Effects of ML-7 on the Actomyosin Networks of the Avian Crystalline Lens

Abstract

Assemblies of contractile proteins such as actin, myosin and myosin light chain kinase (MLCK) are found on the posterior surface of the lens fibre cells. The mechanical properties of chicken lenses, as well as their focal lengths, were found to be affected by the disruption of the network using inhibitors such as 1-(5-Iodonaphthalene-1sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7). The purpose of this study is to determine if ML-7, a MLCK inhibitor influences actomyosin organization in the chicken lens, which may have lead to the reported changes in stiffness and focal lengths. The Nearest Neighbour Distance (NND) values will be used to test the following hypotheses: ML-7-treated lenses have further NNDs compared to their vehicle counterparts, and that the NNDs of the 100μM-treated lenses would be lower than the lenses treated with lower concentrations (1μM and 10μM) of ML-7.

Eyes of 7-day old white leghorn chickens (gallus gallus domesticus) were obtained. The anterior segment of one eye is treated with 1μM, 10μM, or 100μM of ML-7, and the other, with vehicle. The lenses were stained for actin and myosin. The NNDs for actin-actin, actin-myosin, myosin-actin and myosin-myosin were determined from confocal images of the networks to quantify the network distribution.

Myosin-myosin NNDs of 10μM-treated lenses showed significantly lower values compared to 100μM-treated lenses. However, it is uncertain whether the significant change observed was indeed due to ML-7 activity or due to variation between birds. Additionally, only myosin-actin NNDs showed a significant reduction in treated lenses. These results suggest that actomyosin interactions affected by ML-7 may have been too subtle to detect, or compensation by other kinases occurred. Thus, this study was unable to determine if the stiffness and focal length changes in ML-7-treated lenses observed in previous studies were related to changes that ML-7 imparts on the actomyosin networks.