Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin.

Abstract

Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.

• Authors: Hailong Lu, Patricia M Fagnant, Carol S Bookwalter, Peteranne Joel, Kathleen M Trybus
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 112 Issue 31 Pg. E4168-77 (Aug 04 2015) ISSN: 1091-6490 [Electronic] United States
• PMID: 26153420 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• ACTA2 protein, human
• Actin Depolymerizing Factors
• Actins
• Gelsolin
• Mutant Proteins
• Profilins
• Tropomyosin
• Green Fluorescent Proteins
• Actomyosin
• Deoxyribonucleases
• Myosins

Topics

• Actin Cytoskeleton (metabolism)
• Actin Depolymerizing Factors (metabolism)
• Actins (genetics)
• Actomyosin (metabolism)
• Animals
• Chickens
• Deoxyribonucleases (metabolism)
• Electrophoresis, Polyacrylamide Gel
• Gelsolin (metabolism)
• Green Fluorescent Proteins (metabolism)
• Mice
• Microscopy, Fluorescence
• Models, Biological
• Models, Molecular
• Muscle, Smooth, Vascular (metabolism, pathology)
• Mutant Proteins (metabolism)
• Mutation (genetics)
• Myosins (metabolism)
• Polymerization
• Profilins (metabolism)
• Protein Binding
• Protein Stability
• Sf9 Cells
• Tropomyosin (metabolism)
• Vascular Diseases (genetics)