Myosin X is involved in the reorganization of the actin cytoskeleton and protrusion of filopodia. Here we studied the molecular mechanism by which bovine myosin X is regulated. The globular tail domain inhibited the motor activity of myosin X in a Ca(2+)-independent manner. Structural analysis revealed that myosin X is monomeric and that the band 4.1-ezrin-radixin-moesin (FERM) and pleckstrin homology (PH) domains bind to the head intramolecularly, forming an inhibited conformation. Binding of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P(3)) to the PH domain reversed the tail-induced inhibition and induced the formation of myosin X dimers. Consistently, disruption of the binding of PtdIns(3,4,5)P(3) attenuated the translocation of myosin X to filopodial tips in cells. We propose the following mechanism: first, the tail inhibits the motor activity of myosin X by intramolecular head-tail interactions to form the folded conformation; second, phospholipid binding reverses the inhibition and disrupts the folded conformation, which induces dimer formation, thereby activating the mechanical and cargo transporter activity of myosin X.

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli.

Myosin VIIA is an unconventional myosin, responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Here, we studied the molecular mechanism of regulation of myosin VIIA, which is currently unknown. Although it was originally thought that myosin VIIA is a dimeric myosin, our electron microscopic (EM) observations revealed that full-length Drosophila myosin VIIA (DM7A) is a monomer. Interestingly, the tail domain markedly inhibits the actin-activated ATPase activity of tailless DM7A at low Ca 2؉ but not high Ca 2؉ . By examining various deletion constructs, we found that deletion of the distal IQ domain, the C-terminal region of the tail, and the N-terminal region of the tail abolishes the tail-induced inhibition of ATPase activity. Singleparticle EM analysis of full-length DM7A at low Ca 2؉ suggests that the tail folds back on to the head, where it contacts both the motor core domain and the neck domain, forming an inhibited conformation. We concluded that unconventional myosin that may be present a monomer in the cell can be regulated by intramolecular interaction of the tail with the head. molecular motor ͉ regulation ͉ unconventional myosin ͉ Usher syndrome type 1B ͉ EM images

Myosin VIIA, thought to be involved in human auditory function, is a gene responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Recent studies have suggested that it can move processively if it forms a dimer. Nevertheless, it exists as a monomer in vitro, unlike the well-known two-headed processive myosin Va. Here we studied the molecular mechanism, which is currently unknown, of activating myosin VIIA as a cargo-transporting motor. Human myosin VIIA was present throughout cytosol, but it moved to the tip of filopodia upon the formation of dimer induced by dimer-inducing reagent. The forced dimer of myosin VIIA translocated its cargo molecule, MyRip, to the tip of filopodia, whereas myosin VIIA without the forced dimer-forming module does not translocate to the filopodial tips. These results suggest that dimer formation of myosin VIIA is important for its cargo-transporting activity. On the other hand, myosin VIIA without the forced dimerization module became translocated to the filopodial tips in the presence of cargo complex, i.e., MyRip/Rab27a, and transported its cargo complex to the tip. Coexpression of MyRip promoted the association of myosin VIIA to vesicles and the dimer formation. These results suggest that association of myosin VIIA monomers with membrane via the MyRip/Rab27a complex facilitates the cargo-transporting activity of myosin VIIA, which is achieved by cluster formation on the membrane, where it possibly forms a dimer. Present findings support that MyRip, a cargo molecule, functions as an activator of myosin VIIA transporter function.

Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.