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Kulandaiappan Varadaraj, Sindhu S Kumari; Involvement of beaded filament proteins in the regulation of Aquaporin 0, which is critical for lens transparency. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3564.
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We showed previously that Aquaporin 0 (AQP0) expressed in the fiber cells could play a critical role in establishing lens refractive index gradient (RING) through its interaction with other lenticular proteins (Kumari and Varadaraj, 2014, BBA, 1840: 2862-2877). The present study was undertaken to investigate the possible regulatory role of beaded filament (BF) proteins, CP49 and filensin, on AQP0, which is necessary for lens transparency, RING and prevention of spherical aberration (SA) in the constantly growing lens.
AQP0 heterozygous knockout (AQP0+/-) mice were developed in two different strains, FVB mice lacking BF proteins and C57BL/6J (C57) expressing BF proteins. To generate the mouse models, AQP0 knockout mice in a mixed background (hybrid 129S5/C57BL/6J-albino; Shiels et al., 2001, Physiol. Genomics, 7:179-186) were back-crossed with wild type (WT) C57 (Jackson Lab.) or FVB mice (Charles River Labs.) for 15 generations. Genomic PCR was used to analyze CP49 mutation. Expression of CP49 and filensin was studied by immunostaining and Western blotting using BF protein-specific antibodies. Colocalization was evaluated using coimmunoprecipitation. Lens transparency and SA were assessed and quantified.
Genomic PCR confirmed the deletion mutation in CP49 gene of FVB mice and the presence of CP49 in C57 mice. Immunostaining and Western blotting detected CP49 and filensin in C57 lenses in contrast to FVB lenses. Both BF proteins colocalized with AQP0. Lenses of WT FVB showed a slight increase in light scattering compared to those of WT C57. AQP0+/- lenses of FVB mice lacking CP49 incurred substantial reduction in filensin protein and exhibited significant loss of transparency and increase in SA compared to the lenses of C57 AQP0+/- mice.
Data suggest that lens-specific BF proteins, CP49 and filensin, interact with AQP0. Even though AQP0+/- lenses in both strains have only 50% of AQP0 and develop cataract, severity of cataract and SA is more intense in FVB mice that lack BFs, compared to C57 AQP0+/- mice with BFs. BF-cytoskeleton could be assisting in the distribution of AQP0 at the plasma membrane and regulating its functions, which are critical for maintaining lens transparency, RING and homeostasis. This study signifies for the first time the involvement of BF proteins, CP49 and filensin, in the regulation of AQP0, using in vivo animal models.
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