Document Type


Date of Degree

Summer 2012

Degree Name

MS (Master of Science)

Degree In


First Advisor

Washington, M Todd

Second Advisor

Bartlett, Heather L

First Committee Member

Fuentes, Ernesto

Second Committee Member

Dagle, John


More than 30 missense mutations in the ACTA2 gene, which encodes α–smooth muscle (α–SM) actin, cause thoracic aortic aneurysms and dissections (TAAD). Aortic cell samples obtained from patients harboring the R256H mutation, the third most common of this group, reveal a lack of α–SM organization. However, the biochemical mechanisms contributing to this disorganization have yet to be elucidated. Biochemical analysis of these mutations is critical to understanding the mechanisms underlying this disease; however, this goal proves difficult due to the inability to obtain diseased aortic tissue, the difficulty in purifying pure mutant actin from smooth muscle tissue, and the absence of a sufficient animal model. The yeast Saccharomyces cerevisiae has a single actin-encoding gene, ACT1, that shares 86% homology with human α–SM actin, making it a viable model system for analysis of TAAD mutations. Regulation of actin function by actin binding proteins is conserved between yeast and humans and plays a key role in cytoskeletal assembly and disassembly events. Two such actin binding proteins are cofilin and Aip1p, which work together to facilitate filament disassembly. Cofilin severs actin filaments, an action enhanced by actin interacting protein 1 (Aip1p) through mechanisms that are not well understood. Normal regulation of filament disassembly is important for cell proliferation and migration. In vivo data obtained by lab colleagues reveal that, in the absence of Aip1p, cells have abnormal cytoskeleton and organelle morphologies and poor growth. This thesis summarizes the investigation into the regulation of R256H mutant actin by Aip1p. S. cerevisiae was used to express R256H actin as the sole actin in the cell, and the effect of the mutation was assessed in vitro. The mutant actin exhibited decreased thermal stability indicative of an effect on monomer integrity. Filament stability was also affected as evidenced by aberrant polymerization kinetics, and high critical concentration and phosphate release rates. While the mutant actin was more sensitive to cofilin-mediated severing, it was less sensitive to disassembly in the presence of Aip1p with incomplete oligomer breakdown at varying Aip1p concentrations. Collectively, these data suggest an alteration in the filament in the presence of the R256H mutation that interferes with proper actin–Aip1p interaction. The biochemical effects observed with this mutant suggest how its presence leads to α–SM cytoskeletal disorganization and TAAD.


Actin, Aip1p, Cofilin, TAAD


ix, 52 pages


Includes bibliographical references (pages 47-50).


Copyright 2012 Nicole Danielle Vanderpool

Included in

Biochemistry Commons