High-speed AFM reveals subsecond dynamics of cardiac thin filaments upon Ca ²⁺ activation and heavy meromyosin binding

Abstract

High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin–troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

Visualizing dynamic actin cross‐linking processes driven by the actin‐binding protein anillin

“…A). The usefulness of this lipid bilayer has been demonstrated in Hs‐AFM studies on the interactions between actin filaments and several actin‐binding proteins . First, we visualized actin filaments and confirmed that they were right‐handed double‐helical filaments (Fig.…”

Thermal Activation of Thin Filaments in Striated Muscle

“…When [Ca 2+ ] i is increased, Ca 2+ -bound TnC interacts with TnI, and the carboxyl-terminal domain of TnI is dissociated from actin. The Tn conformational changes result in displacement of Tm on actin, which subsequently induces myosin binding to actin and force generation (e.g., Haselgrove, 1973;Huxley, 1973;Lehman et al, 1994;Vibert et al, 1997;Xu et al, 1999;Fukuda et al, 2009;Risi et al, 2017;Matusovsky et al, 2019). It has been reported that during the shift of the thin filament state from "off " to "on, " strongly bound myosin cooperatively enhances binding of neighboring myosin molecules that have ATP and thereby potentially produce force (Greene and Eisenberg, 1980;Trybus and Taylor, 1980).…”

Review: Cantilever-Based Sensors for High Speed Atomic Force Microscopy

“…Yu et al [ 260 ] applied the high-speed atomic force microscope with ultra-short cantilevers to unfold the individual bacteriorhodopsin molecules in a native lipid bilayer. Matusovsky and co-workers [ 261 ] studied the 3-state model of activation of cardiac thin filaments isolated as a complex and deposited on a mica-supported lipid bilayer. They realized that the successful imaging of the regulatory proteins tropomyosin and troponin complexes is dependent on the force applied by the cantilever tip because of their low affinity to F-actin.…”