Regulation of Lens Fiber Cell Structure and Function by Post Translational Modification of Intermediate Filaments

项目总结/摘要 眼透镜是研究细胞和分子老化的极好模型。该透镜 排列成一系列同心壳，每个壳1个纤维细胞厚。在整个生命过程中， 将新的shell添加到现有的shell中。因为没有细胞会随着年龄的增长而丢失， 按出生日期按确切的时间顺序排列，最年长的在中心，最年轻的在 表面上在人类中，这大约是2,500个同心壳。更值得注意的是， 保留它们所有的膜细胞器，直到它们完成新壳的形成， 在这一点上，他们被摧毁了。在人类晶状体中，2,500层中只有外层100层左右 有细胞器其他2,400层不能合成蛋白质， 没有蛋白质替代的生物体。尽管不能合成新的蛋白质， 细胞经历了巨大的结构变化，远远超过了它们失去细胞器的点。 根据初步数据和先前发表的工作，我们假设这些 结构变化由翻译后修饰（PTM）的进展提供动力， 晶状体特异性中间丝（IF）蛋白。我们假设这些PTM 蛋白质功能，然后协调结构变化。透镜生物学领域揭示了 在缺乏生物合成潜力的细胞中进行的大量类似过程。我们 我相信这个建议至少可以直接测试原因（PTM）和影响（结构变化） 一组这些变化，并做到这一点与细胞级的分辨率。成功完成本 该提案将建立一种机制，通过这种机制，细胞可以影响可预测的 随着时间的推移，在没有蛋白质合成的情况下，结构发生了变化。为此，我们将确定 PTM，相对于结构变化定位它们，然后通过以下直接测试PTM的效果： 使用CRISPR对斑马鱼IF PTM位点进行遗传修饰，然后将关键观察结果 从斑马鱼到小鼠模型。

Project Summary/Abstract The ocular lens is an excellent model in which to study cellular and molecular aging. The lens is arranged as a series of concentric shells each 1 fiber cell thick. Lenses grow throughout life by addition of new shells to existing shells. Because no cells are lost with age, the shells are arranged in exact chronological order by birthdate, with the oldest at the center, and youngest at the surface. In humans this is about 2,500 concentric shells. Even more remarkable, fiber cells retain all their membranous organelles only until they complete formation of a new shell, at which point they are destroyed. In human lenses, only the outer 100 or so of the 2,500 layers has organelles. The other 2,400 layers are incapable of protein synthesis, and live the lifetime of the organism without protein replacement. Despite the inability to synthesize new protein, fiber cells undergo dramatic structural changes well past the point at which they have lost organelles. On the basis of preliminary data, and previously published work, we hypothesize that these structural changes are powered by a progression of post translational modifications (PTMs) to lens-specific intermediate filament (IF) proteins. We hypothesize that these PTMs change protein function which then orchestrates structural change. The field of lens biology is revealing a large number of similar processes that progress in cells lacking biosynthetic potential. We believe this proposal can directly test the cause (PTM) and effect (structural change) for at least one set of these changes, and do so with cell‐level resolution. Successful completion of this proposal will establish a mechanism by which cells can effect a progression of predictable structural changes, over time, in the absence of protein synthesis. To do this, we will identify PTMs, localize them with respect to structural change, then test the effect of the PTM directly by using CRISPR to genetically modify zebrafish IF PTM sites, and then translate key observation from zebrafish, into a mouse model.