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Rupalatha Maddala, Nikolai Skiba, Paul FitzGerald, Vasantha P. Rao; A Possible Role for Mouse lens Proteins which Co-Immunoprecipitate with Ankyrin-B in Membrane Organization and Stability. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1306.
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© ARVO (1962-2015); The Authors (2016-present)
Lens fiber cell membrane organization and integrity are considered to be essential for transparency and function of the ocular lens. Little is known however, about the role/identity of scaffolding proteins in regulating membrane cytoskeletal network organization and stability. To explore this question, we identified and characterized ankyrin-B associated macromolecular complexes from the mouse lens since this scaffolding protein has been found to be essential for lens architecture and transparency.
Lenses obtained from 3 to 4 week-old BL6 strain mice were used to isolate Ankyrin B-associated proteins by immunoprecipitation (IP) using Ankyrin-B monoclonal antibody. Protein immunocomplexes were separated by gradient SDS-PAGE (4-20%) and proteins extracted from the gel were analyzed by mass spectrometry (Waters SYNAPT G2 MS) to confirm identity. Additionally, immunoblot and immunofluorescence-based colocalization analyses were performed for select proteins.
Two independent analyses of ankyrin-B antibody derived IP complexes from detergent solubilized mouse lens homogenates revealed abundant amounts of several proteins including spectrin (both α and β), ankyrin B, plectin-1, filensin and phakinin. Proteins detected in minor amounts included vimentin, synemin, Hsc70, HSP90-beta, NrCAM, kinectin, actin, aquaporin-0, Armadillo repeat protein, desmoplakin, plakoglobin, Beta-B1-crystallin, catenin-2, caprin-2, ponsin, catenin-beta1, cadherin-2, ezrin, filamin A, 14-3-3, lengsin, periaxin, myosin 1b and Nck-associated protein. Protein identification was based on a minimum of two independent peptides and protein confidence score > 99% (Mascot). Many of these proteins were also confirmed to present in the Ankyrin-B immunoprecipitates by immunoblot analysis. Further, Filensin was found to colocalize with Ankyrin-B and β-spectrin in mouse lens fibers at discrete regions. Ongoing studies are focused on determining the nature of Ankyrin-B interaction with filensin, and on characterizing the stability and organization of several of the proteins which were found to co-exist with Ankyrin-B, using Ankyrin-B and Filensin KO mouse lenses.
These ongoing studies aimed at elucidating the molecular mechanisms regulating lens fiber cell membrane organization and stability identify ankyrin-B as a crucial scaffolding protein which serves to link and organize various cytoskeletal proteins, adaptor proteins, cell adhesive proteins and channel proteins to the fiber cell plasma membrane.
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