Inhibitor-Treated Avian Crystalline Lenses. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4046. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
A hexagonal lattice of proteins consisting of actin and myosin cradles the posterior surface of the avian crystalline lens. In previous work, lenses exposed to actin and myosin inhibitors softened, but the optical quality of these lenses was unknown. In the present study, the optical quality of inhibitor-exposed lenses was measured.
Lenses of 7-day-old White Leghorn chickens (Gallus gallus domesticus) were treated (15 min) with 10 µM of either an inhibitor or vehicle (dimethyl sulfoxide). The inhibitors used were 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7, a myosin light chain kinase (MLCK) inhibitor; n=4), 1-phenyl-1,2,3,4-tetrahydro-4-hydroxypyrrolo[2.3-b]-7-methylquinolin-4-one (blebbistatin, a myosin II inhibitor; n=5), latrunculin (an actin inhibitor; n=5). Optical quality was assessed using a scanning laser monitor which measures the focal lengths (FLs) of lenses. 4th order polynomial regressions were fitted to FLs to calculate the FL errors. Western blot analysis was used to confirm the effectiveness of the inhibitors (anti-G-actin for latrunculin; anti-ATPase for blebbistatin; anti-MLCK for ML-7). Posterior surfaces of the lenses were analyzed for changes to the actin distributions using phalloidin and confocal microscopy.
The inhibitors did not affect optical quality; latrunculin-, blebbistatin-, ML-7-treated lenses were similar to their respective vehicle-treated lenses (p=0.957, p=0.232, p=0.623, respectively). Western blot analysis indicated that all inhibitors were effective; compared to vehicle-treated lenses, latrunculin-treated lenses expressed a relatively higher concentration of G-actin (133.26%), whereas blebbistatin- and ML-7-treated lenses expressed a lower concentration of ATPase (44.36%) and phosphomyosin (21.14%), respectively. Confocal microscopy indicated that actin distributions in latrunculin-treated lenses were disorganized compared to those in vehicle-treated lenses which maintained their regular repeated array.
All three inhibitors were previously shown to lead to softer lenses and this study confirmed that the optical quality was not adversely affected by the inhibitors (which was validated to be effective). Immunocytochemistry results showing a redistribution of actin with latrunculin treatment suggest that the hexagonal lattice may be necessary for maintaining structural integrity of the lens as a whole.
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