(A) Domain organization of the myosin heavy chain and myosin fragments used to study the biochemical properties of myosin. The top panel shows the myosin hexamer composed of two myosin heavy chains (green), two ELCs (light blue) and two RLCs (gray). The myosin motor domain, the light chain binding neck and the tail domain of the heavy chain are indicated. The bottom panel shows the double headed HMM fragment. The rational for the different myosin fragments lies in the different biochemical properties: Full-length myosin forms filaments, sediments at high speed and can be used in the in vitro motility assay. The HMM fragment is soluble under physiological salt concentrations and suitable to study kinetic properties, the interaction with actin, and regulatory properties including RLC phosphorylation. (B) Multiple sequence alignment of RLCs from different model organisms and RLC mutants. (Top) Sequence alignment showing the high degree of conservation between RLCs from different model organisms. Identical amino acids are colored brown. The primary phosphorylation site of MLCK corresponds to Serine-21 (orange), the secondary phosphorylation site to Threonine-20 (yellow) of the Drosophila RLC. (Bottom) Alignment showing the mutant RLCs used in this study and the respective amino acid substitutions at the sites corresponding to Threonine-20 and Serine-21 of the Drosophila RLC. Alanine replacements are shown in blue, Glutamate replacements in red. Alanine replacement in RLC-AA is expected to mimic the unphosphorylated RLC state, Glutamate replacement in RLC-EE the di-phosphorylated RLC state. RLC-AE is expected to mimic the mono-phosphorylated RLC state that is independent from upstream regulation, whereas RLC-AS and RLC-TA are phosphorylatable and coupled to upstream signaling in vitro and in vivo. Abbreviations used: Mm: Mus musculus, Myl9 (NP_742116.1): Rn: Rattus norvegicus, Myl9 (XP_006235463.1); Hs: Homo sapiens, Myl9 (CAG33124.1); Xl: Xenopus laevis, Myl9 (NP_001087016.1); Dr: Danio rerio, Myl9b (NP_998377.1); Dm: Drosophila melanogaster, Sqh (NP_511057.1). (C) Actin-activated ATP hydrolysis rate of RLC-TS and RLC mutants. Gray bars indicate the ATP hydrolysis rate at an actin concentration of 100 µM in the absence of RLC phosphorylation. Orange bars indicate the ATP hydrolysis rate at an actin concentration of 100 µM after RLC phosphorylation with MLCK, also indicated by a lowercase p. Note that the myosin activity in these assays are even lower than shown since direct actin-mediated hydrolysis of ATP likely accounts for a substantial portion of the ATPase of the unphosphorylated and mutant samples. (D) In vitro motility assay using RLC-TS and RLC mutants. No significant movement was observed for RLC-TS in the absence of MLCK phosphorylation and RLC-AA. n = 47–227 tracked filaments per RLC mutant. The data were corrected for the stage drift. (E) Actin cosedimentation assays of RLC-TS and RLC-AE, supernatants (S) and pellets (P) from mixtures of myosin, actin, and ATP as indicated. In the absence of actin, RLC-TS and RLC-AE remain in the supernatant. Binding of RLC-TS and RLC-AE to actin in the absence of ATP results in pelleting of the actomyosin complex. The presence of ATP disassembles the actomyosin complex and myosin mostly remains in the supernatant. The sedimentation of actin is independent of ATP. Soluble HMM fragments were used in this assay since myosin filaments sediment under the assay conditions. Red numbers indicate lanes.