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.
This proposal lays out a series of experiments intended to reveal how cells can orchestrate change in cell structure and function, in the absence of biosynthetic capability. The ocular lens is used as the model system, because of all vertebrate tissue it is the most amenable to high resolution mapping of cause (post translational modification of intermediate filament proteins) and effect (changes in cell structure), and because its cells are arranged in exact chronological order, so the changes can be mapped as a function of cellular age.
