Related Experiment Video
Updated: Mar 9, 2026

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays
Published on: February 4, 2021
Actomyosin interaction at low ATP concentrations
Manuela Maffei1, Emanuela Longa2, Antonio Sabatini3
1Department of Molecular Medicine, University of Pavia, Via Forlanini 6, 27100, Pavia, Italy.
Abstract:
In vitro motility assay (IVMA) experiments were performed to analyze the movement of actin filaments sliding on a pavement of myosin molecules at different [ATP] and [ADP]. In standard experimental conditions at [ATP] = 2 mM, about 80% of the actin filaments move in unloaded conditions with a constant velocity. However, a fraction of at least 20% static actin filaments is always present. The accepted explanation is the occurrence of damaged "rigor"-like myosin heads that do not undergo the normal ATP-dependent cycling motion. However, in a series of IVMA experiments performed at different [ATP] we observed that the mobility of actin filaments increased with lowering [ATP]. We investigated the influence of [ATP] on the number of mobile actin filaments. IVMA experiments were performed at controlled nucleotide concentrations and the percentage of mobile filaments accurately determined by specific operator-guided software. The value of ΔG ATP involved was determined. Results showed that the number of mobile actin filaments sliding on type 2B heavy meromyosin isoform (2B HMM) increased at very low [ATP] accompanied by less negative ΔG ATP values. Similar results were obtained by increasing [ADP]. Performing experiments at the same [ATP] with different myosin types, we found a higher number of mobile actin filaments on slow type 1 HMM with respect to type 2B HMM while the highest number of mobile actin filaments was found on single-head myosin (S1 fraction). We also found that [ATP] did not influence the percentage of mobile actin filaments sliding on S1. Our results reveal novel aspects of actomyosin interaction.
Related Concept Videos
Actin and Myosin in Muscle Contraction
ATP Synthase: Structure
Overview of Myosin Structure and Function
ATP Synthase: Mechanism
Cross-bridge Cycle
Actin Treadmilling

