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Catherine Cheng, Roberta B. Nowak, Junyuan Gao, Xiurong Sun, Sondip K Biswas, Woo-Kuen Lo, Richard T Mathias, Velia M Fowler; Lens ion homeostasis and connexin 46 gap junction plaque assembly rely on the actin-spectrin and beaded intermediate filament networks. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3560.
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© ARVO (1962-2015); The Authors (2016-present)
Life-long lens homeostasis and transparency depend on a microcirculation system facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50). We investigated the roles of the actin-spectrin membrane skeleton network and lens-specific beaded filaments in the lens microcirculation pathway and gap junction plaque assembly in mouse lenses.
We determined lens ionic homeostasis in mice lacking tropomodulin1 (Tmod1), an actin pointed-end capping protein, CP49, a lens-specific intermediate filament protein, or lacking both Tmod1 and CP49 by evaluating gap junction coupling conductance, intracellular hydrostatic pressure and sodium ion concentration as a function of distance from the lens center. Cx46 and Cx50 protein levels were tested by western blotting. Immunofluorescence and electron microscopy were used to evaluate the assembly of Cx46 and Cx50 plaques on broad sides of differentiating lens fibers.
Surprisingly, we found that the simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, causes increases in gap junction coupling resistance, hydrostatic pressure and sodium concentration in double knockout (DKO) lenses. There were no changes in the protein levels of Cx46 and Cx50 in DKO lenses, and normal gap junctions were observed by electron microscopy. However, immunostaining and quantitative analysis of 3D confocal images showed that Cx46, but not Cx50, gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. Interestingly, we also observed that Cx46 and Cx50 gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network.
Our work demonstrates that the mere presence of gap junctions at the cell membrane is insufficient for normal lens fiber cell coupling and that assembly of large micron-size Cx46 gap junction plaques depends on two biochemically linked cytoskeletal systems, the actin-spectrin membrane skeleton and the beaded intermediate filament network. Since only Cx46 gap junction plaque assembly is affected in DKO lenses, Cx46 and Cx50 likely form homomeric channels in the lens. This is the first work to reveal that proper gap junction plaque localization and size are required for normal lens coupling conductance.
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