␤-and ␥-nonmuscle actins differ by 4 amino acids at or near the N terminus and distant from polymerization interfaces. ␤-Actin contains an Asp 1 -Asp 2 -Asp 3 and Val 10 whereas ␥-actin has a Glu 1 -Glu 2 -Glu 3 and Ile 10 . Despite these small changes, conserved across mammals, fish, and birds, their differential localization in the same cell suggests they may play different roles reflecting differences in their biochemical properties. To test this hypothesis, we established a baculovirus-driven expression system for producing these actins in isoform-pure populations although contaminated with 20 -25% insect actin. Surprisingly, Ca-␥-actin exhibits a slower monomeric nucleotide exchange rate, a much longer nucleation phase, and a somewhat slower elongation rate than ␤-actin. In the Mg-form, this difference between the two is much smaller. Ca-␥-actin depolymerizes half as fast as does ␤-actin. Mixing experiments with Caactins reveal the two will readily co-polymerize. In the Ca-form, phosphate release from polymerizing ␤-actin occurs much more rapidly and extensively than polymerization, whereas phosphate release lags behind polymerization with ␥-actin. Phosphate release during treadmilling is twice as fast with ␤-as with ␥-actin. With Mg-actin in the initial stages, phosphate release for both actins correlates much more closely with polymerization. Calcium bound in the high affinity binding site of ␥-actin may cause a selective energy barrier relative to ␤-actin that retards the equilibration between G-and F-monomer conformations resulting in a slower polymerizing actin with greater filament stability. This difference may be particularly important in sites such as the ␥-actin-rich cochlear hair cell stereocilium where local mM calcium concentrations may exist.

Background: Tropomodulins (Tmods) cap pointed ends of actin filaments in a tropomyosin (TM)-dependent manner. Results: Tmod1 and Tmod3 similarly cap actin filaments with diverse TM and actin isoforms, but only Tmod3 sequesters ␤-and ␥ cyto -actin monomers. Conclusion: Isoform-specific actin monomer sequestration by Tmod3 may provide a mechanism for actin remodeling in TM-deficient regions of cells. Significance: Defining the actin-regulatory activities of Tmods illuminates cytoskeletal dynamics.

Background:The biochemical mechanisms underlying ␣-smooth muscle actin-mediated vascular disease are unknown. Results: The R256H mutation in actin alters polymerization kinetics and causes misregulation by the nucleation factor, formin. Conclusion: Mutation-based changes in conformation affect filament stability and regulation of polymerization. Significance: The Arg-256 residue stabilizes the actin helix and maintains filament conformation required for formin regulation.

Twenty-two missense mutations in ACTA2, which encodes ␣-smooth muscle actin, have been identified to cause thoracic aortic aneurysm and dissection. Limited access to diseased tissue, the presence of multiple unresolvable actin isoforms in the cell, and lack of an animal model have prevented analysis of the biochemical mechanisms underlying this pathology. We have utilized actin from the yeast Saccharomyces cerevisiae, 86% identical to human ␣-smooth muscle actin, as a model. Two of the known human mutations, N115T and R116Q, were engineered into yeast actin, and their effect on actin function in vivo and in vitro was investigated. Both mutants exhibited reduced ability to grow under a variety of stress conditions, which hampered N115T cells more than R116Q cells. Both strains exhibited abnormal mitochondrial morphology indicative of a faulty actin cytoskeleton. In vitro, the mutant actins exhibited altered thermostability and nucleotide exchange rates, indicating effects of the mutations on monomer conformation, with R116Q the most severely affected. N115T demonstrated a biphasic elongation phase during polymerization, whereas R116Q demonstrated a markedly extended nucleation phase. Allele-specific effects were also seen on critical concentration, rate of depolymerization, and filament treadmilling. R116Q filaments were hypersensitive to severing by the actinbinding protein cofilin. In contrast, N115T filaments were hyposensitive to cofilin despite nearly normal binding affinities of actin for cofilin. The mutant-specific effects on actin behavior suggest that individual mechanisms may contribute to thoracic aortic aneurysm and dissection.

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a "parked" conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by approximately 5 degrees per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by approximately 1 degrees per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.