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（由申请方提供）：脊椎动物透镜的透明度取决于连接蛋白组装成间隙连接，这是透镜离子稳态和代谢物循环所需的。人类和小鼠中纤维细胞连接蛋白（Cx46、Cx 50）的突变导致透镜透明度的丧失和白内障，这是全世界失明的主要原因。在分化的纤维（DF）细胞，连接蛋白组装成大斑块的纤维细胞宽侧，而在无细胞器的成熟纤维（MF）细胞，斑块的碎片和分散在膜周围。血影蛋白和肌动蛋白丝（F-肌动蛋白）的交联网络附着于NrCAM和N-钙粘蛋白，在透镜纤维细胞中产生微米级膜子域。F-actin连接体由原调节蛋白1（Tmod 1）封端，并被原肌球蛋白（？）TM），稳定血影蛋白-肌动蛋白网络的完整性。我们已经证明，小鼠透镜中Tmod 1的缺失导致F-肌动蛋白分解和血影蛋白-肌动蛋白网络破坏，沿着异常的细胞形状。Tmod 1-/-晶状体是透明的，但Tmod 2水平升高，表明部分代偿。最近，在与Rick Mathias的合作中，我们发现Tmod 1-/-晶状体中的间隙连接耦合电导降低到正常的一半左右，与静水压力和细胞内Na+升高约2倍一致。Cx46和Cx 50的总水平没有变化，但共聚焦显微镜显示，在Tmod 1-/-晶状体中，DF晶状体宽侧上的大Cx46和Cx 50斑块分散到较小的斑点中。我们假设，一个长距离的血影蛋白-肌动蛋白网络连接到其附着蛋白（NrCAM，N-钙粘蛋白）的组装形成了一个纤维细胞膜亚结构域，不包括连接蛋白，从而“corraling”的连接蛋白到大的间隙连接斑块所需的最佳纤维细胞耦合DF。我们将使用小鼠敲除模型来验证这一假设，以使F-肌动蛋白不稳定并削弱血影蛋白-肌动蛋白网络连接（Tmod 1-/-，Tmod 2-/-，？TM-/-），和一个敲入模型，以加强网络连接（钙蛋白酶/半胱天冬酶抗性突变体）。具体目标是：1）通过对Tmod 1-/-和Tmod 1-/-; Tmod 2-/-晶状体的分析，探讨Tmods在DF细胞缝隙连接斑块中Cx46和Cx 50组装和功能中的作用。2)通过对纤维连接蛋白的分析，探讨TM对纤维连接蛋白-肌动蛋白网络稳定性、连接蛋白组装和间隙连接偶联的调控作用。TM？外显子9D-/-晶状体。3)研究具有钙蛋白酶/半胱天冬酶抗性的<$II-血影蛋白突变体的MF细胞中的间隙连接，该突变体预计会阻止MF中正常血影蛋白-肌动蛋白网络的分解（与J. Morrow一起）。包含连接蛋白或血影蛋白-肌动蛋白网络的纤维细胞膜亚结构域将通过共聚焦和TIRF显微镜来确定，蛋白质缔合通过生物化学方法来确定，间隙连接斑块形态通过冷冻断裂TEM（与W. K. Lo）和透镜离子稳态以及整个晶状体的电生理耦合（用R. Mathias）。这些研究将为透镜透明性的缝隙连接调控奠定基础，并为白内障的防治提供新的思路。