Pseudo-obstruction–inducing ACTG2(R257C) alters actin organization and function

Actin γ 2, smooth muscle (ACTG2) R257C mutation is the most common genetic cause of visceral myopathy. Individuals with ACTG2 mutations endure prolonged hospitalizations and surgical interventions, become dependent on intravenous nutrition and bladder catheterization, and often die in childhood. Currently, we understand little about how ACTG2 mutations cause disease, and there are no mechanism-based treatments. Our goal was to characterize the effects of ACTG2(R257C) on actin organization and function in visceral smooth muscle cells. We overexpressed ACTG2(WT) or ACTG2(R257C) in primary human intestinal smooth muscle cells (HISMCs) and performed detailed quantitative analyses to examine effects of ACTG2(R257C) on (a) actin filament formation and subcellular localization, (b) actin-dependent HISMC functions, and (c) smooth muscle contractile gene expression. ACTG2(R257C) resulted in 41% fewer, 13% thinner, 33% shorter, and 40% less branched ACTG2 filament bundles compared with ACTG2(WT). Curiously, total F-actin probed by phalloidin and a pan-actin antibody was unchanged between ACTG2(WT)- and ACTG2(R257C)-expressing HISMCs, as was ultrastructural F-actin organization. ACTG2(R257C)-expressing HISMCs contracted collagen gels similar to ACTG2(WT)-expressing HISMCs but spread 21% more and were 11% more migratory. In conclusion, ACTG2(R257C) profoundly affects ACTG2 filament bundle structure, without altering global actin cytoskeleton in HISMCs.