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Abstract
Hirano bodies are intracytoplasmic structures that are characterized by the presence ofparacrystalline arrays of actin filaments. Hirano bodies have been found to be associated with avariety of neurodegenerative diseases; however, most of what is known about Hirano bodies hascome from studies using autopsy derived tissue samples. Therefore little is known about theformation of Hirano bodies and their relationship to disease. Recently, a Hirano body cell modelsystem has been developed based on expression of altered forms of the Dictyostelium 34 kDaactin-bundling protein. The goals of the studies presented in my dissertation were to: (1)determine the biochemical and structural features required for Hirano body formation; and (2)identify the important intermolecular interactions between 34 kDa protein and actin. In the firststudy presented, I used F-actin cosedimentation assays to characterize a novel, mutant form of 34kDa protein, E60K, and I showed that it had calcium-sensitive but activated actin binding. I alsoshowed that E60K protein was able to crosslink and bundle actin filaments in vitro.Furthermore, in vivo expression of E60K protein in Dictyostelium cells resulted in the formationof Hirano bodies. Lastly, I used the F-actin depolymerizing drug, latrunculin B anddemonstrated that actin filaments within the Hirano bodies were resistant to depolymerization.In the second study presented here, I used chemical crosslinking and mass spectrometry toidentify the intermolecular interactions involved at the 34 kDa protein-F-actin binding interface.My results show that the full length 34 kDa protein contacts actin at sites within all threepreviously described actin binding regions (residues 1-123, 193-254, and 279-295). I alsoidentified two distinct 34 kDa protein binding sites on F-actin and show that each of these sitesare comprised of at least two actin subunits. Based on the results of my studies, I hypothesizethat the formation of Hirano bodies requires both actin filament stabilization and crosslinking ofactin filaments into ordered arrays.