Abstract
Non-muscle myosin IIB (NMIIB) is a cytoplasmic conventional myosin, which plays an important role in development of the brain and heart, and in directed growth cone motility by maintaining cortical tension in motile cells. It forms short bipolar filaments with ∼14 myosin molecules on each side of the bare zone. NMIIB is a very slow myosin both in terms of actin-activated ATPase activity and actin translocation capability. Our previous studies showed that the NMIIB is a moderately high duty ratio (at least 20-25%) motor. The ADP release step (∼0.35 s-1), of NMIIB is only ∼3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s-1). Because of its slow ADP off-rate, acto-NMIIB has the highest ADP-affinity reported so far for the myosin superfamily (<0.15 μM). To examine the mechanics and kinetics of NMIIB at the single-molecule level we used a dual-beam optical tweezer to perform the three-bead assay. The surface-immobilized bead was coated with recombinantly engineered single-headed heavy meromyosin-like (NMIIB-SH-HMM) molecules. We measured the lifetimes of unitary actomyosin interactions and determined the actin-detachment kinetics with varying ATP concentrations. Results showed that at physiological ATP concentration (1 mM), the rate of detachment of acto-NMIIB-SH-HMM interactions was ∼0.51 s-1, similar to the ADP release rate and steady-state ATPase rate reported from solution kinetic studies. Decreasing the ATP concentration to 1 μM did not alter this rate of detachment (∼0.47 s-1). Also, our results showed that the power-stroke of NMIIB-SH-HMM was ∼8 nm. We will discuss our single-molecule results from the perspective of the essential cellular functions of NMIIB in cell locomotion, tension generation and maintenance.
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