The ability to coordinate the timing of motor protein activation lies at the center of a wide range of cellular motile processes including endocytosis, cell division, and cancer cell migration. We show that calcium dramatically alters the conformation and activity of the myosin-VI motor implicated in pivotal steps of these processes. We resolved the change in motor conformation and in structural flexibility using single particle analysis of electron microscopic data and identified interacting domains using fluorescence spectroscopy. We discovered that calcium binding to calmodulin increases the binding affinity by a factor of 2,500 for a bipartite binding site on myosin-VI. The ability of calcium-calmodulin to seek out and bridge between binding site components directs a major rearrangement of the motor from a compact dormant state into a cargo binding primed state that is nonmotile. The lack of motility at high calcium is due to calmodulin switching to a higher affinity binding site, which leaves the original IQ-motif exposed, thereby destabilizing the lever arm. The return to low calcium can either restabilize the lever arm, required for translocating the cargobound motors toward the center of the cell, or refold the cargo-free motors into an inactive state ready for the next cellular calcium flux.unconventional myosin | electron microscopy | calmodulin I n human cells, cytoskeletal motor proteins move along microtubules and actin filaments to generate complex cellular functions that require a precise timing of motor activation and inactivation. Myosin-VI is thought to have unique properties because it is the only myosin in the human genome shown to move toward the minus end of actin filaments (1). Apart from its roles in the formation of stereocilia in cells of the auditory system (2, 3), membrane internalization (4-6), and delivery of membrane to the leading edge in migratory cells (7), myosin-VI is an early marker of cancer development, aggressiveness, and cancer-cell invasion because of its dramatically up-regulated expression in breast, lung, prostate, ovary, and gastresophagus carcinoma cells (7-11). How this motor might promote cancercell migration, proliferation, and survival is unknown.In migrating cells, localized calcium transients (∼50 nM to ∼10 μM) (12, 13) have been reported to play a multifunctional role in steering directional movement (14), cytoskeleton redistribution, and relocation of focal adhesions (15). The effect of calcium transients on the mobilization and cargo binding of myosin-VI and on its mechanical activation, however, are not understood. In the current model, the catalytic head domain hydrolyzes ATP, whereas the tail domain anchors the motor to specific compartments. In vitro studies have shown that calcium affects myosin-VI binding to phospholipids (6), as well as the kinetics and motility rate of the motor (16, 17). The underlying molecular mechanisms, however, are unknown. It has also been discussed that myosin-VI might be able to adopt an inactive folded state (18,19), perha...

Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor's oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite.unconventional myosin | motor properties

N-terminal extension that shows no similarity to any known protein domain. Here, we show that the human myosin-18B motor domain binds to F-actin with an affinity of 4 mM. The isolated motor domain binds ATP but has no intrinsic ATPase activity. The large N-terminal extension is shown to directly bind to F-actin with an affinity of 7 mM. This interaction is nucleotideindependent but shows strong ionic strength dependence, which is indicative for a charge-mediated actin binding mechanism. We further analyzed the molecular function of the N-terminal extension by means of actin polymerization assays and found that the myosin-18B N-terminus inhibits F-actin assembly in vitro. Myosin-18B has previously been shown to be located in the cytoplasm of undifferentiated myoblasts. At later stages of differentiation it accumulates in myonuclei. Furthermore, it has been shown that cardiomyocytes display a partial sarcomeric pattern of myosin-18B alternating that of a-actinin-2. Based on our data, we propose a role for myosin-18B in the regulation of muscle sarcomere architecture during differentiation and the regulation of the nuclear actin pool.