Characterization of the Functional Diversity of the Synthetic Nanomachine Powered by Different Muscle Myosin Isoforms

Abstract

The functional diversity of two ortholog isoforms of the fast-skeletal muscle myosin (amphibian and mammalian) can be precisely defined using a synthetic nanomachine and a simulation model able to interface the output of the nanomachine to that of the muscle of origin. The nanomachine is made by an ensemble of HMM fragments, carried on a piezoelectric nanopositioner and brought to interact with an actin filament attached with the correct polarity to a bead trapped into the focus of a Dual Laser (Pertici et al., Nat Commun 9:3532, 2018). In the present version, the bead-actin filament assembly is prepared with the Ca2+-insensitive gelsolin fragment TL40, to make [Ca2+] an independent parameter and allow future studies on the Ca2+-dependence of the sarcomeric proteins action. In this way, even in the simple actin-myosin assay, we reveal a class-specific Ca2+-sensitivity of the amphibian myosin performance. In 2 mM ATP, following the attainment of the isometric plateau force (F0), up to five different isotonic force-velocity points for each interaction are recorded by switching the control from position- to force-feedback. The different sensitivity to temperature of mammalian and frog fast-skeletal muscle performance is tested comparing the force-velocity relations of the two isoforms at the same temperature (∼24°C). Just like for the muscle of origin, the unloaded shortening velocity of the nanomachine powered by mammalian isoform is ∼1/2 that of the amphibian. Model simulation with a unique actin-myosin kinetic scheme allows interfacing in situ and in vivo performances, demonstrating the ability of the nanomachine to recapitulate the emergent properties of the half-sarcomere of the muscle of origin and identify, at the level of a specific protein, the functional diversity of muscles in vivo. Supported by IIT-SEED, Genova and Fondazione CR Firenze, Italy.