The molecular motor, Myo1c, a member of the myosin family, is widely expressed in vertebrate tissues. Its presence at strategic places in the stereocilia of the hair cells in the inner ear and studies using transgenic mice expressing a mutant Myo1c that can be selectively inhibited implicate it as the mediator of slow adaptation of mechanoelectrical transduction, which is required for balance. Here, we have studied the structural, mechanical and biochemical properties of Myo1c to gain an insight into how this molecular motor works. Our results support a model in which Myo1c possesses a strain-sensing ADPrelease mechanism, which allows it to adapt to mechanical load.
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...
DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.
Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/ extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein's mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s −1 ) is similar to the maximum ATPase activity (∼12-14 s −1 ) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircaselike processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.actomyosin | stable single alpha-helix | optical trapping | myosin X | myosin-5a
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