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: 10630274
• 负责人: Velia M Fowler
• 金额: $ 34.73万
• 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/10630274
• 关键词: Actin-Binding Protein; Actinin; Actins; Address; Affect; Age; Anterior eyeball segment structure; Apical; Architecture; Behavior; Binding; 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; Elasticity; Elements; Epithelial Cells; Epithelium; 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; Physiological; Play; Presbyopia; Primates; Property; Protein Isoforms; Radial; Regulation; Reporter; Retina; Role; Scanning Electron Microscopy; Shapes; Stress Fibers; Structure; Testing; Thinness; Tissues; Tropomyosin; Visualization; Work; age related; confocal imaging; crosslink; fiber cell; flexibility; fluorescence imaging; gene function; lens; lens induction; lens transparency; mouse model; multiphoton imaging; multiphoton microscopy; nanoscale; non-muscle myosin; pharmacologic; 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 终生透明的晶状体和灵活的形状，是将光线聚焦到视网膜所必需的，依赖于上皮细胞 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：侧细胞连接、基础应力纤维和独特的顶端多边形 数组。这些网络由稳定F-肌动蛋白和非肌肉的原肌球蛋白(TPM)亚型组成 肌球蛋白IIA(NMIIA)，被认为能产生收缩或张力力以稳定上皮变形 和整个镜片形状变化过程中的完整性，但这还没有经过测试。上皮细胞分化为 细长的纤维细胞，形成复杂的膜互锁突起和改变的桨状结构域 成熟而有深度。纤维细胞膜突起由F-肌动蛋白网络支持， 纤维细胞Tpm3.5，它调节F-肌动蛋白交联物。在TPM3.5耗尽的镜片中，柔性交联剂a- 膜上的肌动蛋白1增加，而硬质交联剂纤毛蛋白(纤溶酶原)减少。Tpm3.5- 耗尽的晶状体降低了整个晶状体的硬度和弹性，这表明更灵活的F-肌动蛋白 网络允许更大的纤维细胞膜变形，从而导致更柔软的晶状体。然而，机械论 F-肌动蛋白网络、膜变形、细胞结构和整个晶状体形状变化之间的联系 都不清楚。这项建议的目的是确定F-肌动蛋白在上皮细胞和纤维细胞中的网络 控制薄膜变形和细胞形状，赋予整个镜头透明度和灵活性。至 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，并使用灵长类晶状体作为人类晶状体的模型 形状改变。目标1将检验不同的F-肌动蛋白和NMIIA网络控制上皮细胞的假设 在整个透镜形状变化过程中的变形和稳定性。与上皮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和细胞膜将被可视化 活细胞共聚焦显微镜研究整个晶状体形状过程中的网络动力学和细胞变形 改变。F-肌动蛋白网络功能将以药理学(小鼠和灵长类)或遗传学为靶点 (鼠标)接近。目标2将验证Tpm3.5调节的纤维细胞中的F-肌动蛋白网络提供 在整个晶状体形状变化过程中，膜的变形和晶状体的柔韧性随深度变化。 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)的标记膜，扫描电子显微镜观察膜结构 在变形下固定的镜片(老鼠和灵长类动物)，以及作为以下函数测量的整个镜片硬度 镜头年龄。这项工作将阐明F-肌动蛋白网络建立晶状体上皮细胞的基本基础 稳定性和纤维细胞变形性，终身保持镜片的透明度和灵活性。身份识别 F-肌动蛋白网络中控制晶状体灵活性的分子靶点将为设计未来提供候选 减轻年龄相关性白内障和老花眼的策略，这与年龄相关的晶状体僵硬有关。