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 终生的透镜透明度和灵活的形状，需要将光线聚焦到视网膜上，依赖于上皮细胞。 以及纤维细胞，其形状和组织取决于丝状（F-）肌动蛋白网络。上皮细胞 包含三种不同的F-肌动蛋白网络：侧细胞连接，基底应力纤维和独特的顶端多边形 阵这些网络由稳定F-肌动蛋白的原肌球蛋白（Tpm）异构体以及非肌肉细胞组成。 肌球蛋白IIA（NMIIA），并被认为产生收缩力或张力以稳定上皮变形 以及在整个透镜形状变化期间的完整性，但这还没有被测试。上皮细胞分化为 长而细的纤维细胞，形成复杂的膜联锁突起和桨状域， 成熟和深度。纤维细胞膜突起由F-肌动蛋白网络支撑， 纤维细胞Tpm 3.5，其调节F-肌动蛋白交联剂。在Tpm 3.5耗尽的透镜中，柔性交联剂，a- 肌动蛋白1在膜上增加，而刚性交联剂膜（plastin）减少。Tpm3.5- 耗尽晶状体的整个透镜硬度和弹性降低，表明更灵活的F-肌动蛋白 网状结构允许更大的纤维细胞膜变形，从而导致更软的透镜。然而，机械 F-肌动蛋白网络、膜变形、细胞结构和整个透镜形状变化之间的联系 不清楚。本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 控制膜变形和蜂窝形状以赋予整个透镜透明度和柔韧性。到 为了解决这个问题，我们将使用小鼠晶状体来测试基因功能，并将灵长类晶状体作为人类透镜的模型 形状变化。目的1将检验不同的F-肌动蛋白和NMIIA网络控制上皮细胞的假说。 在整个透镜形状变化期间的变形和稳定性。与上皮细胞F-肌动蛋白相关的Tpm亚型 网络将被确定，荧光标记的Tpms，F-肌动蛋白，NMIIA和细胞膜可视化， 活细胞共聚焦显微镜用于研究整个透镜形状期间的网络动力学和细胞变形 变化F-肌动蛋白网络功能将通过药理学（小鼠和灵长类动物）或遗传学（如， （老鼠）接近。目的2将检验Tpm 3.5调节的纤维细胞中的F-肌动蛋白网络赋予纤维细胞中的F-actin网络的假设。 在整个透镜形状改变期间以依赖于深度的方式的膜变形和透镜柔性。 纤维细胞在机械应变下的形状变形将通过荧光的多光子成像来可视化。 活体晶状体（小鼠）中的标记膜，通过扫描电子显微镜检查的膜结构 在变形下固定的晶状体（小鼠和灵长类动物），以及作为以下函数测量的整个透镜刚度 透镜年龄。这项工作将阐明F-肌动蛋白网络建立透镜上皮的基本基础 以维持终身透镜透明度和柔韧性。鉴定 控制透镜灵活性的F-肌动蛋白网络中的分子靶标将为设计未来的 减轻与年龄相关的白内障和老花眼的战略，老花眼与年龄相关的透镜硬化有关。