Direct visualization of human myosin II force generation using DNA origami-based thick filaments

Area of Science:

• Muscle physiology
• Biophysics
• Molecular motor mechanics

Background:

• The sarcomere is the fundamental contractile unit of muscle, comprising myosin thick filaments and actin thin filaments.
• Understanding myosin's force generation mechanism within the sarcomere's geometric constraints is crucial but remains challenging.
• Existing synthetic filaments do not fully replicate the native bipolar structure of sarcomeric thick filaments.

Purpose of the Study:

• To engineer a novel system for directly visualizing single myosin head dynamics during force generation.
• To investigate how geometric restrictions within the sarcomere influence myosin's mechanical cycle.
• To elucidate the precise steps and motion of myosin heads under confinement.

Main Methods:

• Development of DNA origami-based synthetic thick filaments incorporating human muscle myosin.
• Direct visualization of myosin head movement during force generation within the engineered restricted environment.
• Analysis of myosin head diffusion, binding, and lever-arm swing dynamics.

Main Results:

• Myosin heads exhibit Brownian ratchet behavior, weakly interacting with actin before strong binding.
• Strong binding preferentially occurs in the forward direction of motion.
• The myosin lever-arm swing, a two-step process, predominantly halts at the first step and can occasionally reverse.
• The engineered system effectively recapitulates myosin behavior in a restricted space.

Conclusions:

• DNA origami provides a powerful platform for dissecting motor protein mechanisms in confined environments.
• Myosin force generation is significantly influenced by the geometric restrictions of the sarcomere.
• The observed halting and occasional reversal of the lever-arm swing offer new insights into muscle contraction dynamics.