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

项目摘要/摘要 人工晶状体是研究细胞和分子衰老的极佳模型。镜片是 排列成一系列同心的壳，每个壳有1个纤维细胞厚。晶状体在一生中都会生长 在现有壳的基础上添加新壳。因为细胞不会随着年龄的增长而丢失，所以贝壳 按照出生日期的确切时间顺序排列，最大的在中心，最小的在 表面。在人类身上，这大约是2500个同心壳。更值得注意的是，纤维细胞 仅保留它们的所有膜细胞器，直到它们完全形成新的壳为止，在 在这一点上他们被摧毁了。在人类的晶状体中，只有2500层中的100层左右 有细胞器。其他2400层不能合成蛋白质，它们的寿命是 没有蛋白质替代的有机体。尽管无法合成新的蛋白质、纤维 细胞经历了巨大的结构变化，远远超过了细胞器丢失的程度。 根据初步数据和之前发表的工作，我们假设这些 结构变化是由翻译后修饰(PTM)的过程推动的 晶状体特异的中间丝(IF)蛋白。我们假设这些PTM会改变 蛋白质的功能，然后协调结构的变化。晶状体生物学领域很有启发性 在缺乏生物合成能力的细胞中进行的大量类似的过程。我们 我相信这一建议至少可以直接检验因果关系(PTM)和结果(结构变化) 一组这样的更改，并以细胞级别的分辨率进行更改。成功完成这项工作 提案将建立一种机制，通过该机制，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，结构会发生变化。要做到这一点，我们将确定 PTM，根据结构变化进行本地化，然后通过以下方式直接测试PTM的效果 利用CRISPR对斑马鱼IF PtM位点进行遗传修饰，然后将关键观察结果转化为 从斑马鱼，变成老鼠模型。

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.