The ocular lens is responsible for fine focusing of light onto the retina. Age-related changes in lens mechanics are linked to presbyopia, a reduction in the lens' ability to change shape during focusing (accommodation). While mouse lenses do not accommodate, they do show age-dependent stiffening like primates and other mammals, and provide a genetic model system to elucidate cytoskeletal regulation of cellular architecture, transparency and mechanics during aging. The lens consists of a bulk of elongated, hexagonally packed fiber cells, which undergo organelle loss and compaction to minimize light scattering, with formation of complex interlocking membrane protrusions at cell-cell interfaces. Lens fibers contain a spectrin-actin network with two F-actin-stabilizing proteins, tropomodulin1 (Tmod1) and tropomyosin (?TM). Tmod1-/- mouse lenses have reduced stiffness and abnormal fiber cell interlocking domains, and ?TM-/- lenses have reduced stiffness with progressive anterior cataracts. The lens also contains nonmuscle myosin IIA (NMIIA), and human lenses with mutations in the NMIIA heavy chain, termed MYH9-related disorders (MYH9-RD), develop cataracts. This proposal will test the hypotheses that F-actin stability mediated by Tmod1 and ?TM, and tensile forces mediated by actomyosin contractility, function to confer distinct fiber cell architectures during differentiation and maturation, thereby determining lens optical and mechanical properties that vary radially with fiber cell maturation, and temporally with aging.
Aim 1 will elucidate the relationship between lens mechanics, fiber cell architecture and F-actin networks in wild-type mouse lenses as a function of aging. Detailed lens biomechanical properties will be measured with an Instron instrument, and responses of fiber cell shapes and F-actin networks to compression (and release) evaluated by immunostaining, confocal microscopy and scanning electron microscopy across the lens radius, and biochemistry of whole lenses. We predict that age- dependent cytoskeletal reorganization at the fiber-cell level contributes to age-dependent stiffening and loss of resilience of the mouse lens at the whole-organ level.
Aim 2 will test the role of F-actin stability by analyzing lenses from Tmod1-/-, ?TM-/- or Tmod1/?TM double knockout mice. We predict that Tmod1 and ?TM cooperate to stabilize the spectrin-actin network in interlocking domains of mature fibers, thereby regulating age-dependent lens transparency, mechanical stiffness and resilience.
Aim 3 will test the role of actomyosin contractility by analyzing lenses from transgenic knock-in mouse models with mutations in the NMIIA motor domain (R702C) or rod domain (D1424N, E1841K) that phenocopy human MYH9-RD. We predict that loss of F-actin stability, or impaired actomyosin contractility, result in distinct cytoskeletal and cellular reorganizations at the fiber-cell level that contribute to age-dependent stiffening and loss of resilience of the mouse lens at the whole-organ level. This project is the first to use a multidisciplinary, integrative approach to link age-dependent changes in lens biomechanical properties to alterations in fiber cell architecture and cytoskeletal organization.

Public Health Relevance

The eye lens is a transparent organ responsible for fine focusing of light onto the retina: two common age- related lens pathologies are 1) presbyopia, a reduction in the lens' ability to change shape during focusing, and, by extension, the need for reading glasses, and 2) cataracts, defined as any opacity in the lens, which are the leading cause of blindness in the world. This project will use mouse models to study the genetic basis of age-dependent changes in lens stiffness, which has been associated with presbyopia, employing a multidisciplinary, integrative approach to link lens biomechanical properties to defects in transparency and alterations in cellular architecture and cytoskeletal organization. These studies will provide mechanistic insights into the molecular and cellular basis for age-dependent declines in lens optical and mechanical properties, and contribute to understanding the pathogenesis of inherited human cataracts.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY017724-10
Application #
9319267
Study Section
Biology of the Visual System Study Section (BVS)
Program Officer
Araj, Houmam H
Project Start
2008-09-01
Project End
2020-06-30
Budget Start
2017-07-01
Budget End
2018-06-30
Support Year
10
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Cheng, Catherine; Fowler, Velia M; Gong, Xiaohua (2017) EphA2 and ephrin-A5 are not a receptor-ligand pair in the ocular lens. Exp Eye Res 162:9-17
Cheng, Catherine; Gokhin, David S; Nowak, Roberta B et al. (2016) Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses. J Vis Exp :
Cheng, Catherine; Nowak, Roberta B; Biswas, Sondip K et al. (2016) Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells. Invest Ophthalmol Vis Sci 57:4084-99
Cheng, Catherine; Nowak, Roberta B; Gao, Junyuan et al. (2015) Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells. Am J Physiol Cell Physiol 308:C835-47
Yamashiro, Sawako; Gokhin, David S; Kimura, Sumiko et al. (2012) Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types. Cytoskeleton (Hoboken) 69:337-70
Nowak, Roberta B; Fowler, Velia M (2012) Tropomodulin 1 constrains fiber cell geometry during elongation and maturation in the lens cortex. J Histochem Cytochem 60:414-27
Gokhin, David S; Nowak, Roberta B; Kim, Nancy E et al. (2012) Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens. PLoS One 7:e48734
Leonard, Michelle; Zhang, Liping; Zhai, Ni et al. (2011) Modulation of N-cadherin junctions and their role as epicenters of differentiation-specific actin regulation in the developing lens. Dev Biol 349:363-77
Nowak, Roberta B; Fischer, Robert S; Zoltoski, Rebecca K et al. (2009) Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens. J Cell Biol 186:915-28