Electron Microscopic Evidence for the Cross-Bridge Lever Arm Mechanism in Living Muscle Thick Filaments Obtained using the Gas Environmental Chamber

Abstract

We have succeeded in recording the ATP-induced cross-bridge recovery stroke in living bipolar muscle thick filaments using the gas environmental chamber (EC) (Sugi et al., PNAS 105:17396, 2008). It is generally believed that the distal part of the cross-bridge (catalytic domain) is rigid, while its proximal part acts as a lever arm moving around the hinge to produce force and motion in muscle. To ascertain the validity of this mechanism by our experimental methods, we prepared three different antibodies, directed to the peptide in the cross-bridge catalytic domain (antibodiy 1), to the reactive lysine residue at interface between the catalytic and lever arm domains (antibody 2), and to the peptide in the cross-bridge lever arm domain (antibody 3), respectively. These antibodies, attached to the cross-bridges on the thick filaments, were position-marked with colloidal gold particles.The peak amplitude of the ATP-induced movement of the cross-bridges with antibody 1 (5∼7.5nm) did not differ significantly from that of the cross-bridges with antibody 2, being consistent with the idea that the cross-bridge catalytic domain remains rigid during the cross-bridge stroke. On the other hand, the amplitude of the ATP-induced movement in the cross-bridges with antibody 3 was found to be extremely small and in most cases just barely detectable (2.5nm or less), indicating that the proximal part of the cross-bridge (close to the lever arm hinge region) does not move appreciably during the ATP-induced cross-bridge stroke. These results may constitute the first direct evidence for the cross-bridge lever arm mechanism in muscle contraction.