The ocular lens is comprised of successive layers of extremely long thin fiber cells whose shapes, ordered hexagonal packing and regular membrane structure are critical for lens transparency and focusing. The broad, long-term objectives of this research are to elucidate the role of the spectrin-based membrane skeleton in fiber cell morphogenesis, lens optical quality and mechanical properties, and how defects in the membrane skeleton may contribute to progressive, age-dependent declines in lens functions. This proposal will study tropomodulins (Tmods), a family of actin regulatory proteins that function cooperatively with tropomyosins (TMs) to cap actin filament pointed ends, regulating actin filament turnover and stability in the membrane skeleton, contributing to cell shapes, interactions and mechanical properties. The mouse lens contains two Tmods, Tmod1 and Tmod3, which are both associated with fiber cell membranes. To investigate Tmod functions in vivo in the lens, we have generated knockout mice with targeted deletions in Tmod1 and Tmod3. Recent work from our laboratory has shown that lenses lacking Tmod1 develop normally but demonstrate striking defects in their cortical fiber cells, including abnormal fiber cell shapes with disordered cellular packing in the anterior cortex. Levels of a membrane-associated, short 3-TM isoform are reduced selectively in the absence of Tmod1, concomitant with depolymerization of actin filaments and reduction of 12-spectrin on membranes. It is hypothesized that Tmod1 and the short 3-TM function to stabilize the actin filament linkers in the membrane skeleton, thus controlling its integrity and long-range organization. This is expected to be critical for anchoring of adhesion receptors to control fiber cell shapes, interactions and ordered packing. Increased levels of Tmod3 in embryonic and postnatal but not adult lenses indicates that Tmod3 may compensate for the absence of Tmod1 in lens development and initial growth, leading to age-dependent progression of fiber cell disorder. Further, Tmod3 (but not Tmod1) is proteolysed in adult lens fiber cells, suggesting a unique role for Tmod3 degradation in membrane skeleton remodeling in fiber cell elongation and maturation. The specific aims are: (1) To investigate the role of Tmod1 in controlling membrane skeleton assembly, stability and associations with adhesion receptors by biochemical and morphological analyses of lenses from Tmod1 knockout mice, (2) To investigate the expression and function of Tmod3 in lens fiber cells and its role in compensating for absence of Tmod1 by molecular, biochemical and morphological analyses of lens development and growth in Tmod1 and Tmod3 knockout mice, (3) To investigate the consequences of loss of Tmod1 or Tmod3 for lens optical quality and mechanical stiffness by functional assays on living lenses ex vivo. Completion of these studies will elucidate the molecular and cellular basis for membrane skeleton regulation of fiber cell shapes and interactions during lens development, growth, and ageing, and how this influences lens optical quality and mechanical properties. PUBLIC HEALTH RELEVANCE: This project seeks to elucidate the molecular basis for membrane skeleton regulation of lens fiber cell shape, interactions and ordered hexagonal packing, and how this contributes to maintenance of lens transparency and focusing ability. Our studies will utilize transgenic mouse models in which deficiencies in specific membrane skeleton components lead to developmental or age-dependent abnormalities in lens fiber cell structures and interactions. These studies are expected to provide mechanistic insights into the molecular and cellular basis for normal lens optical quality and mechanical functions. Our studies on mouse lenses may also aid in understanding the causes of inherited human cataracts and loss of accommodative ability with age.