Myosin IIIA (MYO3A) is a molecular motor thought to play an important role in formation of the stereocilia of inner ear hair cells (IHC). Its load-dependent properties are thought to be critical in sensing and regulating membrane tension during IHC formation; however, these properties have not been characterized. Therefore, we investigated the effect of load on the single molecule kinetics of MYO3A in a three-bead laser trap assay using a single-headed (S1) 2IQ construct at 1 mM ATP. Load was varied by modulating the stiffness of the laser traps, from 0.02 to 0.2 pN.nm -1 . This approach allowed the myosin to experience forces ranging from ±5 pN as it bound to and translocated the trapped actin filament. MYO3A displayed a symmetric load-dependence about 0 pN of force such that at higher forces its rate of detachment from actin significantly ( p < 0.05) increased in response to both assistive (negative) and resistive (positive) forces. Fitting these detachment rates vs. force to the Bell equation (k det = k 0 × exp(F d /kT), which relates kinetics to load, quantified the degree of load sensitivity with the d value being 2 nm. This parameter was similar for both assistive and resistive forces. This symmetry of the load-dependence is distinctly different from the asymmetric load-dependence displayed by other myosins (e.g., myosin II and myosin Va). Subsequent experiments at low ATP (15μM) revealed that the detachment rate from actin was significantly slower at all forces and was unaffected by either the direction or the magnitude of the load. This suggests that assistive and resistive loads accelerate the rate of ADP release. This uniquely symmetrical load-dependence may be necessary for MYO3A’s critical role in mediating the length and width of inner ear hair cell stereocilia during development.
Boos et al. (Sun,) studied this question.