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Phalloidin and fluorescently labeled phalloidin analogs are established reagents to stabilize and mark actin filaments for the investigation of acto-myosin interactions. In the present study, we employed transient and steady-state kinetic measurements as well as in vitro motility assays to show that phalloidin perturbs the productive interaction of human non-muscle myosin-2A and -2C1 with filamentous actin. Phalloidin binding to F-actin results in faster dissociation of the complex formed with non-muscle myosin-2A and -2C1, reduced actin-activated ATP turnover, and slower velocity of actin filaments in the in vitro motility assay. In contrast, phalloidin binding to F-actin does not affect the interaction with human non-muscle myosin isoform 2B and Dictyostelium myosin-2 and myosin-5b.

Heterozygous dominant mutations in the ubiquitously produced cytoskeletal β-actin isoform lead to a broad range of human disease phenotypes, which are currently classified as three distinct clinical entities termed Baraitser-Winter-Cerebrofrontofacial syndrome (BWCFF), ACTB-associated pleiotropic malformation syndrome with intellectual disability (ACTB-PMSID), and ACTB-associated syndromic thrombocytopenia (ACTB-AST). The latter two are distinguishable from BWCFF by the presence of milder craniofacial features and less pronounced developmental abnormalities, or the absence of craniofacial features in combination with a characteristic thrombocytopenia with platelet anisotropy. Production and correct function of β-actin is required for multiple essential processes in all types of cells. Directed cell migration, cytokinesis and morphogenesis are amongst the functions that are supported by β-actin. Here we report the recombinant production and biochemical characterization of the ACTB-AST mutant p.S368fs, resulting in an altered sequence in the C-terminal region of β-actin that includes a replacement of the last 8 residues and an elongation of the molecule by 4 residues. The mutation affects a region important for actin polymerization and actin-profilin interaction. Accordingly, we measured markedly reduced rates of nucleation and polymerization during spontaneous actin assembly and lower affinity of p.S368fs for human profilin-1. The reduced affinity is also reflected in the lower propensity of profilin-1 to extend the nucleation phase of p.S368fs. While localized in close proximity to actin-cofilin and actin-myosin interfaces, we determined only minor effects of the mutation on the interaction of mutant filaments with cofilin and myosin family members. However, allosteric effects on sites distant from the mutation manifest themselves in a 7.9 °C reduction in thermal denaturation temperature, a 2-fold increase in the observed IC

Myosins are actin-based motor proteins that hydrolyze ATP to generate force and movement. Class I myosins are single-headed and can be subdivided into short and long-tailed isoforms. Myosin 1c is a short-tailed isoform involved in the insulin-dependent transport of GLUT4-storage vesicles and the adaptation response of the stereocilia in the inner ear. Both these processes are Ca2+-regulated. Here, we show how myosin 1c motor activity is regulated by calcium ion binding to calmodulin at the first IQ motif. We present the crystal structure of the human myosin 1c motor domain in complex with calmodulin bound to the first IQ motif in the pre-power stroke state at 2.7 A. The structure reveals a lever arm orientation that positions the Ca2+-binding lobes of calmodulin closely to the HO-linker of the motor domain. Deletion of a unique 6 amino acids insertion in the HO-linker abolished Ca2+-regulation of myosin 1c, as evidenced by a detailed comparative kinetic analysis of wild-type and deletion mutants. Our findings reveal an allosteric communication pathway, where the extended HO-linker senses Ca2+-induced changes in calmodulin. These changes are transduced via the HO-helix and the central β-sheet to residues that mediate coupling between the actin and nucleotide binding sites.