Membrane Skeleton Regulation of Cell Shape and Interactions in Lens Development

描述(申请人提供)：脊椎动物晶状体的透明度取决于缝隙连接蛋白的组装，缝隙连接是晶状体离子平衡和代谢物循环所必需的。人类和小鼠的纤维细胞连接蛋白(Cx46，CX50)突变导致晶状体透明度丧失和白内障，这是全球失明的主要原因。在分化纤维(DF)细胞中，连接蛋白在纤维细胞的宽面上聚集成大的斑块，而在无细胞器的成熟纤维(MF)细胞中，连接蛋白斑块碎裂并分散在膜周围。一个由血影蛋白和肌动蛋白细丝组成的交联网络(F-肌动蛋白)附着在NrCAM和N-钙粘蛋白上，在晶状体纤维细胞中创建微米级的膜亚结构域。F-肌动蛋白连接子由原模蛋白1(Tmod1)封端，并被原肌球蛋白(？TM)包裹，稳定了血影蛋白-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-肌动蛋白的分解和血影蛋白-肌动蛋白网络的破坏，并伴随着异常的细胞形状。Tmod1-/-晶状体是透明的，但Tmod2水平升高，提示部分代偿。最近，我们与Rick Mathias合作，发现Tmod1-/-晶状体的缝隙连接耦合电导降低到大约一半正常，与静水压和细胞内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，位于DF细胞宽侧的大的Cx46和CX50斑块分散在较小的斑点上。我们假设，一个与其附着蛋白(NrCAM，N-cadherin)相连的长程光谱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结构域，从而将连接蛋白‘聚集’到DF中最佳的纤维细胞连接所需的大间隙连接斑块中。我们将使用小鼠基因敲除模型来验证这一假设，该模型可以破坏F-肌动蛋白的稳定性和削弱血影蛋白-肌动蛋白的网络连接(Tmod1-/-、Tmod2-/-、？TM-/-)，并使用敲入模型来加强网络连接(Calain/caspase抗性的II-血影蛋白突变体)。1)通过对Tmod1-/-和Tmod1-/-；Tmod2-/-晶状体的分析，探讨Tmods在Df细胞Cx46和CX50组装中的作用及其在缝隙连接斑块中的作用。2)通过对TM外显子9D-/-晶状体的分析，研究TM对DF中光影蛋白-肌动蛋白网络稳定性、连接蛋白组装和缝隙连接偶联的调节作用。3)研究带有钙蛋白/半胱氨酸天冬氨酸氨基转移酶抗性突变体的MF细胞中的缝隙连接，该突变体有望防止MF中正常的幽门蛋白-肌动蛋白网络分解(与J.Morrow)。含有连接蛋白或光谱蛋白-肌动蛋白网络的纤维细胞膜亚结构域将通过共聚焦显微镜和TIRF显微镜来确定，蛋白质关联通过生化方法来确定，缝隙连接斑块的形态通过冷冻-断裂电子显微镜(W.K.Lo)来确定，晶状体离子的动态平衡和偶联通过整个晶状体的电生理来确定(与R.Mathias)。这些研究将建立缝隙连接控制晶状体透明度的基本机制，并为预防白内障的治疗提供新的见解。

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.