Qdot Labeled Actin Super-Resolution Motility Assay Measures Step-Size for a Low Duty Cycle Muscle Myosin

Abstract

Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves the relative sliding of myosin and actin filaments that is sometimes modeled in vitro with a motility assay quantitating actin filament translation over a myosin coated surface. Skeletal actin filaments were fluorescence labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdot's were imaged in time as they translated with actin over the skeletal heavy meromyosin (HMM) coated surface using total internal reflection fluorescence microscopy. Qdots were spatially localized to a few nanometers using a super-resolution algorithm and tracked over time. Average Qdot-actin velocity matched measurements with rhodamine-phalloidin labeled actin. The Qdot-actin velocity histogram contained low velocity events corresponding to actin translation in quantized steps of ∼5 nm. The quantization was independent of myosin surface concentration. Skeletal or cardiac myosin is a low duty cycle motor presenting challenges for a single molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay enables modestly more actomyosin interactions to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space then characterized using super-resolution. The approach opens the way for quick, quantitative, and inexpensive step-size measurement in low duty cycle muscle myosins.This research was supported by NIH Institutes: NIAMS R01 AR049277 and NHLBI HL095572 and by the Mayo Foundation.