Phosphate Enhances Actin Filament Velocity at Low pH in an in vitro Motility Assay

Abstract

<0.15 mM). 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 mM did not alter this rate of detachment (~0.43 s-1). In case of NMIIB-HMM the detachment rates werẽ 0.43 s-1 (1 mM ATP) and~0.28 s-1 (1 mM 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 doubleheaded 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.