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

通过中间丝的翻译后修饰调节晶状体纤维细胞结构和功能

基本信息

批准号：
9217353
负责人：
PAUL Gillespie FITZGERALD
金额：
$ 39.93万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-03-01 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9217353
关键词：
Affect Age Aging Alpha Cell Antibodies Appearance Atomic Force Microscopy Biological Models Biology Caliber Caspase Cell Differentiation process Cell Maturation Cell physiology Cells Cellular Structures Chronology Clustered Regularly Interspaced Short Palindromic Repeats Crystalline Lens Data Electron Microscopy Event Genes Genetic Engineering Genetic Transcription Human Intermediate Filament Proteins Intermediate Filaments Lens Fiber Life Light Lipids Maps Masks Mass Spectrum Analysis Mechanics Mediating Mitosis Modeling Modification Molecular Mus Mutation Neonatal Optics Organelles Organism Phenotype Phosphorylation Phosphorylation Site Post-Translational Modification Site Post-Translational Protein Processing Process Protein Biosynthesis Proteins Publishing Regulation Reporting Resistance Resolution Rest Series Site Structure Surface Testing Thick Time Tissues Translating Translations Work Zebrafish cell type experimental study fiber cell lens mechanical properties mouse model mutant programs protein function temporal measurement

项目摘要

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.

项目概要/摘要目镜是研究细胞和分子衰老的绝佳模型。镜头是排列成一系列同心壳，每个壳有 1 个纤维细胞厚。晶状体在一生中都会不断增长将新的 shell 添加到现有的 shell 中。因为细胞不会随着年龄的增长而丢失，所以外壳会按照出生日期的确切时间顺序排列，最年长的在中心，最小的在表面。人类有大约 2,500 个同心壳。更值得注意的是，纤维细胞仅保留所有膜细胞器，直到它们完成新壳的形成，他们被摧毁的那一点。在人类晶状体中，只有 2,500 层中的外部 100 层左右有细胞器。其他 2,400 层无法合成蛋白质，其寿命为没有蛋白质替代的生物体。尽管无法合成新的蛋白质、纤维细胞经历剧烈的结构变化，远远超过它们失去细胞器的程度。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一系列翻译后修饰（PTM）驱动的晶状体特异性中间丝 (IF) 蛋白。我们假设这些 PTM 发生了变化然后协调结构变化的蛋白质功能。晶状体生物学领域正在揭示在缺乏生物合成潜力的细胞中进行大量类似的过程。我们相信这个提案至少可以直接测试原因（PTM）和结果（结构变化）一组这些变化，并以细胞级分辨率进行。顺利完成本次该提案将建立一种机制，通过该机制细胞可以影响可预测的进展在没有蛋白质合成的情况下，随着时间的推移，结构会发生变化。为此，我们将确定 PTM，根据结构变化对它们进行本地化，然后直接通过以下方式测试 PTM 的效果：使用 CRISPR 对斑马鱼 IF PTM 位点进行基因修饰，然后转化关键观察结果从斑马鱼到小鼠模型。