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: 8680237
• 负责人: Velia M Fowler
• 金额: $ 45.25万
• 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/8680237
• 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（由适用提供）：脊椎动物镜头的透明度取决于将连接蛋白组装到间隙连接中，这是晶状体离子稳态和代谢物循环所需的。人类和小鼠中纤维细胞中的突变（CX46，CX50）导致透明度和白内障的丧失，这是全球失明的主要原因。在区分纤维（DF）细胞中，连接蛋白在纤维细胞宽边的大斑块中组装成大斑块，而在无细胞器的成熟纤维（MF）细胞中，斑块碎片和膜周围分散。谱蛋白和肌动蛋白丝（F-肌动蛋白）的交联网络连接到NRCAM和N-钙粘着蛋白，从而在透镜纤维细胞中产生微米尺度的膜亚域。 F-肌动蛋白的接头由tropomodulin1（TMOD1）盖住，并涂有肌球蛋白（？tm），稳定光谱蛋白 - 肌动蛋白网络完整性。我们已经表明，小鼠透镜中缺少TMOD1会导致F-肌动蛋白拆卸和光谱 - 肌动蛋白网络破坏以及异常的细胞形状。 TMOD1 - / - 透镜是透明的，但TMOD2水平升高，表明部分补偿。最近，与Rick Mathias合作，我们发现TMOD1 - / - 透镜中的间隙连接偶联电导降低到正常的一半，与静水压力和细胞内NA+的高度相一致。 CX46和CX50的总水平不变，但是共聚焦显微镜表明，DF Cel的宽面上的大型CX46和CX50斑块分散在TMOD1 - / - 镜头中的较小点。我们假设组装与其附着蛋白（NRCAM，N-钙粘着蛋白）相关的远程光谱 - 肌动蛋白网络形成了一个纤维细胞膜子域，该子膜子域排除了连接蛋白，从而“将”连接蛋白“ conroll” connexins connexins connexins connexins connexins connexins connexins所需我们将使用小鼠敲除模型检验该假设，以破坏F-肌动蛋白和弱谱 - 肌动蛋白网络链接（TMOD1 - / - ，TMOD2 - / - ，？TM - / - ），并增强网络链接（CALPAIN/CASPase/CASPase-CASPase-caspase-caspase-caspase-耐药»II-SPECTRIN突变）。具体目的是：1）通过分析TMOD1 - / - 和TMOD1 - / - ; TMOD2-/ - 镜头，研究TMOD在CX46和CX50组装中的作用以及功能在DF细胞的间隙连接板中的作用。 2）通过分析？ 3）用钙蛋白酶/caspase耐药»II-谱蛋白突变体研究MF细胞中的间隙连接，这有望防止MF（与J. Morrow）中的正常光谱 - 肌动蛋白网络拆卸。含有连接蛋白或光谱 - 肌动蛋白网络的纤维细胞膜子域将通过共焦和TIRF显微镜，通过生化方法的蛋白质关联，通过Freeze-fracture tem（W.-K。LO）和镜片稳态和镜头couplence（用coupplesias couppleysias coupplesias）来定义。这些研究将建立透镜透明度的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。