Thesis (Ph.D.)--State University of New York at Stony Brook, 2001
During retinal growth and optic axon regeneration, the differential expression of the neuronal intermediate filament (nIF) proteins plasticin and gefiltin in the goldfish visual pathway suggests that these proteins support programmed axonal growth. To investigate plasticin and gefiltin during axonogenesis, I turned to the zebrafish, a system that is more amenable to mutational analysis. As a first step, I demonstrated that the intermediate filament composition of goldfish and zebrafish are similar. In addition, the cDNAs for zebrafish plasticin and gefiltin were cloned and characterized. I showed increased mRNA levels for these proteins following optic nerve crush. Furthermore, in the unoperated eye of experimental fish, there is a moderate increase in the levels of plasticin and gefiltin mRNA suggesting that soluble factors influence the expression of these proteins. The successive expression of plasticin and gefiltin suggests that these nIF proteins are integral components of axonogenesis
The assembly characteristics of the nIF protein plasticin were studied in SW13 cells in the presence and absence of a cytoplasmic filament network. Full length plasticin is unable to polymerize into homopolymers in filament-less SW13c1.2Vim<super>−</super> cells, but efficiently coassembles with vimentin in SW13c1.1Vim<super>+</super> cells. The assembly properties of plasticin monomers bearing point mutations analogous to the keratin mutations that cause Epidermolysis Bulosa Simplex were assayed. A point mutation in the helix termination sequence resulted in complete filament aggregation when coexpressed with vimentin, but showed limited coassembly with neurofilament-low (NF-L) and neurofilament-medium (NF-M). A point mutation in the first heptad of the alpha-helical coil region equally formed filaments, aggregates, and a mixture of filaments and aggregates in transfected SW13c1.1Vim<super> +</super> cells. Moreover, a shift toward aggregation was observed when coexpressed with NF-L and NF-M
The effects of mutant plasticin expression on the development of the early zebrafish nervous system were analyzed using microinjection methodologies. Surprisingly, axons expressing ectopic plasticin had normal calibers and proper pathfinding trajectories
I propose that plasticin expression may be a strategy to increase axonal plasticity during development, and regeneration, when elongating neurons encounter environments that demand increased axonal flexibility
