Characterization of Single- and Double-Headed Non-Muscle Myosin IIB Heavy Meromyosins using Single Molecule Techniques

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Non-muscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays important role in cell motility by maintaining cortical tension. It forms bipolar filaments with ∼14 myosin molecules on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (∼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 s-1), and as a result acto-NMIIB has the highest ADP-affinity reported so far for the myosin superfamily (<0.15 μM). To examine the kinetics of NMIIB at single-molecule level we used a dual-beam optical tweezers. A surface-immobilized bead was coated with single- (NMIIB-SH-HMM) and double-headed (NMIIB-HMM) heavy meromyosin-like molecules. We measured the lifetimes of unitary actomyosin interactions and determined the actin-detachment kinetics at two 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 (∼0.43 s-1). In case of NMIIB-HMM the detachment rates were ∼0.43 s-1 (1 mM ATP) and ∼0.28 s-1 (1 μM ATP). Also, we found that the power-stroke of NMIIB-SH-HMM and NMIIB-HMM were about 8 nm. No signs of processive stepping were observed in case of NMIIB-HMM. The detachment rates calculated from landing assays using NMIIB-SH-HMM-GFP and NMIIB-HMM-GFP were 0.58 and 0.57 s-1 at 1 mM ATP. The double-headed molecules were not motile, but we observed robust motility of minifilaments of full-length NMIIB-GFP. We will discuss our single-molecule results from the perspective of the essential cellular functions of NMIIB in cell locomotion, tension generation and maintenance.