Actin cytoskeleton regulation of lens architecture, transparency and mechanics

肌动蛋白细胞骨架对晶状体结构、透明度和力学的调节

• 批准号: 10405108
• 负责人: Velia M Fowler
• 金额: $ 33.69万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10405108
• 关键词: Actin-Binding Protein; Actins; Address; Affect; Age; Anterior eyeball segment structure; Apical; Architecture; Behavior; Biomechanics; Blindness; Cataract; Cell Differentiation process; Cell Shape; Cell Surface Extensions; Cell membrane; Cell-Matrix Junction; Cells; Cellular Morphology; Complement; Complex; Confocal Microscopy; Crosslinker; Crystalline Lens; Cytoskeleton; Data; Elements; Epithelial; Epithelial Cells; F-Actin; Family; Filament; Fimbrin; Future; Genetic; Goals; Growth; Human; Intercellular Junctions; Knowledge; Label; Lateral; Light; Link; Macaca; Measures; Mechanics; Mediating; Membrane; Microfilaments; Modeling; Molecular; Molecular Target; Morphology; Mus; Nonmuscle Myosin Type IIA; Organ; Pathology; Pathway interactions; Peripheral; Pharmacology; Physiological; Play; Presbyopia; Primates; Property; Protein Isoforms; Radial; Regulation; Reporter; Retina; Role; Scanning Electron Microscopy; Shapes; Stress Fibers; Structure; Testing; Thinness; Tissues; Tropomyosin; Work; age related; alpha Tropomyosin; confocal imaging; crosslink; fiber cell; flexibility; fluorescence imaging; gene function; lens; lens transparency; mouse model; multiphoton imaging; multiphoton microscopy; nanoscale; non-muscle myosin; plastin; predicting response; prevent; recruit; resilience; response

Project Summary Lifelong lens transparency and flexible shape, required for focusing light onto the retina, relies upon epithelial and fiber cells whose shapes and organizations depend on filamentous (F-) actin networks. Epithelial cells contain three distinct F-actin networks: lateral cell junctions, basal stress fibers, and unique apical polygonal arrays. These networks consist of tropomyosin (Tpm) isoforms that stabilize F-actin, as well as non-muscle myosin IIA (NMIIA), and are thought to generate contractile or tensile forces to stabilize epithelial deformation and integrity during whole lens shape changes, but this has not been tested. Epithelial cells differentiate into long, thin fiber cells that form complex membrane interlocking protrusions and paddle-like domains that change with maturation and depth. Fiber cell membrane protrusions are supported by F-actin networks stabilized by fiber cell Tpm3.5, which regulates F-actin cross-linkers. In Tpm3.5-depleted lenses, the flexible crosslinker, a- actinin1, is increased on membranes, whereas the stiff crosslinker fimbrin (plastin) is decreased. Tpm3.5- depleted lenses have decreased whole lens stiffness and resiliency suggesting that more flexible F-actin networks allow greater fiber cell membrane deformation to result in softer lenses. However, the mechanistic links between F-actin networks, membrane deformation, cellular architecture, and whole lens shape change are unclear. The objective of this proposal is to determine how the F-actin networks in epithelial and fiber cells control membrane deformations and cellular shapes to confer whole lens transparency and flexibility. To address this, we will use mouse lenses to test gene function and primate lenses as a model for human lens shape change. Aim 1 will test the hypothesis that distinct F-actin and NMIIA networks control epithelial deformation and stability during whole lens shape changes. Tpm isoforms associated with epithelial F-actin networks will be determined, and fluorescent-tagged Tpms, F-actin, NMIIA and cell membranes visualized by live cell confocal microscopy to investigate network dynamics and cell deformation during whole lens shape changes. F-actin network functions will be targeted by pharmacological (mouse and primate) or genetic (mouse) approaches. Aim 2 will test the hypothesis that Tpm3.5-regulated F-actin networks in fiber cells confer membrane deformation and lens flexibility in a depth-dependent fashion during whole lens shape change. Fiber cell shape deformations under mechanical strain will be visualized by multiphoton imaging of fluorescent- labeled membranes in live lenses (mouse), membrane structures examined by scanning electron microscopy of lenses fixed under deformation (mouse and primate), and whole lens stiffness measured as a function of lens age. This work will elucidate the fundamental basis by which F-actin networks establish lens epithelial stability and fiber cell deformability to sustain lifelong lens transparency and flexibility. Identification of molecular targets in F-actin networks that control lens flexibility will provide candidates to devise future strategies to mitigate age-related cataracts and presbyopia, which is linked to age-dependent lens stiffening.

项目摘要 将光聚焦到视网膜上所需的终身镜头透明度和柔性形状，依赖于上皮 以及纤维细胞的形状和组织依赖于丝状（F-）肌动蛋白网络。上皮细胞 包含三个不同的F-肌动蛋白网络：横向细胞连接，基础应力纤维和独特的顶点多边形 数组。这些网络由稳定F-肌动蛋白以及非肌肉的型肌球蛋白（TPM）组成 肌球蛋白IIA（NMIIA），被认为会产生收缩或拉伸力来稳定上皮变形 和整个晶状体变化的完整性，但尚未进行测试。上皮细胞分化为 长而细的纤维细胞形成复杂的膜互锁突起和类似桨状的结构域，这些结构域改变了 随着成熟和深度。纤维细胞膜突起由F-肌动蛋白网络稳定支持 纤维细胞TPM3.5，调节F-肌动交联。在tpm3.5删除的镜头中，柔性交联，A- 肌动蛋白1在膜上增加，而刚性交联纤维蛋白（Plastin）降低。 TPM3.5- 耗尽的镜头降低了整个晶状体的刚度和弹性，表明F-肌动蛋白更柔软 网络允许更大的纤维细胞膜变形，从而导致较软的镜片。但是，机械 F-肌动蛋白网络，膜变形，蜂窝结构和整个晶状体形状变化之间的联系 不清楚。该提案的目的是确定上皮细胞和纤维细胞中的F-肌动蛋白网络 控制膜变形和细胞形状，以赋予整个晶状体透明度和柔韧性。到 解决这个问题，我们将使用鼠标透镜测试基因功能和灵长类动物镜头作为人镜的模型 形状变化。 AIM 1将检验以下假设：不同的F-肌动蛋白和NMIIA网络控制上皮 整个晶状体形状变化的变形和稳定性。与上皮F-肌动蛋白相关的TPM同工型 将确定网络，并通过荧光标记的TPM，F-肌动蛋白，NMIIA和细胞膜可视化 活细胞共聚焦显微镜研究整个晶状体形状期间网络动力学和细胞变形 更改。 F-肌动蛋白网络功能将由药理学（小鼠和灵长类动物）或遗传学 （鼠标）方法。 AIM 2将检验以下假设，即纤维细胞中TPM3.5调节的F-肌动蛋白网络会赋予 在整个晶状体形状变化过程中，膜变形和晶状体柔韧性以深度依赖性方式变化。 机械应变下的纤维细胞形状变形将通过荧光的多光子成像可视化 活镜片（小鼠）中的标记膜，通过扫描电子显微镜检查的膜结构 固定在变形下（小鼠和灵长类动物）的透镜，以及整个晶状体的刚度 镜头年龄。这项工作将阐明F-肌动蛋白网络建立镜头上皮的基本基础 稳定性和纤维细胞变形性，以维持终身透镜透明度和灵活性。识别 控制镜头灵活性的F-肌动蛋白网络中的分子靶标将为候选者设计未来 减轻与年龄相关的白内障和老花皮的策略，这与年龄依赖性晶状体僵硬有关。