Characterization of the interactions of the Dictyostelium 34 kDa actin-bundling protein with actin filaments and the requirements for formation of Hirano bodies
Griffin, Paul Andrew
MetadataShow full item record
Hirano bodies are intracytoplasmic structures that are characterized by the presence of paracrystalline arrays of actin filaments. Hirano bodies have been found to be associated with a variety of neurodegenerative diseases; however, most of what is known about Hirano bodies has come from studies using autopsy derived tissue samples. Therefore little is known about the formation of Hirano bodies and their relationship to disease. Recently, a Hirano body cell model system has been developed based on expression of altered forms of the Dictyostelium 34 kDa actin-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 first study presented, I used F-actin cosedimentation assays to characterize a novel, mutant form of 34 kDa protein, E60K, and I showed that it had calcium-sensitive but activated actin binding. I also showed 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 formation of Hirano bodies. Lastly, I used the F-actin depolymerizing drug, latrunculin B and demonstrated that actin filaments within the Hirano bodies were resistant to depolymerization. In the second study presented here, I used chemical crosslinking and mass spectrometry to identify 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 three previously described actin binding regions (residues 1-123, 193-254, and 279-295). I also identified two distinct 34 kDa protein binding sites on F-actin and show that each of these sites are comprised of at least two actin subunits. Based on the results of my studies, I hypothesize that the formation of Hirano bodies requires both actin filament stabilization and crosslinking of actin filaments into ordered arrays.
Showing items related by title, author, creator and subject.
A functional genomic and genetic analysis of subclass III Actin Depolymerizing Factor (ADF) proteins in Arabidopsis thaliana King, Lori Marie (uga, 2011-12)The Actin Depolymerizing Factor (ADF)/Cofilin family of proteins are essential actin-binding proteins found in all eukaryotes. ADF proteins modulate actin filament dynamics by severing monomers from the pointed end of the ...
Molecular evolution and functional characterization of the Arabidopsis thaliana actin-depolymerizing factor gene family Roy-Zokan, Eileen Melanie (uga, 2012-12)The actin cytoskeleton is an essential component of eukaryotic cells and has had a profound impact on the evolution of multicellular organisms. The Actin-Depolymerizing Factor/Cofilin (ADF/CFL) gene family encode for a ...
Davis, Richard C (uga, 2002-12)Hirano Bodies are cytoplasmic rod-shaped paracrystalline F-actin containing structures that have been reported in association with neurodegenerative diseases as well as other conditions including alcoholism,diabetes,and ...