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Vasanth Rao, Ariana Allen, Rupalatha Maddala; Ankyrin-B is required for lens fiber cell lateral membrane organization, hexagonal symmetry, tension and function. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2236.
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© ARVO (1962-2015); The Authors (2016-present)
To understand the cellular and molecular basis of Ankyrin-B (AnkB) deficiency- associated lens opacification in postnatal (P16) mice.
To determine whether lens opacification under AnkB deficiency is preceded by changes in protein degradation, apoptosis and fiber cell organization. Lenses derived from P12, P14 and P16 AnkB cKO (maintained on C57BL/6J genetic back ground) and littermate control (AnkB floxed) mice were analyzed by SDS-PAGE (for protein profile of soluble and insoluble fractions), TUNEL assay and wheat germ agglutinin staining, respectively. Lens fiber cell morphology and membrane organization was evaluated using scanning and transmission electron microscopy. Individual lens fibers (isolated single cells) immunolabeled for specific proteins of interest were imaged by high resolution microscopy.
AnkB cKO mouse lenses develop normally, exhibit a significant decrease in size and intense opacity in P16 mice without detectable changes in protein profile or apoptosis. Lens fiber cell hexagonal shape, membrane organization and lateral membrane ball and socket interdigitations however, were found to be dramatically disrupted together with decreased levels of membrane associated β-spectrin, actin, NrCAM, N-cadherin, periaxin, dystrophin, myosin1b, Piezo 1 and αN-catenin in AnkB cKO mice (P16) compared to control lenses. Single cell lens fibers isolated from the AnkB cKO mice (P16) were found to be fragile, swollen and stunted with significantly reduced lateral membrane protrusions associated with impaired membrane organization of actin, β-spectrin, NrCAM, N-cadherin, CaV1.3 L-type calcium channel, Piezo 1 pressure sensitive ion channel, tension sensitive myosin1b and connexin-50 as compared to control lens fibers. While fiber cells from control lenses found to be retractile during single cell separation, cells from the AnkB deficient lenses failed to exhibit this property.
This study demonstrates that AnkB, the well characterized membrane scaffolding protein, plays a vital role in maintaining lens fiber cell cytoarchitecture, hexagonal shape, lateral membrane organization and function, and that AnkB deficiency induces a discrete and dramatic lens phenotype after stage P14.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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