Membrane Skeleton Regulation of Cell Shape and Interactions in Lens Development

晶状体发育中细胞形状和相互作用的膜骨架调节

• 批准号: 8511653
• 负责人: Velia M Fowler
• 金额: $ 43.87万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8511653
• 关键词: Abnormal Cell; Actins; Affect; Age; Binding; Biochemical; Biochemistry; Blindness; Blood Circulation; Calpain; Caspase; Cataract; Cell Communication; Cell Differentiation process; Cell Shape; Cell membrane; Cells; Cleaved cell; Collaborations; Complex; Confocal Microscopy; Connexins; Coupling; Crystallins; Data; Development; Electrophysiology (science); Engineering; Equipment and supply inventories; Evaluation; Exons; F-Actin; Fiber; Financial compensation; Freeze Fracturing; Gap Junctions; Homeostasis; Human; Hydrostatic Pressure; Ions; Knock-in Mouse; Knock-out; Knockout Mice; Lead; Lens Fiber; Link; Liquid substance; Measures; Membrane; Microfilaments; Modeling; Molecular; Morphogenesis; Morphology; Mus; Mutation; N-Cadherin; Organelles; Pathway interactions; Phenotype; Precipitation; Prevention; Protein Isoforms; Proteins; Proteolysis; Regulation; Regulation of Cell Shape; Resistance; Role; Shapes; Side; Spectrin; Testing; Total Internal Reflection Fluorescent; Tropomyosin; Western Blotting; base; crosslink; fiber cell; induced pluripotent stem cell; insight; lens; lens transparency; membrane skeleton; mutant; prevent; programs; scaffold; solute

DESCRIPTION (provided by applicant): The transparency of the vertebrate lens depends on assembly of connexins into gap junctions that are required for lens ionic homeostasis and metabolite circulation. Mutations in fiber cell connexins (Cx46, Cx50) in humans and mice result in loss of lens transparency and cataracts, which are a major cause of blindness worldwide. In differentiating fiber (DF) cells, connexins assemble into large plaques on fiber cell broad sides, whereas in organelle-free mature fiber (MF) cells, the plaques fragment and disperse around the membranes. A cross-linked network of spectrin and actin filaments (F-actin), attaches to NrCAM and N-cadherin, creating micron-scale membrane subdomains in lens fiber cells. The F-actin linkers are capped by tropomodulin1 (Tmod1), and coated with tropomyosin (?TM), stabilizing spectrin-actin network integrity. We have shown that absence of Tmod1 in the mouse lens leads to F-actin disassembly and spectrin-actin network disruption, along with abnormal cell shapes. Tmod1-/- lenses are transparent, but Tmod2 levels are elevated, suggesting partial compensation. Recently, in collaboration with Rick Mathias, we found that gap junction coupling conductance in Tmod1-/- lenses is reduced to about half normal, consistent with ~2-fold elevations in hydrostatic pressure and intracellular Na+. Total levels of Cx46 and Cx50 are unchanged, but confocal microscopy reveals that the large Cx46 and Cx50 plaques on the broad sides of DF cels are dispersed to smaller puncta in Tmod1-/- lenses. We hypothesize that assembly of a long-range spectrin-actin network linked to its attachment proteins (NrCAM, N-cadherin) forms a fiber cell membrane subdomain that excludes connexins, thereby 'corralling' the connexins into the large gap junction plaques required for optimal fiber cell coupling in DF. We will test this hypothesis using mouse knock-out models to destabilize F-actin and weaken spectrin-actin network linkages (Tmod1-/-, Tmod2-/-, ?TM-/-), and a knock-in model to strengthen network linkages (calpain/caspase-resistant ¿II-spectrin mutant). The Specific Aims are: 1) to investigate the roles of Tmods in Cx46 and Cx50 assembly and function in gap junction plaques of DF cells by analysis of Tmod1-/- and Tmod1-/-;Tmod2-/- lenses. 2) To investigate TM regulation of spectrin-actin network stability, connexin assembly, and gap junction coupling in DF by analysis of ?TM?exon9d-/- lenses. 3) To investigate gap junctions in MF cells with a calpain/caspase- resistant ¿II-spectrin mutant that is expected to prevent normal spectrin-actin network disassembly in MF (with J. Morrow). Fiber cell membrane subdomains containing connexins or the spectrin-actin network will be defined by confocal and TIRF microscopy, protein associations by biochemical approaches, gap junction plaque morphology by freeze-fracture TEM (with W.-K. Lo) and lens ionic homeostasis and coupling by electrophysiology of whole lenses (with R. Mathias). These studies will establish basic mechanisms for gap junction control of lens transparency, and provide new insights for therapies in cataract prevention.

描述（由申请人提供）：脊椎动物晶状体的透明度取决于连接蛋白在晶状体离子稳态和代谢物循环所需的间隙连接中的组装。人类和小鼠纤维细胞连接蛋白（Cx46, Cx50）的突变会导致晶状体透明度下降和白内障，这是全世界失明的主要原因。在分化纤维（DF）细胞中，连接蛋白聚集成纤维细胞宽侧的大斑块，而在无细胞器的成熟纤维（MF）细胞中，斑块破碎并分散在膜周围。一个由谱蛋白和肌动蛋白丝（F-actin）组成的交联网络附着在NrCAM和n-钙粘蛋白上，在晶态纤维细胞中形成微米级的膜亚域。f -肌动蛋白连接体被原调节蛋白1 （Tmod1）覆盖，并被原肌凝蛋白(？TM)，稳定谱-肌动蛋白网络的完整性。我们已经证明，小鼠晶状体中缺乏Tmod1会导致f -肌动蛋白分解和谱蛋白-肌动蛋白网络破坏，以及异常的细胞形状。Tmod1-/-透镜透明，但Tmod2水平升高，提示部分代偿。最近，我们与Rick Mathias合作，发现Tmod1-/-透镜的缝隙结耦合电导降低到正常的一半左右，与静水压力和细胞内Na+升高2倍一致。Cx46和Cx50的总水平没有变化，但共聚焦显微镜显示，在Tmod1-/-晶状体中，DF细胞宽侧的大Cx46和Cx50斑块分散到更小的点上。我们假设，连接到其附着蛋白（NrCAM， n-钙粘蛋白）的远程谱蛋白-肌动蛋白网络的组装形成了一个排除连接蛋白的纤维细胞膜亚域，从而将连接蛋白“围捕”到DF中最佳纤维细胞偶联所需的大间隙连接斑块中。我们将使用小鼠敲除模型来验证这一假设，以破坏f -肌动蛋白的稳定并削弱谱蛋白-肌动蛋白网络连接(Tmod1-/-, Tmod2-/-, ？TM-/-)，以及加强网络连接的敲入模型（calpain/caspase抗性¿II-spectrin突变体）。具体目的是：1)通过分析Tmod1-/-和Tmod1-/-，探讨tmodds在DF细胞间隙连接斑块中Cx46和Cx50组装中的作用和功能；Tmod2 - / -镜头。2)通过分析？来研究TM对DF中谱蛋白-肌动蛋白网络稳定性、连接蛋白组装和间隙连接耦合的调控。TM吗?exon9d - / -镜头。3)研究具有calpain/caspase抗性的- II-spectrin突变体的MF细胞中的间隙连接，该突变体有望阻止MF中正常的spectrin-actin网络的分解（与J. Morrow合作）。含有连接蛋白或谱蛋白-肌动蛋白网络的纤维细胞膜亚结构域将通过共聚焦和TIRF显微镜来定义，通过生化方法来定义蛋白质关联，通过冷冻断裂TEM(与w - k。Lo)和晶状体的离子稳态和整个晶状体的电生理耦合（with R. Mathias）。这些研究将建立晶状体透明间隙连接控制的基本机制，并为预防白内障的治疗提供新的见解。