Transparency and accommodation are crucial determinants of image focusing by the ocular lens and depend upon precise hexagonal geometry, radial packing, mechanical properties and membrane organization of fiber cells. The broad goals of this research project are to identify and characterize plasma membrane cytoskeletal scaffolding proteins regulating fiber cell shape, adhesion, tensile properties, membrane organization, and to determine how dysregulation of membrane cytoskeletal scaffolding activity impacts these vital characteristics of lens. This proposal will investigate th coordinated and interdependent scaffolding function(s) of Periaxin (PRX) and its interacting proteins- Ankyrin-B (AnkB) and the dystrophin- glycoprotein complex (DGC) in differentiating and mature fiber cells in the context of lens function. Our recent observations show that PRX and AnkB not only co-immunoprecipitate in a reciprocal manner but also exhibit a close spatial overlap of distribution profile in fiber cells. Importantly, mice with PRX null or heterozygous for AnkB null mutations (AnkB-/+) revealed prominent and similar disruptions in fiber cell shape, alignment and membrane clustering of connexin-50, filensin, spectrin, NrCAM and WAVE-2. Moreover, in AnkB-/+ lenses, PRX failed to anchor to the plasma membrane and in a vice versa manner, AnkB levels were decreased in PRX null lenses. Significantly in these lenses, membrane organization of the DGC was dramatically impaired indicating a close interaction among PRX, AnkB and the DGC. Although both PRX null and AnkB-/+ mouse lenses remained largely transparent, lens stiffness was found to be significantly decreased. These multiple and significant preliminary observations prompted us to hypothesize that the fiber specific PDZ- domain protein PRX serves as a key scaffolding protein via direct interaction with AnkB and the DGC, to maintain fiber cell hexagonal shape, radial and compact alignment, membrane domain organization and mechanical properties, which are critical determinants of lens transparency and accommodation. To determine the functional importance of the coordinated and essential scaffolding role(s) played by the PRX-AnkB-DGC axis in lens tissue, we will pursue the following three specific aims: 1. Detailed in vitro characterization of the interdependent scaffolding activities of PRX, AnkB and the DGC in mouse lens fibers, 2. Detailed in vivo analysis of how absence of PRX, AnkB and impaired DGC activity affects lens optical quality, fiber cell shape, adhesion and the membrane skeletal network, using gene targeted mice, and 3. Determine the role(s) of PRX-AnkB-DGC axis in organization and regulation of lens fiber cell channel protein membrane domains and activity, and in maintenance of lens mechanical properties using gene targeted mice. The completion of these unstudied aspects of fiber cells is expected to provide novel and important insights into how membrane cytoskeletal scaffolding interactions regulate the unique structure-function attributes of ocular lens in the context of its optical properties an accommodation.
Transparency and accommodation are essential for ocular lens function, and rely on the precise geometry, alignment, membrane domain organization and deformability of hexagonal fiber cells. This application will examine the coordinated membrane cytoskeletal scaffolding activities of Periaxin and its interacting proteins- Ankyrin-B and the Dystrophin-Dystroglycan Complex in establishing the unique hexagonal geometry and radial packing, channel protein membrane domain organization and activity in lens fiber cells, and in maintenance of lens clarity and mechanical integrity, using mice with null mutations for the respective genes. This project is expected to provide significant and novel insights into how membrane cytoskeletal scaffolding interactions impact optical clarity, architecture and mechanical properties of the normal lens, and tie into the etiology of age-related cataract and loss of accommodation, which are a global heath burden.
